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Publications
Banday AR, Azim S, Tabish M. Alternative promoter usage and differential expression of
multiple transcripts of mouse Prkar1a gene. Mol Cell Biochem. 2011 Jun 3. [Epub ahead
of print]
Banday AR, Azim S, Tabish M. Differentially expressed three non-coding alternate exons
at 5' UTR of regulatory type I beta subunit gene of mouse. Mol Biol Rep. 2011 Jun 26.
[Epub ahead of print]
Azim S, Banday AR, Tabish M. Alternatively Spliced Three Novel Transcripts of gria1 in
the Cerebellum and Cortex of Mouse Brain. Neurochem Res. 2011 Sep 17. [Epub ahead of
print]
Banday AR, Azim S, Tabish M. Two Novel N-terminal coding exons of Prkar1b gene of
mouse- Identified using novel in silico pipeline confirmed by molecular biology
techniques. Communicated
Banday AR, Azim S, Tabish M. Identification and expression analysis of three novel
splice variants of PKA catalytic beta subunit gene in mouse: combinatorial in silico and
molecular biology approaches. Communicated
Banday AR, Azim S, Tabish M. Prediction and identification of new splice variant of
Prkaca gene in mouse. Communicated
Banday AR, Azim S, Ola M, Tabish M. PKA at interfaces of different disease status: an
overview. Communicated
Abstracts in international conferences
Banday AR, Azim S, Tabish M. Novel transcript variants arise from alternate promoter
usage and alternative splicing in 5' regions of cAMP-dependent Protein kinase A subunit
genes in mouse FASEB J March 17, 2011 25:900.3. Presented paper in Experimental
Biology-2011, April 9-13, Walter E. Washington Convention Center, Washington, DC.
Banday AR, Azim S, Tabish M “Computational and molecular analysis of gene encoding
PKA Cα-subunit in mouse” Abstract No. 105 (Page 23). The Eight Asia-Pacific
Bioinformatics Conference, January 18-21, 2010. J.N. Tata Auditorium, Indian Institute of
Science, Bangalore
Azim S, Banday AR, Tabish M. Alternative splicing of chrng gene generates Novel
Isoforms with different N-terminals FASEB J March 17, 2011 25:900.2
Alternative promoter usage and differential expression of multipletranscripts of mouse Prkar1a gene
Abdul Rouf Banday • Shafquat Azim •
Mohammad Tabish
Received: 14 February 2011 / Accepted: 19 May 2011
� Springer Science+Business Media, LLC. 2011
Abstract Prkar1a gene encodes regulatory type 1 alpha
subunit (RIa) of cAMP-dependent protein kinase (PKA)
in mouse. The role of this gene has been implicated in
Carney complex and many cancer types that suggest its
involvement in physiological processes like cell cycle
regulation, growth and/or proliferation. We have identi-
fied and sequenced partial cDNA clones encoding four
alternatively spliced transcripts of mouse Prkar1a gene.
These transcripts have alternate 50 UTR structure which
results from splicing of three exons (designated as E1a,
E1b, and E1c) to canonical exon 2. The designated
transcripts T1, T2, T3, and T4 contain 50 UTR exons as
E1c, E1a ? E1b, E1a, and E1b, respectively. The tran-
script T1 corresponded to earlier reported transcript in
GenBank. In silico study of genomic DNA sequence
revealed three distinct promoter regions namely, P1, P2,
and P3 upstream of the exons E1a, E1b, and E1c,
respectively. P1 is non-CpG-related promoter but P2 and
P3 are CpG-related promoters; however, all three are
TATA less. RT-PCR analysis demonstrated the expres-
sion of all four transcripts in late postnatal stages; how-
ever, these were differentially regulated in early postnatal
stages of 0.5 day, 3 day, and 15 day mice in different
tissue types. Variations in expression of Prkar1a gene
transcripts suggest their regulation from multiple pro-
moters that respond to a variety of signals arising in or
out of the cell in tissue and developmental stage-specific
manner.
Keywords Regulatory type 1 alpha subunit � Alternate
promoter � Differential expression � Transcription factors �Alternative splicing
Abbreviations
PKA Cyclic AMP-dependent protein kinase
R1a Regulatory type 1 alpha subunit
PN Postnatal
T Transcript
P Promoter
RT-PCR Reverse transcriptase polymerase chain
reaction
RACE Rapid amplification of cDNA ends
Introduction
Cyclic AMP-dependent protein kinase (PKA) is the
enzyme that phosphorylates the downstream molecules on
activation by secondary messenger called cAMP. This
phosphorylation plays a key regulatory role in different
cellular events like transcription, metabolism, cell cycle
progression, and apoptosis. The PKA holoenzymes contain
two catalytic (C) subunits bound to homo or heterodimers
of either regulatory type I (RI) or regulatory type II (RII)
subunit. There are four different R subunits (Ia,Ib and
IIa,IIb) and three different C subunits (Ca,Cb, and Cc),
each encoded by a separate gene. These holoenzymes and
individual R subunits show different biochemical proper-
ties and also play specific physiological functions.
Among all the subunits of PKA, RIa is studied with
immense importance in cancer cases. Increase in the
expression of RIa has been found to be associated with
A. R. Banday � S. Azim � M. Tabish (&)
Department of Biochemistry, Faculty of Life Sciences,
A.M. University, Aligarh, Uttar Pradesh 202002, India
e-mail: [email protected]
123
Mol Cell Biochem
DOI 10.1007/s11010-011-0897-z
neoplastic cell growth in case of cancers of breast [1],
ovary [2], lung [3], and colon [4]. Downregulation of RIaexpression with sequence-specific oligonucleotide results
in inhibition of growth and modulation of cAMP signaling
in cancer cells like LS-174T, HCT-15, and Colo-205 colon
carcinoma cells; A-549 lung carcinoma cells; LNCaP
prostate adenocarcinoma cells; Molt-4 leukemia cells; and
Jurkat T lymphoma cells. Also inhibition of RIa expression
has shown compensatory changes in expression of the
isoforms of R and C subunits and cAMP signaling in a cell
type-specific manner [5]. Recently, in case of cholangio-
carcinoma (CCA), abrogation of PRKAR1A gene (gene
symbol for human) expression showed significant CCA
cell growth inhibition, oncogenic signaling, and coupled
apoptosis induction, suggesting potential of this gene as a
drug target for CCA therapy [6]. In Carney complex,
mutations leading to the loss of function of one RIa allele
give a phenotype displaying variable defects in cellular
growth control [7]. In common variable immunodeficiency
(CVI) patients, hyperactivation of PKA type I contribute to
T cell dysfunction [8]. One of the studies has demonstrated
that isoform-specific antagonists of PKA regulatory sub-
units may be targeted for the novel drug discovery to be
used in many cancer types [9].
Therefore, the study of isoforms of PKA regulatory
subunits would be significant for designing of isoform-
specific drugs. Alternative splicing mechanism is one of the
main sources for generation of isoforms. For RIa subunit
gene, 11 splice variants in Drosophila [10, 11] and three in
human [12] have been studied as per records available in
GenBank. To analyze the isoforms resulting from alterna-
tive splicing, we have studied the mouse PKA RIa subunit
gene as mouse is the best model for correlating gene
structure and function in human. In GenBank single tran-
script was reported for mouse Prkar1a gene with accession
number NM_021880 [13]. However, partial sequences of 50
UTR exons have been previously described [14] which
were shown to be different by our methodology using 50
RACE kit and further supported by EST data. In this arti-
cle, we have demonstrated multiple transcripts that arise
due to alternative splicing and alternative promoter usage
in 50 UTR of Prkar1a gene. We detected three novel tran-
script variants of Prkar1a gene of mouse, which differ from
the previously reported transcript in first exon and studied
their expression profile in brain, heart, skeletal muscle, and
liver across different postnatal development stages. Three
promoter regions were also characterized. The results
provide evidence for a complex regulation of Prkar1a
expression from three promoters which showed consider-
able differences for transcription factor-binding sequence
motifs. We have also found that the transcripts of RIasubunit gene of human and mouse share less homology for
50 UTR exons, could be considered as different, but have
the same coding exons.
Materials and methods
Materials
Mice (C57BL/6J) were purchased from NII (National
Institute of Immunology), New Delhi, India and bred in-
house. All the animals were housed according to the
Institutional Animal Care and Use Committee and Guide-
lines of the Committee on Care and Use of Laboratory
Animal Resources, National Research Council (Depart-
ment of Health, Education and Welfare; National Institutes
of Health). The total RNA extraction kit was purchased
from iNtRON Biotechnology, Inc. (Gyeonggi-do, Korea).
M-MuLV-Reverse Transcriptase, PCR nucleotide mix,
1 kb DNA ladders were purchased from Fermentas Life
Sciences, USA. The TOPO-TA cloning kit II was obtained
from Invitrogen Corp. (Carlsbad, CA). The Plasmid DNA
miniprep kit and Qiaquick PCR gel purification kits were
purchased from Qiagen, Inc. (Santa Clarita, CA). The 50
RACE kit was purchased from Clontech (Palo Alto, CA).
Primers were custom synthesized from MWG Biotech, Pvt.
Ltd., India. All the other chemicals used in the experiments
were of molecular biology grade.
Preparation of RNA from different tissues of mouse
Total cellular RNA was isolated from mouse tissues like
brain, liver, skeletal muscle, and heart using RNA extrac-
tion kit according to manufacturer’s instructions. Finally,
the RNA was re-dissolved in diethyl pyrocarbonate-treated
water and quantitated spectrophotometrically. Integrity of
RNA was confirmed by ethidiumbromide staining after
denaturing agarose gel electrophoresis. RNA prepared was
either used immediately or stored at -80�C.
Primers
The following oligonucleotide primers were used:
PF1: 50-GCGACTCGGTCTTTAATCTTCCACTCTA
(from exon E1a)
PF2: 50-AGTCGCCCACCTGTCACCGAATCGT (from
exon E1b)
PF3: 50-CTATCGCAGAGTGGTAGTGAGGCT (from
exon E1c)
PR1: 50-CTCCTTGATCAATCACATAGAAGTTATCC
(from exon E6)
PR2: 50-AATGTCACTTCTCTCGTTATCATCAAGGT
(from exon E4/E5 junction)
Mol Cell Biochem
123
PR3: 50-AACAGCACATTCTTTTCGATGGCCTTGGC
(from exon E4)
50-Rapid amplification of cDNA ends (RACE)
50-RACE reactions were performed with 3 lg of total RNA
using a 50 RACE kit. The total RNA was annealed with
gene-specific reverse primer 50-AACAGCACATTCTTTT
CGATGGCCTTGGC-30 (PR3) according to the instruc-
tions provided with the kit. 50-RACE followed by PCR was
performed with cycling conditions as follows: after an
initial denaturation for 3 min at 94�C, 35 cycles at 94�C for
30 s, 62�C for 30 s, and 72�C for 30 s were performed,
followed by a final extension at 72�C for 7 min in an
Amplicator Block (Taurus Scientific, USA). The RACE
product was visualized on 1.5% agarose gels. Several
bands were excised from the gel, purified, and subcloned
into the TOPO-TA vector (a plasmid vector). E. coli
JM109-competent cells were transformed. Transformed
colonies were grown overnight at 37�C. Plasmid DNA was
purified using a plasmid purification kit. Plasmids con-
taining the insert were sequenced using an automatic
sequencer using either M13 forward or reverse primers.
Reverse transcriptase (RT)-PCR
To confirm the RACE data, we performed RT-PCR anal-
ysis in touchdown mode. Tissue-specific total RNA (2 lg)
was primed with gene-specific primer PR1 for the synthesis
of cDNA using M-MuLV-Reverse Transcriptase at 42�C
for 1 h in a total volume of 20 ll. Subsequently, 2 ll of the
single-stranded cDNA thus synthesized was amplified by
touchdown PCR with an appropriate set of primers using
PCR master mix in a total volume 50 ll. PCR was per-
formed for 35 cycles as denaturation at 94�C for 4 min, 1
cycle; 93�C, 30 s; annealing 65�C, 30 s with a decrease of
0.3�C per cycle, extension 72�C for 45 s, with final
extension at 72�C for 8 min. The PCR products obtained
were subjected to electrophoresis on a 1.5% (w/v) agarose
gel, stained with ethidium bromide, and photographed on a
UV transilluminator.
Semi-nested RT-PCR
To further validate the genuine PCR product, semi-nested
PCR was performed. After the RT reaction, as described
above, PCR amplification was carried out for 30 cycles (as
above) using gene-specific internal reverse primer PR2 and
first-exon-specific primers as down and upstream primers,
respectively. The resulting RT-PCR product (1 ll) was
used as a template for further amplification by PCR using
the same upstream primer but downstream primer from the
junction of exon-4 and exon-5. The resulting semi-nested
PCR product (8 ll) was then subjected to electrophoresis
on a 1.5% (w/v) agarose gel, stained with ethidium bro-
mide and photographed on a UV transilluminator.
Subcloning and sequencing
The PCR amplified products were electrophoresed on 1.5%
(w/v) agarose gels. Anticipated ethidium-bromide-stained
bands were cut from the gels and DNA was recovered
using a Qiaquick PCR gel purification kit. The purified
DNA was subcloned using the plasmid cloning vector. E.
coli JM109-competent cells were transformed. Trans-
formed colonies were grown overnight at 37�C and plas-
mid DNA was purified for sequencing. Plasmid containing
the insert was sequenced using an automatic sequencer
using either M13 forward or reverse primers.
Bioinformatics tools and databases used
Homology and similarity searches of the nucleotide
sequences were performed using the BLASTN non-red-
undant database (http://www.ncbi.nlm.nih.gov/BLAST).
Gene/Exon finding tools were used at HMMgene (http://
www.cbs.dtu.dk/services/HMMgene), Genescan (http://genes.
mit.edu/GENSCAN.html), GeneSplicer (http://cbcb.umd.
edu/software/GeneSplicer/), FGENESH (http://www.soft
berry.com/berry.phtml). Alignment analysis was carried
out using the Gene stream Align tool (http://www2.igh.cnrs.
fr/bin/align-guess.cgi) and ClustalW tool available at www.
ebi.ac.uk/clustalw. Promoter regions were characterized
using tools such as TFSEARCH (http://www.cbrc.jp/
research/db/TFSEARCH.html), TFBIND (http://tfbind.hgc.
jp/), SIGSCAN (http://www-bimas.cit.nih.gov/molbio/
signal/), CpG Island Searcher (http://www.uscnorris.com/
cpgislands2/cpg.aspx) and CpGPlot (http://www.ebi.ac.uk/
Tools/emboss/cpgplot/index.html). Expressed sequence tag
search was done at DNA bank of Japan (DDBJ) using
BLASTN program (http://blast.ddbj.nig.ac.jp/top-e.html).
Results
Four transcripts of mouse Prkar1a gene that differ
in the 50 UTR exons
The 50 UTR of mouse regulatory type 1 alpha gene is
complex having multiple upstream non-coding exons.
Using 50-RACE followed by cloning and subsequent
sequencing, we found four unique clones having the het-
erogeneous structure of non-coding exons present upstream
of first coding exon (designated as E2). Sequence analysis
indicated that all of these clones contain a common
Mol Cell Biochem
123
sequence from exon E2. Aligning these sequences with the
available genomic DNA sequence of the RIa gene down-
loaded from MGI database (MGI: 104878, Fig. 1a), con-
firmed that these sequences are part of the genomic DNA
of the RIa gene representing its 50 UTR. Three exons
designated as E1a, E1b, and E1c were located upstream of
first coding exon E2 according to the organization in DNA
sequence of RIa gene (Fig. 1a). The non-coding exon, E1c
corresponded to the non-coding exon of previously
described transcript available in pubmed with GenBank
accession no. NM_021880. Further analysis revealed that
the four clones are the result of alternate exons in the 50
UTR and presence of multiple transcription start sites.
However, one of the newly identified transcript contains
Fig. 1 Schematic diagram of genomic structure of the mouse Prkar1a
gene and mRNA transcripts expressed in adult mouse brain, heart,
liver, and skeletal muscle as determined by 50-RACE, RT-PCR and
sequencing. a The exon–intron organization on gene and position of
gene on chromosome 11. Boxes refer to exons (size of boxes indicate
relative sizes of exons) and interconnecting lines as introns. The 50
UTR of the Prkar1a gene consists of at least three different exon 1
variants: E1a, E1b, and E1c (size and genomic positions relative to
ATG at position ?1 are shown). The small starches of nucleotides
below indicate the nucleotides present at exon–intron boundaries.
b These exon 1 variants are spliced (alone or in combination) to a
common exon 2 splice acceptor region (position –6, relative to ATG
at position ?1). ORF is located in exon 2. c Four transcripts were
detected in brain, heart, liver, and skeletal muscle of mouse: T1, T2,
T3, and T4. In transcript isoform T2, exon E1a is spliced with exon
E1b and then to the common splice acceptor region of exon 2. d 50
RACE of mouse mRNA. 50 Rapid amplification of cDNA ends was
independently performed three times using RIa-subunit encoding
gene-specific internal primer and RNA isolated from mouse tissues.
The products were resolved on a 1.5% agarose gel and stained with
ethidium bromide. M indicates DNA size marker, ?RT indicates
reverse transcriptase was added, and -RT indicates no reverse
transcriptase was added to the reaction mixture. Excised products
were subcloned into TOPO-TA plasmid and sequenced. Same band
pattern was reproduced from all four tissues. e, f RT-PCR and semi-
nested PCR amplified products electrophoresed on 1.5% agarose gel
using exon-specific forward primers. Lane 1 indicates marker (I kb
ladder). Lanes 2, 3, and 4 represent amplified products obtained using
forward primers for exons E1a, E1b, and E1c, respectively. Second
lane shows two bands, above one represents transcript T2 and below
one transcript T3. Anticipated sizes of the products obtained in RT-
PCR are 796 bp and 612 bp (lane 2), 719 bp (lane 3), and 643 bp
(lane 4) using exon-specific forward primers PF1, PF2, and PF3,
respectively with reverse PR1. Internal reverse primer PR2 was used
for semi-nested PCR and bands of anticipated size were obtained.
Arrows indicate size of marker and corresponding product size. RT-
PCR products were subcloned in TOPO-TA plasmid and both strands
were sequenced using M13 forward and M13 reverse primers.
g Alignment of nucleotide sequence of four transcripts. The 50 UTR
exons are indicated with the name of exon. The accession numbers
are given at the end of each transcript variant. The sequence shown is
up to the sixth exon. Sequence from exon-2 onward is the same in all
the transcripts. Translational initiation codon is bold and underlinedin exon-2
Mol Cell Biochem
123
Fig. 1 continued
Mol Cell Biochem
123
two non-coding exons in which E1a is spliced with E1b
and then spliced to the common acceptor site in exon 2
(Fig. 1b, c). The new transcripts were named as T2, T3,
and T4 considering published transcript as T1 as seen in 50
RACE experiment (Fig. 1d).
In order to further validate the presence of these newly
identified exons with transcripts, RT-PCR was done using
forward primers specific to these 50 UTR exons and reverse
primers from downstream known exons to maximize the
length of transcript. Three bands (lanes 2 and 3 marked
with arrows) were obtained at indicated sizes using the
forward primers FP1 (corresponding to the exon E1a) and
FP2 (corresponding to the E1b), with reverse primer PR1
(Fig. 1e—lanes 2 and 3). As a control, we used the forward
primer from exon E1c (FP3) with the same reverse primer
PR1, and found a single band of the anticipated size
(Fig. 1e, lane 4). Two bands were obtained in lane 2 with
forward primer FP1 as expected, one corresponding to
transcript T2, and other corresponding to transcript T3
validating the presence of splicing event between exons
E1a and E1b. The new transcript T2 was the largest one
containing both E1a and E1b upstream exons. The semi-
nested PCR also proved the presence of these transcripts.
The expected products were obtained using another reverse
primer (PR2) internal to the previous reverse primer
(Fig. 1f). The presence of these new transcripts having
different exon structures at the 50 end was confirmed by
three independent RT-PCR experiments performed using
different RNA preparations, and by cloning and sequencing
the products. The cDNA sequences of these new transcripts
were submitted in the GenBank and the assigned acces-
sions numbers are T2 (HQ412666), T3 (HQ412667), and
T4 (HQ412668).
The two new exons E1a and E1b have TG/GT locus for
splicing donor sites indicating minor variation from con-
sensus splice donor site, like AG/GT (Fig. 1a). However,
the first two and last two bases in the intron between E1a
and E1b are GT and AG, respectively. Also, a splice
acceptor site at the 50 end of first coding exon is always
CAGA which indicated that the same protein product is
generated from all the three transcripts. This is also clear
from multiple sequence alignment of four transcripts which
showed 100% sequence homology from exon 2 (Fig. 1g).
Mouse Prkar1a gene is expressed from three distinct
promoter regions
Using an alternative approach of bioinformatics tools, three
different promoter regions were characterized in the 50
UTR of Prkar1a gene designated as P1, P2, and P3
(Fig. 2a). Earlier it has been established that promoter
regions of the RIb and RIIb subunit genes of PKA are
TATA less and G/C rich, and initiation of transcription
occurs at multiple sites [15, 16]. Using CpG island detec-
tion tools such as CpG Island Searcher and CpGPlot, we
found that no CpG island is present in or near upstream of
exon E1a; however, there is the presence of CpG islands in
and near upstream of exon E1b and E1c (Fig. 2b). These
CpG islands have an average GC content of about 65–70%
and observed/expected CpG ratio [60%. It is noteworthy
that all the three promoter regions are TATA less, but have
CAAT window upstream of transcription start site that
matches with conventional CAAT box. The position of
CAAT window varies upstream of transcription start site of
exons E1a, E1b, and E1c at positions -346, -414, and-
273, respectively. These findings supported the occurrence
of transcript T4 having first exon as E1b. An interesting
fact is that E1b exon has E-box consensus sequence of
CACGTG spanning from position ?40 to ?45 downstream
of transcription start site. This E-box may act either as an
enhancer or a silencer depending on the binding protein. It
is well established that Max/Myc dimer binding with E-box
activates transcription whereas the Max/Mad dimer sup-
presses transcription of these genes [17]. The transcription
from P2 promoter might also be regulated by Max/Myc
dimer.
In order to visualize the transcription regulation from
promoter regions, in silico analysis was performed by using
tools like TFSEARCH, TFBIND, and SIGSCAN for find-
ing transcription factor-binding consensus sequence motifs
(Table 1). Analysis has identified a complex array of rec-
ognition sequences that are involved in different cellular
processes while responding to variety of signals in and out
of a cell. The most important observation was that all three
promoters have some binding sites in common, e.g., for
Sp1 (steroidogenic factor 1) which is expressed in all
mammalian cells and is involved in regulating the tran-
scriptional activity of genes implicated in most cellular
processes [18, 19], CREB (cAMP response element-bind-
ing) which decreases or increases transcription [20], GR
(glucocorticoid receptor) responds to cortisol, and other
glucocorticoids [21], LF-A1 (Liver factor A1) binds pro-
moter regions of several liver-specific genes [22], NF-I
(Nuclear factor I) recognizes the sequence CAAT box and
is implicated in eukaryotic transcription, as well as DNA
replication [23], etc. However, there are different binding
sites for different transcription factors specific to each
promoter, e.g., AP-2 (Activator protein-2), which is known
to have its roles in vertebrate development, apoptosis, and
cell-cycle control [24], can bind to promoter P3; TFIID
(Transcription factor II D), is one of several general
transcription factors that make up the RNA polymerase II
pre-initiation complex [25], can bind to promoter P1; and
F2F (Footprint II-binding factor) is believed to be a
Mol Cell Biochem
123
transcriptional repressor [26] can bind to promoter P2.
These observations clearly describe the diverse regulations
of Prkar1a gene transcription.
Differential expression of Prkar1a gene in mouse heart,
brain, skeletal muscle, and liver
To give some insights into the regulatory behavior of
alternate promoters, the expression patterns of the alter-
native transcript isoforms were analyzed in four major
organs (heart, brain, skeletal muscle, and liver) by RT-PCR
at different postnatal development stages of mouse like
0.5 day, 3 day, 15 day, and 60 day named as PN0.5, PN3,
PN15, and PN60, respectively. The identity of the resulting
amplicons was confirmed by DNA sequencing (not shown).
Only few tissues from early and late postnatal stages were
studied because it was beyond the scope of the study pre-
sented here to study all tissues at different developmental
stages of mouse. The RT-PCR analysis showed ubiquitous
pattern across these organs in adult stages of development.
However, earlier stages of postnatal development showed
variation in their expression patterns (Fig. 3). Under our
experimental conditions, at PN60 all four transcripts were
amplified from the RNAs isolated from heart, brain, skel-
etal muscle, and liver. The transcript T3 showed the most
apparent expression at all postnatal stages which may be
because its transcription was under the promoter P1 that
possesses binding site for transcription factor TFIID. Dur-
ing the developmental stages, the transcript T1 was found
to be very weakly expressed at PN0.5 and PN3 stages;
however, in brain and liver at stage PN15, it showed rel-
atively higher expression. In adult, at stage PN60, all four
tissues examined, i.e., heart, brain, skeletal muscle, and
liver, showed higher expression of transcript T1. Interest-
ingly, at early postnatal stages, transcript T4 was amplified
from muscle tissue only. The transcript T2 was not
detected from early postnatal stages. However, both T2 and
T4 transcripts were detected at late postnatal stage PN60 in
all four tissue types examined (Fig. 3). This indicated that
the splicing event between exons E1a and E1b occurs
mostly in adult mice, the consequence of which is
unknown. The absence of CpG Islands in promoter P1 may
be the reason behind the ubiquitous type of expression
patterns of transcripts expressed from this promoter. In
Fig. 2 a Schematic
representation of promoters
upstream of 50 exons.
Transcription start sites (TSS)
are depicted as ?1. All three
promoters have CAAT window
located at position -346,-414
and -273 upstream of TSS of
exons E1a, E1b and E1c,
respectively. Hanging linesshow E box sequence in exon
E1b spanning from ?40 to ?45
downstream of TSS.
b CPGPLOT generated by CpG
Plot tool available at EBI used
for CpG Island prediction.
Query sequence of 2021 bp was
used as input covering genomic
nucleotide sequence from
promoter P1 to exon E1c.
Double headed arrows show
relative position of promoter
regions. CpG Islands are present
in regions of promoter P2 and
P3 with observed/expected
ratio [0.60 and G?C
percentage [55.00
Mol Cell Biochem
123
order to further identify tissue and developmental stage-
specific expression, ESTs were searched for all four tran-
script isoforms using DDBJ blast program. We retrieved
several ESTs that showed considerable match with these
transcript isoforms. The accession numbers of ESTs, along
with their presence in tissue types and respective devel-
opmental stages are given in Table 2. The EST data sup-
ported our observations of differential and ubiquitous type
of Prkar1a gene expression in temporal manner. However,
due to lack of EST coverage from all tissues, it was not
conclusive to correlate data comparatively. No EST was
found against transcript T3 belonging to embryonic
developmental stages which may be due to the absence of
or very rare expression of T3 transcript during embryonic
development. In brain, the role of R1a subunit might be
more diverse and complex because all four transcripts were
expressed in adult mice brain tissue types (visual cortex,
cerebellum, lateral ventricle wall, corpora quadrigemina,
etc.). One of the most important observations from EST
data was that the splicing event between exon E1a and E1b
occurs not only in adult mice, but also in embryo. Among
all four transcripts, a large number of ESTs with many
tissue types were retrieved for transcript T1 which clearly
indicated its abundant expression. These facts evidently
suggest complex regulation of transcription of Prkar1a
from multiple promoter regions that we envisaged during
in silico analysis of these promoter regions.
Discussion
Our results confirmed the presence of four RIa transcript
variants in different tissues of adult mouse like brain, liver,
heart and skeletal muscle. These transcripts of RIa gene
arise due to alternate promoter usage and alternative RNA
splicing of three distinct non coding exons in 50 UTR. The
consensus sequences at the splice-donor/acceptor site are
believed to be the structural features that determine whe-
ther a specific splice site is used although these consensus
sequences may not be always present [27]. Analysis of the
exon–intron junction region of all new exons indicated
structural conservation however, splice donor site varies
slightly from consensus where A is replaced by T (TG/GT
instead of AG/GT). The presence of more than 75% con-
sensus sequences in the splice-donor/acceptor site provides
evidence for the described splicing events. Thus, our data
indicated that there are four possible transcript variants for
mouse Prkar1a gene including one reported earlier, which
are expressed in heart, liver, brain, and skeletal muscle.
Interestingly, all four transcript variants differ only in the
Fig. 3 Relative RT-PCR products of mouse heart, brain, liver, and
skeletal muscle mRNA at postnatal stages PN0.5, PN3, PN15, and
PN60. Primers used were same as given in ‘‘Methods’’ section with
forward primer exon specific and same reverse primer from internal
exon. 1.5% agarose gels in row represent tissue and, in column,
represent postnatal stage. Each lane across column indicates same
RT-PCR product. Names of exon and respective transcript (given in
curved bracket) amplified are shown above each lane. At PN60 [lane
E1a, E1a ? E1b (T3, T2)] two products representing transcripts T2
and T3 are amplified using forward primer PF1 (from exon E1a).
However, at PN0.5, PN3, and PN15, only one band appeared that
represent transcript T3. Few lanes have unexpected extra bands that
are due to non-specific binding of primers. Marker (1 kb DNA ladder)
was always run to correlate the amplified product size (not shown
here)
Mol Cell Biochem
123
non-coding exons. In all variants, coding exons are present
unchanged encoding identical proteins. The 50 RACE and
bioinformatics analysis has revealed multiple transcription
sites controlled by distinct promoter regions. The initiation
of transcription at several sites, generating transcripts dif-
fering in their 50 UTR could render the expression of this
important gene less vulnerable to mutations which could
modify its expression. Also the sequence composition in
the upstream region for promoters were found TATA less
and G/C rich, which is in accordance with the promoter
regions of the genes for mouse RIb, and mouse/rat RIIb as
described previously [15, 16]. However, all three promot-
ers have varying GC content ratio making P1 as non-CpG-
related promoter while P2 and P3 as CpG-related pro-
moters. The presence of CpG islands may play an impor-
tant role in gene regulation, for example, in causing gene
silencing or transcriptional repression by hypermethylation
of the CpG Island [28, 29]. It is noteworthy that in silico
study revealed characteristic signature sequences in all the
three promoter regions which are potential binding sites for
different transcription factors. This supports the existence
of multiple promoters for Prkar1a gene. The existence of
multiple promoters may be due to the need for the
expression of this gene in many but not all cell types and/or
at different times during embryogenesis. The individual
promoters can be regulated independently so that the gene
can be transcribed in different and overlapping cell types or
in response to different signaling systems. In silico analysis
of the promoter elements together with expression profiling
have shown diagnostic differences; however, in vitro
analysis is needed to sort out and establish consensus over
the significance of these differences. Another consequence
of using different promoters is the addition of a different 50
UTR sequence to the mRNA. The sequences present in
E1a, E1b, and E1c could also potentially play a very
selective role in translational regulation of Prkar1a gene
and/or may contain recognition sites for RNA binding
proteins involved in mRNA translocation, degradation, or
stability [30]. Particularly, the transcript T2 that contains
two 50 exons spliced to each other could play role in post-
transcriptional regulation of translation, functions that rely
on a combination of primary and secondary structures of
the RNA, such as containing a susceptible site for miRNA
or hairpin formation [31].
RIa is the only regulatory subunit which is crucial for
the progression of embryogenesis in the mouse [32]. We
have examined the expression profile of these transcripts at
different postnatal development stages. The results showed
variation in expression of these transcripts across tissues at
different postnatal development stages. For example, the
transcript T3 containing exon E1a was almost expressed
ubiquitously at all postnatal development stages. However,
Table 1 Comparative in silico
prediction of different
transcription factor binding sites
of promoter regions and their
position relative to transcription
start sites using TFSEARCH,
TFBIND, SIGSCAN tools
Signs (-,?) indicates position
upstream or downstream of
transcription start site
Transcription factor Target sequence P1 P2 P3
AP-2 TGGGGA -92
ATF TGACGT -9
CAC-binding-pro GGTGG -71,-378 -95,?33
EBP45 GTTTGCTTT -198
GATA-1 TTATCT -256,-82
GATA-2 AGATAA -193
CREB TGACG -189 -378 -148,-10
F2F TAAAAT -527
GR CAGAG -229,?83 -25,?19
GR TGTCCC -519
GR GACACA -296
H4TF-1 GATTTC -428,-207
LF-A1 GGGCA -237,-29 -350 -177
MEP-1 TGCGCTC -103
NF-1 GCCA -46
NF-1 TCCA -284 ?278
NF-1/L TGGCA -108, -386
NF-E CTGTC -303 -394,?23
PEA3 AGGAAA -148 -236
Sp1 CCGCCC -41 -34
Sp1 GGGCGG -236
TFIID TTCAAA -364
Mol Cell Biochem
123
transcripts like T1, T2, and T4 showed differential
expression where as T2 was heavily expressed in adult
mice (Fig. 3). In later stages, expression of all four tran-
scripts was ubiquitous that appeared unchanged. These
results suggest developmental stage-specific expression of
Prkar1a gene. Expressed sequence tags matching these
transcripts also supported our RT-PCR analysis. However,
EST data revealed that transcript T2 is also expressed in
embryo and that transcript T1 is abundantly present at
different embryonic developmental stages. These findings
Table 2 Accession numbers,
tissues and developmental
stages of expressed sequence
tags matching transcript variants
Transcripts EST Accession No. Tissues (cell types) Developmental stages
T1 CX238268 Lateral ventricle wall Adult
CN676072 Embryonic stem (ES) cell Embryo
BY350563 Whole joints Adult
BY325193 Synovial fibroblasts Adult
BY295910 Visual cortex Adult
BY171043 Bone marrow macrophage Adult
BY099397 Kidney Adult
BU922352 Retina 14.5 days embryo
BE850859 Mammary gland Adult
BB665656 Oviduct 2 days pregnant adult female
BB654792 Whole embryo 9 days
CJ182565 Rathke’s pouches 14.5 days embryo
CA894890 Neural stem cell Differentiated
BY782095 Whole body 17.5 days embryo
BY747531 Thymus thymic cells 2 days neonate
BY736666 Blastocyst Embryo
BY726571 Corpora quadrigemina Adult
BY705244 Liver Adult
BB610002 Lung Adult
BY350231 Whole joints Adult
BY333602 Synovial fibroblasts Adult
CN663942 Whole embryo 13.5 days embryo
CA540754 Whole Embryo 7.5 days embryo
BY229339 Olfactory sensory neurons Adult
BY064378 Kidney 17 days embryo
BB850806 Inner ear Adult
BB864160 B lymphocyte (B cells) Adult
BY036616 Liver 14 days embryo
CF167794 Germ Cell Embryonic
T2 BB610165 Cerebellum Adult
BY132531 Whole body 17.5 days embryo
BY153772 Pancreas islet cells Adult
BB567873 Head 12 days embryo
T3 DN174114 Lateral Ventricle Wall Adult
BY129491 Brain Adult
BB610165 Cerebellum Adult
T4 BU709493 Whole brain Develop. stage: embryo 15.5
CB194067 Embryonic limb, maxilla
and mandible (pooled)
Embryo day 12.5, 13.5,
14.5, and 15.5
BI657582 Tumor, gross tissue 5 months
BY267453 Visual cortex Adult
BY026492 Mammary gland Adult
BY153772 Pancreas islet cells Adult
Mol Cell Biochem
123
further support the importance of RIa gene for progression
of embryogenesis in mouse.
In human RIa, gene initiation at different transcription
start sites lead to three distinct, alternatively spliced
transcripts coding for identical proteins [12]. Multiple
sequence alignment of human and mouse R1a gene tran-
scripts showed that 50 UTR exons of mouse and human are
different and share less homology (Fig. 4). However, all of
these transcripts have almost 100% homology from exon 2.
This observation indicated two possibilities that either 50
UTR of human and mouse R1a gene are less conserved in
sequence or that the 50 UTR exons that we reported here
have not been identified in human and vice versa. The pair-
wise alignment of human R1a 50 UTR exons with genomic
DNA of mouse and that of mouse R1a 50 UTR exons with
genomic DNA of human using NCBI BLAST program
showed no significant match at exon level. This fact
excluded the possibility of unreported transcripts in human
of the same type of sequence that we found in mouse. Also,
the 50 UTR of human and mouse R1a gene showed 68%
homology at nucleotide level which indicates less conser-
vation of nucleotide sequence. It was also found that no
splicing event is reported between human R1a 50 UTR
exons similar to that of mouse where such an event pro-
duces transcript T2.
These data suggest complex regulation of R1a subunit
gene transcription in mouse and human to produce the
same protein product. The study of transcripts belonging to
mouse and human R1a gene will be helpful while inves-
tigation of diseases and other functional aspects in mouse
as model, in particular to those cases which pertain to
regulation of this gene. This study seeks to understand the
mechanism and significance of splicing event that gives
rise to transcript T2 in mouse and also whether such
splicing event is present in human R1a subunit gene.
Acknowledgments The authors are thankful to the Department of
Biotechnology (DBT), the Ministry of Science and Technology, New
Delhi, India for providing generous funding to project No. BT/PR-
10917/BID/07/254/2008. The necessary facility provided by A.M.U.,
Aligarh, India is also thankfully acknowledged.
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1 23
Molecular Biology ReportsAn International Journal onMolecular and Cellular Biology ISSN 0301-4851 Mol Biol RepDOI 10.1007/s11033-011-1108-4
Differentially expressed three non-codingalternate exons at 5# UTR of regulatorytype I beta subunit gene of mouse
Abdul Rouf Banday, Shafquat Azim &Mohammad Tabish
1 23
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Differentially expressed three non-coding alternate exonsat 50 UTR of regulatory type I beta subunit gene of mouse
Abdul Rouf Banday • Shafquat Azim •
Mohammad Tabish
Received: 23 May 2011 / Accepted: 17 June 2011
� Springer Science+Business Media B.V. 2011
Abstract Prkar1b gene encodes regulatory type I, beta
subunit (RIb) of cAMP dependent protein kinase A in
mouse. Among the various isoforms of regulatory and
catalytic subunits that comprise mammalian PKA, RIbsubunit is considered to be one of the important subunits
for neuronal functions. This is involved in multiple forms
of synaptic plasticity, and influences memory and learning
by maintaining hippocampal long-term potentiation (LTP).
Deficient expression of this gene has been implicated in
autoimmune disease systemic lupus erythematosus (SLE).
We have identified two novel non-coding exons of the
Prkar1b gene (designated as exon 1A and exon 1B), which
are spliced to the canonical exon 2 and constitute the 50
untranslated region giving rise to three alternative tran-
script isoforms. We have also confirmed the expression of
the previously known first exon (designated as exon 1C)
with known transcript published earlier. The transcripts
containing exons 1A, 1B and 1C are differentially regu-
lated during the development and tissue types. In silico
study of more than 20 kb nucleotide sequence upstream of
known translational initiation codon revealed three distinct
promoter regions named as PA, PB, and PC upstream of
the exon 1A, exon 1B and exon 1C respectively. PB is non-
CpG related promoter but PA and PC are CpG related
promoters, however all three promoters are TATA less.
Further analysis showed that these promoters possess
potential signature sequences for common as well as dif-
ferent transcription factors suggesting complex regulation
of Prkar1b gene.
Keywords Alternative splicing � Alternate promoter �Differential expression � Exon � Regulatory type 1 beta
subunit � Transcription factors � RT-PCR
Introduction
In eukaryotic cells, cAMP regulates many different cellular
functions. Its effects are in most cases mediated by cAMP-
dependent protein kinase A (PKA). PKA enzyme is tetra-
mer that contains two catalytic (C) subunits bound to
homo- or heterodimers of either regulatory type I (RI) or
regulatory type II (RII) subunit. At least four regulatory
(RIa, RIb, RIIa, and RIIb) and three catalytic (Ca, Cb and
Cc) subunits have been characterized in mammals. The
activation of PKA is controlled by the regulatory (R) sub-
units of the holoenzyme. Different anatomical distribution
of the regulatory isoforms may contribute to determine the
specificity of diverse effects of cAMP [1].
Signaling events mediated by PKA are critical for many
neuronal functions [2]. PKA is involved in multiple forms
of synaptic plasticity [3–5], synaptic transmission, memory
and learning [6]. Among the various isoforms of regulatory
and catalytic subunits that comprise mammalian PKA,
the regulatory type I beta (RIb) subunit is considered to be
one of the important subunits for neuronal functions. RIbsubunit has been found to be important for mossy fiber
long-term potentiation (LTP) [7], hippocampal long-term
depression and depotentiation [8, 9], inflammation and
sensitization of primary afferent nociceptors [10]. In
autoimmune disease systemic lupus erythematosus (SLE)
25% of subjects show no detectable RIb protein in their T
cells [11]. One of the studies has demonstrated that isoform
specific antagonists of PKA R subunits may be targeted for
the novel drug discovery to be used in many cancer types
A. R. Banday � S. Azim � M. Tabish (&)
Department of Biochemistry, Faculty of Life Sciences,
A.M. University, Aligarh, UP 202002, India
e-mail: [email protected]
123
Mol Biol Rep
DOI 10.1007/s11033-011-1108-4
Author's personal copy
[12]. Alternative splicing generates variants that are tissue
or developmental stage specific and can be differentially
affected by pharmacological agents to either up regulate or
down regulate the expression as per the requirements of
disease condition [13]. But this is possible only when
spliced variants are known for any given gene. Owing to
this we proposed that there could be alternatively spliced
variants in case of mouse RIb gene which can become
therapeutic targets.
The PKA RIb subunit encoding gene named Prkar1b is
present on chromosome 5 in mouse. To identify the pos-
sible spliced variants arising from RIb subunit encoding
gene, we have studied the mouse Prkar1b gene as mouse is
the best model for correlating gene structure and function
with human. In GenBank, single transcript was reported for
mouse Prkar1b gene. Previously, the promoter region of
mouse Prkar1b gene has been described as TATA-less
flanked by 1.5 kilobases of 50-upstream sequence and 1.8-
kilobase intron [14]. Here, we have demonstrated three
transcripts that arise due to alternative splicing in pre-
mRNA and alternative promoter usage in 50 UTR of
Prkar1b gene. We have also located two more promoter
regions which lead to the expression of three novel tran-
script variants of Prkar1b gene. These variants differ from
the previously reported transcript with respect to first exon.
One of the new exons was found to be part of previously
described promoter (PDP) region [14]. We have also
studied their expression pattern in brain, heart, skeletal
muscle and liver across different postnatal development
stages.
Materials and methods
Materials
Mice (C57BL/6 J) were obtained from NII (National
Institute of Immunology), New Delhi, India and bred in
house. All animals were housed according to the Institu-
tional Animal Care and Use Committee and Guidelines of
the Committee on Care and Use of Laboratory Animal
Resources, National Research Council (Department of
Health, Education and Welfare; National Institutes of
Health). The total RNA extraction kit was purchased from
iNtRON Biotechnology, Inc. (Gyeonggi-do, Korea).
M-MuLV-Reverse Transcriptase, PCR nucleotide mix,
1 kb DNA ladders were purchased from Fermentas Life
Sciences, USA. The TOPO-TA cloning kit was obtained
from Invitrogen Corp. (Carlsbad, CA). The Plasmid DNA
miniprep kit and Qiaquick PCR gel purification kits were
purchased from Qiagen, Inc. (Santa Clarita, CA). The 50
RACE kit was purchased from Clontech (Palo Alto, CA).
Primers were custom synthesized from Sigma-Aldrich
chemicals Pvt. Ltd. (Bangalore, India). All other chemicals
used in the experiments were of molecular biology grade.
Preparation of RNA from different tissues of mouse
Total cellular RNA was isolated from various mouse tis-
sues using RNA extraction kit according to manufacturer’s
instructions. Finally, the RNA was re-dissolved in diethyl
pyrocarbonate-treated water and quantitated spectrophoto-
metrically. Integrity of RNA was confirmed by ethi-
diumbromide staining after denaturing agarose gel
electrophoresis. RNA prepared was either used immedi-
ately or stored at -80�C.
Primers
Primer positions are indicated in parenthesis. The follow-
ing oligonucleotide primers were used:
PF1: 50-CGAA CCT TGA GGG CGA GGC AGA AGA
(from exon 1A)
PF2: 50-ATGGAC TCT GTT TCC ACA GGA GCA
GAT G (from exon 1B)
PF3: 50-CTA AGA GAA GCA AGT GTA ATT GGC
TAG CG (from exon 1C)
PR1: 50-CAC ATA TAC ATC TAC TTC TCC TTG
GTC (from junction of exons 6 and 7)
PR2: 50-ATG TCA CTT CTC TCG TTG TCG TCC
AGG (from junction of exons 4 and 5)
PR3: 50-GTC ATG GTC TTA TAG TCC TTG GGA
ATG (from exon 4)
50 Rapid amplification of cDNA ends (RACE)
50 RACE reactions were performed with 3 lg of total RNA
isolated from mouse brain using a 50 RACE kit. The total
RNA was annealed with gene-specific reverse primer PR3
according to the instructions provided with the kit. 50
RACE products were PCR amplified with cycling condi-
tions as follows: after an initial denaturation for 3 min at
94�C, 30 cycles were performed with denaturation at 94�C
for 30 s, annealing at 66�C for 30 s, extension at 72�C for
30 s followed by a final extension at 72�C for 8 min in an
Amplicator Block (Taurus Scientific, USA). The RACE
product was visualized on 1.3% agarose gels. Several
bands were excised from the gel, purified and subcloned
into the TOPO vector. E. coli JM109-competent cells were
transformed. Transformed colonies were grown overnight
at 37�C. Plasmid DNA was purified using a plasmid puri-
fication kit. Plasmids containing the insert were sequenced
using an automatic sequencer using either M13 forward or
reverse primers.
Mol Biol Rep
123
Author's personal copy
Reverse transcriptase (RT)-PCR
To confirm the RACE data, we performed RT–PCR anal-
ysis in touch down mode. Tissue specific total RNA (2 lg)
was primed with oligo-dT for the synthesis of cDNA using
M-MuLV-Reverse Transcriptase at 42�C for 1 h in a total
volume of 20 ll. Subsequently, 2 ll of the single-stranded
cDNA thus synthesized was amplified by touchdown PCR
with an appropriate set of primers using the PCR master
mix in a total volume of 50 ll. PCR was performed for 30
cycles as; initial denaturation at 94�C for 4 min followed
by denaturation 93�C, 30 s; annealing 70�C, 30 s with a
decrease of 0.3�C per cycle, extension 72�C for 45 s, with
final extension at 72�C for 8 min. The PCR products
obtained were subjected to electrophoresis on a 1.3% (w/v)
agarose gel, stained with ethidium bromide and photo-
graphed on a UV transilluminator.
Semi-nested RT-PCR
To further validate the genuine PCR product, semi-nested
PCR was performed. After the RT reaction, as described
above, PCR amplification was carried out for 30 cycles (as
above) using gene-specific reverse primer (PR1) and first-
exon-specific primers as down- and upstream primers,
respectively. The resulting RT-PCR product (1 ll) was
used as a template for further amplification by PCR for 30
cycles using the same upstream primer but downstream
primer from internal exon (PR2). The resulting semi-nested
PCR product (8 ll) was then subjected to electrophoresis
on a 1.3% (w/v) agarose gel, stained with ethidium bro-
mide and photographed on a UV transilluminator.
Subcloning and sequencing
The PCR amplified products were electrophoresed on 1. 3%
(w/v) agarose gels. Anticipated ethidium-bromide-stained
bands were cut from the gels and DNA was recovered using
a Qiaquick PCR gel purification kit. The purified DNA was
either directly sequenced or subcloned into TOPO vector. E.
coli JM109-competent cells were transformed. Transformed
colonies were grown overnight at 37�C and plasmid DNA
was purified for sequencing. Plasmid containing the insert
was sequenced using an automatic sequencer using either
M13 forward or reverse primers.
Computational analysis
Homology and similarity searches of the nucleotide
sequences were performed using the BLASTN nonredun-
dant database (http://www.ncbi.nlm.nih.gov/BLAST). Gene/
Exon finding tools were used at HMMgene (http://www.cbs.
dtu.dk/services/HMMgene), Genescan (http://genes.mit.edu/
GENSCAN.html), GeneSplicer (http://cbcb.umd.edu/software/
GeneSplicer/), FGENESH (http://www.softberry.com/berry.
phtml). Alignment analysis was carried out using the Gene
stream Align tool (http://www2.igh.cnrs.fr/bin/align-guess.cgi)
and ClustalW tool available at http://www.ebi.ac.uk/clustalw.
Promoter regions were characterized using tools such as
TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.
html), TFBIND (http://tfbind.hgc.jp/), SIGSCAN (http://
www-bimas.cit.nih.gov/molbio/signal/), CpG Island
Searcher (http://www.uscnorris.com/cpgislands2/cpg.aspx)
and CpGPlot (http://www.ebi.ac.uk/Tools/emboss/cpgplot/
index.html).
Results and discussion
Diversity at 50 end of mouse Prkar1b transcripts
It has been reported that mouse Prkar1b consists of 11
exons and 10 introns [14]. The structure of this gene
appears obscure at 50 end as there is large segment of DNA
flanking upstream of known transcription start site not
belonging to any gene. This fact prompted us to analyze the
50 transcriptional regulatory region of this gene. To this end
we sought to define the 50 end of Prkar1b encoding tran-
script by applying the 50 RACE as described in material
and method section. Using 50 RACE followed by cloning
and subsequent sequencing, we found three unique clones
having the heterogeneous structure at the 50 end. Sequence
analysis indicated that 50 region of Prkar1b transcripts is
diverse but all of the transcripts contain a common
sequence from exon 2. Aligning these sequences with the
available genomic DNA sequence of the Prkar1b gene
along with 20 kb flanking sequence of nucleotides
upstream of known transcription site downloaded from
MGI database (MGI: 104878, Fig. 1), confirmed that these
sequences are part of the genomic DNA of the Prkar1b
gene representing its 50 UTR. Three non-coding exons
appearing as variants of exon 1 designated as exon 1A,
exon 1B and exon 1C were located upstream of first coding
exon (exon 2) according to the organization in DNA
sequence of Prkar1b gene (Fig. 1). The non-coding exon
1C corresponded to the non-coding exon of previously
described transcript available in pubmed with GenBank
accession number NM_008923 [14]. The new transcripts
were named as Mr1b-A and Mr1b-B considering published
transcript as Mr1b-C according to the presence of 50 exon 1
variant. The translational initiation codon ‘methionine’ lies
in exon 2 in case of all three transcript variants which are
depicted in Fig. 1. The number of nucleotides in 50 UTR of
each transcript varies from 106 bp to 217 bp as shown in
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Fig. 1. The initiation of transcription at several sites, gen-
erating transcripts differing in their 50 UTR could render
the expression of this important gene less vulnerable to
mutations which could modify its expression.
In order to further validate the presence of newly
identified exons with transcripts by 50 RACE (Fig. 2a); RT-
PCR was done using forward primers specific to newly
identified exons from 50 UTR and reverse primers from
downstream known coding exons to maximize the length
of transcripts. As shown in Fig. 2b, using common reverse
primer PR1, two bands were amplified from forward pri-
mer PF2 (lane 2) and one band was amplified from forward
primer PF1 (lane 3). As a positive control, we used the
forward primer PF3 from the 1st exon published earlier
[14] with same reverse primer, and found a single band of
the anticipated 718 bp size (Fig. 2b, lane 1). One band in
lane 2 was of expected size (770 bp) as anticipated from
the result of 50 RACE however, a second band appeared
below showing a smaller variant. Sequencing the second
band revealed that a product of 688 bp size was amplified
because one more transcript variant existed in which a part
of exon 1B was skipped off. This transcript variant was
named as Mr1b-B’ as being a variant of transcript Mr1b-B.
In order to remove PCR artifacts, we performed semi-
nested PCR as described in materials and methods section.
The semi-nested PCR reaction also resulted in the ampli-
fication of expected size products that were obtained by
using another reverse primer (PR2) internal to the previous
reverse primer PR1 (Fig. 2c). The presence of these new
transcripts having different exon structure at the 50 end was
confirmed by three independent RT-PCR experiments
performed using different RNA preparations, and by
cloning and sequencing the products. The sequences of
these new transcripts were submitted to the GenBank and
were assigned accessions numbers: Mr1b-A (GenBank
accession no. JF720023), Mr1b-B (GenBank accession no.
JF720024) and Mr1b-B’ (GenBank accession no.
JF720025). Multiple sequence alignments of all four tran-
scripts of Prkar1b gene including one published earlier [14]
showed 100% sequence homology from exon 2 (Fig. 2d).
The transcripts of Prkar1b gene arise due to alternate
promoter usage and alternative RNA splicing of three
distinct non coding exons in 50 UTR. The nucleotide
sequences present in exon 1A, exon 1B and exon 1C could
also potentially play a very selective role in translational
regulation of Prkar1b gene and/or may contain recognition
Fig. 1 Schematic diagram of genomic structure of the mouse Prkar1b
gene and mRNA transcripts as determined by 50 RACE, RT-PCR and
sequencing. The exon–intron organization on gene and position of
gene on chromosome 5. Boxes refer to exons (size of boxes indicate
relative size of exons) and interconnecting lines as introns. The
sequence of Prkar1b gene in GenBank runs from 139493258 bp to
139605706 bp on chromosome 5 whereas we have identified the
sequence runs further upstream to 139473009 bp. The 50 UTR of the
Prkar1b gene consists of at least 3 different variants of first exon
namely exon 1A, exon 1B and exon 1C (size and genomic positions
relative to ATG at position ?1 are shown). PA, PB and PC are
promoter regions upstream of exon 1A, exon 1B and exon 1C
respectively. Each of the first exon could splice to a common exon 2
splice acceptor region (position –21, relative to ATG at position ?1)
generating multiple transcripts. Translational initiation codon ‘ATG’
is located in exon 2. Four transcripts were detected in brain namely
Mr1b-A, Mr1b-B, Mr1b-B’ and Mr1b-C. At the 30 end of exon 1B a
sequence of 82 nucleotides was skipped off which resulted in a
shorter variant Mr1b-B’. Previously described promoter (PDP) is
shown in dotted box. The region upstream of position -3505 was
newly identified part of Prkar1b gene in mouse
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sites for RNA binding proteins involved in mRNA trans-
location, degradation or stability [15].
Analysis of the exon–intron junction region of exon 1A
and exon 1B for splicing donor–acceptor sites indicated the
presence of conserved consensus sequences (Fig. 3). The
two new exons 1A and 1B have consensus AG/GT locus
for splicing donor sites (Fig. 3). Also, a splice acceptor site
at the 50 end of first coding exon is always GGAG which
indicated that the same protein product could be generated
from all the three transcripts. The presence of consensus
Fig. 2 50 RACE, RT–PCR and semi-nested PCR. a 50 Rapid
amplification of cDNA ends was independently performed three
times using Prkar1b gene-specific internal primer (PR3) and RNA
isolated from mouse brain. The products were resolved on 1.3%
agarose gel and stained with ethidium bromide. M indicates DNA size
marker, ?RT indicates reverse transcriptase was added and -RT
indicates no reverse transcriptase was added to the reaction mixture.
Excised products were subcloned into TOPO-TA plasmid and
sequenced. b RT-PCR amplified products were electrophoresed on
1.3% agarose gel using exon specific forward primers. These
amplified products were obtained by using forward primers for exon
1A, exon 1B and exon 1C respectively. Two bands were obtained
with forward primer FP2 (exon 1B, lane 2), upper one represented
transcript Mr1b-B and lower one represented transcript Mr1b-B’.
Anticipated sizes of the products obtained in RT-PCR are 635 bp
(lane 3), 770 bp and 688 bp (lane 2) and 718 bp (lane 1) using exon
specific forward primers PF1, PF2 and PF3 respectively with reverse
primer PR1. Second band in lane 2 appears very weak. In lane 4, 1 kb
DNA ladder was run as marker. c Semi-nested PCR analysis was
performed as described above. Using internal reverse primer PR2 all
four bands were obtained with anticipated size. Arrows indicate size
of marker and corresponding product size. RT-PCR products were
subcloned in TOPO-TA plasmid and both strands were sequenced
using M13 forward and M13 reverse primers. d Jalview of Multiple
sequence alignment of partial sequence of four transcripts. The
sequence from exon 2 at position 215 bp onwards is same in all the
transcripts
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sequences in the splice-donor/acceptor site provides evi-
dence for the described splicing events. This is also clear
from multiple sequence alignment of four transcripts which
showed 100% sequence homology from exon 2 (Fig. 2d).
Three distinct promoter regions and expression
of mouse Prkar1b gene
Earlier it was found that regulation of mouse Prkar1b gene
is controlled by a sequence of genomic DNA comprised of
a TATA-less promoter flanked by 1.5 kb of 50-upstream
sequence and 1.8 kb downstream sequence [14]. Align-
ment between the nucleotide sequences of newly found
exons and PDP revealed that exon 1B belongs to PDP as
shown in Fig. 1. Using an alternative approach of
bioinformatics tools the immediate upstream regions of
exon 1A, exon 1B and exon 1C were characterized as
promoter regions designated as PA, PB and PC respec-
tively. Thus PDP contains PB, exon 1B and PC sequence
regions. Using CpG island detection tools like CpG Island
Searcher and CpG Plot, no CpG island was established in
or near upstream of exon 1B, however there was presence
of CpG islands in and near upstream of exon 1A and exon
1C (Fig. 4). These CpG islands have an average GC con-
tent of about 65–70% and observed/expected CpG ratio
greater than 60%. The presence of CpG islands may play
an important role in gene regulation, for example, in
causing gene silencing or transcriptional repression by
hypermethylation of the CpG Island [16, 17]. There was an
E-box consensus sequence of CACGTG at position -538
Fig. 3 DNA sequence of newly defined 50 region of Prkar1b gene.
Exons are indicated in uppercase nucleotides and promoters/introns in
lowercase nucleotides. Transcription start sites are identified by
inverted arrow (;) and translational initiation codon is indicated in
bold letters with a bent arrow on it. TF binding sequence motifs are
shown in blue color and overlapped regions for more than one TF are
underlined with each TF over its binding site. The intron–exon
junctions are also underlined. The part of exon 1B which was skipped
in a shorter transcript is boxed. The previously published sequence is
up to start of PB [14]. Previously, whole region of PB, exon 1B and
PC was considered as a single promoter region. An intron of more
than 18 kb was identified between exon 1A and PB. This intron was
first such large intron of its kind identified in 50 region of cAMP
dependent protein kinase A subunit genes. This newly defined 50 UTR
sequence was submitted to the GenBank and the provided accession
number was JF720022
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upstream of transcription start site of exon 1B. This E-box
may act either as an enhancer or a silencer depending on
the binding protein. It is well established that Max/Myc
dimer binding to E-box activates transcription whereas the
Max/Mad dimer binding to E-box suppresses transcription
of the genes [18]. Thus, in silico studies indicated that
Max/Myc or Max/Mad dimer can regulate transcription of
Prkar1b gene from promoter PB.
As three promoter regions lead to the expression of the
transcript encoding same protein, there may be possibility
of differences in their transcription factor (TF) binding
sites so that they can respond to variety of signals in and
out of the cell. For such analysis we performed in silico
analysis using tools and databases like TFSEARCH,
TFBIND and SIGSCAN. Analysis revealed a complex
array of recognition sequence motifs for different types of
TFs (Fig. 3). It was interesting to observe that the three
promoters PA, PB and PC show discrepancy for TFs
responsible for constitutive and conditional expression of
Prkar1b gene. All three promoters have some binding
sites in common for some of the TFs though number vary
for example the general TF Sp1 (steroidogenic factor 1)
and NF-I (Nuclear factor I) remain constitutively active.
Sp1 is expressed in all mammalian cells and is involved
in regulating the transcriptional activity of genes impli-
cated in most cellular processes [19, 20]. NF-I is involved
in eukaryotic transcription as well as DNA replication
[21]. However, there are different binding sites for dif-
ferent transcription factors specific to each promoter e.g.
signal dependent TF CREB (cAMP response element-
binding) which decreases or increases transcription [22]
has binding site in promoter region PA and this site may
be involved in auto-regulation of transcription of Prkar1b
gene by its catalytic subunit counterpart; GR (glucocor-
ticoid receptor) TF which is a conditionally active and
responds to cortisol and other glucocorticoids [23] has
number of binding sites in promoter region PB; AP-2
(Activator protein-2) which is known to have role in
vertebrate development, apoptosis and cell-cycle control
[24], can bind to promoter PB and PC. The PB promoter
seems to be more regulatory in nature as it has number of
different TF binding sites than PA and PC. The promoter
Fig. 4 CpG Plot generated by tools available at EBI used for CpG
Island prediction. The predicted promoter region sequence was used
for all three promoters. CpG Islands were present in the regions of
promoter PA and PC with observed/expected ratio [0.60 and G ? C
percentage [55.00. However, the putative CpG Islands were found in
PC only
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PB has binding sites for GATA, PIT-1, Hox etc. which
take part in developmental and cell specific expression
and once expressed require no additional activation. This
fact prompted us to find out whether expression from PB
was differentially regulated or not. To this end we studied
the expression patterns of 50 UTR exons by RT–PCR
using exon specific forward primer and reverse primer
(PR2) in four major organs namely heart, brain, skeletal
muscle and liver at three different postnatal development
stages of mouse like 3 day, 15 day, 60 day namely PN3,
PN15, PN60 respectively (Fig. 5). Only few tissues from
early and late postnatal stages were studied because it was
beyond the scope of the work presented here to study all
tissues at different developmental stages of mouse. As
envisaged from in silico analysis the expression from
exon 1B was found to be differentially expressed. The
exon 1B containing transcript was only expressed in brain
at all developmental stages. It was interesting to note that
the transcripts having exon 1B was found in all tissues
tested from PN60 stage mouse. However, the exon 1A
and exon 1C were found to be expressed in all tissues at
all postnatal development stages. Also the expression
patterns revealed that once PB gets activated it then tends
to be constitutively expressed as seen in PN3, PN15,
PN60 stages of brain tissue (Fig. 5). These observations
further support the role of R1b subunit in neuronal tissues
and thus per se addresses why this gene was earlier
considered as neuron specific [14, 25].
Conclusion
Our results confirmed the presence of four Prkar1b tran-
script variants in different tissues of adult mouse like
brain, liver, heart and skeletal muscle where transcript
variants Mr1b-A and Mr1b-C were ubiquitously expressed
however, the transcript variants Mr1b-B and Mr1b-B’
were expressed in brain at PN3 and PN15 only and
expressed in all tested tissues at PN60. The study of
transcripts belonging to mouse will be helpful while
investigation of diseases and other functional aspects in
mouse as model, in particular to those cases which pertain
to regulation of this gene or cloning experiments in
expression vectors with one of the three putative promoter
regions required as per the factors under which expression
has to be investigated.
Fig. 5 RT-PCR products of mouse heart, brain, liver and skeletal
muscle mRNA at postnatal stages PN3, PN15 and PN60. Primers used
were same as given in methods section with forward primer exon
specific and reverse primer RP2 from internal exon. 1.3% agarose
gels in row represent postnatal stage and in column represent tissues.
Each lane across column indicates same RT-PCR product. Exon 1B
containing transcript was amplified only in brain at early PN stages
however, at PN60 (adult mice) exon 1B was expressed in all tissues.
Exon 1A and exon IB were expressed in all tissues at all PN stages
examined. Few lanes have unexpected extra bands that were due to
non specific binding of primers as confirmed by sequencing. Marker
(1 kb DNA ladder) was always run to correlate the amplified product
size
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Acknowledgments The authors are thankful to the Department of
Biotechnology (DBT), Ministry of Science and Technology, New
Delhi, India for providing generous funding to project No. BT/PR-
10917/BID/07/254/2008. The necessary facilities provided by
A.M.U., Aligarh, India are thankfully acknowledged.
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ORIGINAL PAPER
Alternatively Spliced Three Novel Transcripts of gria1in the Cerebellum and Cortex of Mouse Brain
Shafquat Azim • Abdul Rouf Banday •
Mohammad Tabish
Received: 22 July 2011 / Revised: 30 August 2011 / Accepted: 8 September 2011
� Springer Science+Business Media, LLC 2011
Abstract Glutamate receptor type 1 (GluR1) subunit of
a-amino-3-hydroxy-5-methyl-4-isoxazole propionate recep-
tors plays an important role in the expression of long-term
potentiation and memory formation. GluR1 is encoded by
gria1 gene containing 16 exons and 15 introns in mouse.
Previous studies have reported two alternatively spliced
variants of this subunit. These flip and flop variants differ
enormously in their properties as well as expression. In our
studies, we report the presence of three new transcripts of this
gene present in the cerebellum and cortex of mouse brain
produced by alternative splicing at 50 end. Four new exons are
reported; N1 is located in 50 untranslated region, N2 is located
in the 1st intronic region while N3 and N4 are located in the
2nd intronic region. The properties of these new exons
encoding N-terminal variants are highly diverse. N1, N3 and
N4 are coding while N2 is a non-coding exon and results in a
truncated transcript. The existence of N2 exon containing
transcript is further supported by the presence of an Expressed
Sequence Tag from the database. The translated amino acid
sequences of these transcripts differ in the presence of signal
peptide as well as in their phosphorylation and acetylation
pattern. The differences in their properties might be involved
in receptor modulation.
Keywords Glutamate � a-amino-3-hydroxy-5-methyl-4-
isoxazole propionate receptors � Cerebellum � Cortex �Alternative splicing � Flip and flop variants
Introduction
The a-amino-3-hydroxy-5-methyl-4-isoxazole propionate
receptors (AMPARs) are of fundamental importance in the
brain as they are responsible for the majority of fast
excitatory synaptic transmission, synapse formation and
stabilization and synaptic plasticity. AMPARs are com-
prised of four subunits (GluR1–GluR4). Amino acid
sequences for these subunits share approximately 70%
sequence homology. The genes encoding these subunits
may undergo alternative splicing in two distinct regions,
resulting in long or short C-termini, and flip or flop variants
in an extracellular domain [1]. Two variants designated as
flip and flop are produced and they differ in 38 amino acids
that lie between the third and fourth putative transmem-
brane domain [2]. These variants differ in their channel
kinetics, pharmacological properties, sensitivity to certain
allosteric modulators and exhibit regional and cell-specific
expression, developmental regulation, and modification by
physiological insults, lesions and disease [3–11]. AMPARs
are homomers of identical subunits or heteromers of dif-
ferent subunits. The N-terminal is important for receptor
assembly and receptor targeting [12, 13]. Subunits com-
patible only at the NTD interacts with each other and are
functional only if there is compatibility in the C-terminal
half of the subunits. Such hetero-oligomerization produces
functional receptors. This suggests that there might be
diversity in the N-terminus of the subunits which facili-
tates the various combinations of the subunits. Moreover,
AMPARs have widespread and varied distribution in the
brain [14].
The presence of flip-flop variants suggests that alterna-
tive splicing plays an important role in generating diversity
which facilitates their differential expression and varied
subunit combinations. The other family of glutamate
S. Azim � A. R. Banday � M. Tabish (&)
Department of Biochemistry, Faculty of Life Sciences,
Aligarh Muslim University, Aligarh, UP 202002, India
e-mail: [email protected]
123
Neurochem Res
DOI 10.1007/s11064-011-0599-7
receptors includes NMDARs. The NR2B subunit encoding
gene undergoes alternative splicing to produce 5 variants
with different N-termini [15–17]. These subunits belonging
to the different families of the glutamate receptor share
considerable sequence homology. This provided us the
impetus to study the N-terminal variants of gria1 subunit of
AMPAR. In this study we have analyzed the potential new
50 end exons which can splice with the internal exons to
produce new transcript variants of gria1 gene in mouse
brain. The novel exons of these new transcripts might play
a role in differential targeting of the receptor in different
areas of the brain. We have identified three new transcripts
of gria1 gene that arise due to alternative splicing of four
new exons encoding GluR1 subunit with three different
N-termini. These new exons N1, N2, N3 and N4 were
identified from the 50 untranslated region (50UTR), 1st
intron and 2nd intron. They can splice with each other or
internal exons to produce three transcript variants T1, T2
and T3.
Materials and Methods
Materials
Mice (A/J) were bred in house according to the Institu-
tional Animal Care and Use Committee and Guidelines of
the Committee on Care and Use of Laboratory Animal
Resources, National Research Council (Department of
Health, Education and Welfare; National Institutes of
Health). The total RNA extraction kit was purchased
from iNtRON Biotechnology, Inc. (Gyeonggi-do, Korea).
M-MuLV-Reverse Transcriptase, PCR nucleotide mix,
100 bp PCR DNA ladders were purchased from GenScript
(USA). The TOPO-TA cloning kit II was obtained from
Invitrogen Corp. (Carlsbad, CA). The Plasmid DNA
miniprep kit and Qiaquick PCR gel purification kits were
purchased from Qiagen, Inc. (Santa Clarita, CA). Primers
were custom synthesized from Sigma Aldrich Chemicals
Pvt. Ltd., India. All other chemicals used in the experi-
ments were of molecular biology grade.
RNA Preparation
Total cellular RNA from cerebellum and cortex of
2 months old A/J mouse was prepared using the easy
spinTM (DNA free) Total RNA Extraction Kit (iNtRON
Biotechnology, INC) according to the manufacturer’s
instructions. The eluted RNA was quantitated spectropho-
tometrically and RNA integrity was checked by ethidium
bromide staining on denaturing agarose gel electrophoresis.
Primers
Genomic sequence of gria1 gene was downloaded from
NCBI with accession number GenBank ID NM001113325.
Primers were designed using the downloaded sequence.
The following oligonucleotides primers were used:
E1 specific forward
primer
MCTRGRIA1 GCC GTA CAT CTT
TGC CTT TTT CTG
CAC CG
N1 specific forward
primer
MN1GRIA1 CAT TCT ACA GAT
ACA TGC TGA GCT
CAG GG
N2 specific forward
primer
MN2GRIA1 CAT TCC TGG CTT
CCA TCC TCT CAA
CCT G
N3 specific forward
primer
MN3GRIA1 GCT CAG GGC AGA
ATG CAG TTG ATT
GGT C
Reverse primer from
the junction of 5th and
6th exon
MREV1GRIA1 GTC AAT GTC CAT
GAA GCC CAG GTT
GGC
Reverse primer from
5th exon
MREV2GRIA1 GTG GTA CCC GAT
GCC GTT CTT TTC
TAG C
Rapid Amplification of cDNA Ends (50RACE)
The presence of alternatively spliced transcripts was
examined through 50 rapid amplification of cDNA ends
(RACE). 2 lg of total RNA from cerebellum and cortex
were amplified using a 50RACE kit and gria1 specific
reverse primer (MREV1GRIA1) from the junction of exon
5 and exon 6. The amplification was performed according
to the instructions provided with the kit. The RACE
product was fractionated by electrophoresis using 1.2%
agarose gel. Several bands were excised from the gel,
purified and sub-cloned into the TOPO vector, and
sequenced as described later.
Reverse Transcriptase (RT)-PCR
Multiple transcripts encoded by gria1 gene were identified
using RT-PCR. RNA (2 lg) from the cortex and cerebel-
lum were primed with oligo (dT)18 and single-stranded
cDNA was synthesized using the RevertAidTM H-Minus
Reverse Transcriptase at 43�C for 1 h in a total volume
20 ll. 1 ll of the single-stranded cDNA was then added to
PCR system for amplification using appropriate set of
primers in a total vol. 50 ll. PCR product was amplified
using touchdown PCR for 30 cycles. The cycle was per-
formed as; denaturation at 94�C for 4 min, 1 cycle; 93�C,
Neurochem Res
123
30 s; annealing 66�C, 30 s with a decrease of 0.3�C per
cycle, extension 72�C for 45 s, with final extension at 72�C
for 8 min. Final product was subjected to electrophoresis
on a 1.2% agarose gel, stained with ethidium bromide and
photographed on a UV transilluminator.
Semi-Nested RT-PCR
To confirm the results obtained after first round of PCR and
reconfirm the genuine PCR product, semi-nested PCR was
performed. After the RT reaction, as described above, PCR
amplification was carried out for 30 cycles (as above) using
gene-specific reverse primer (MREV1GRIA1 from the
junction of exon 5 and exon 6) and first-exon-specific
primers (based on the sequence obtained from 50RACE
products) as down and upstream primers, respectively. 1 ll
of the resulting PCR product was used as a template for
further amplification by PCR using the same upstream pri-
mer and a downstream primer from exon-5 (MREV2GRIA1
internal to the reverse primer used in 1st PCR). 10 ll of the
semi-nested PCR product was then subjected to electro-
phoresis on 1.2% (w/v) agarose gel, stained with ethidium
bromide and photographed on a UV transilluminator.
Subcloning and Sequencing of RT-PCR Products
The RACE and semi-nested PCR amplified products were
electrophoresed on 1.2% (w/v) agarose gels. Anticipated
ethidium-bromide-stained bands were cut from the gels and
DNA was purified using a PCR gel purification kit. The
purified DNA was subcloned using the plasmid cloning
vector. E. coli JM109-competent cells were transformed.
Transformed colonies were grown overnight at 37�C and
plasmid DNA was purified for sequencing. Plasmids con-
taining the insert were sequenced by automatic sequencer
using either M13 forward or reverse primers [18].
Bioinformatic Analysis
Homology and similarity searches of the obtained nucleo-
tide sequences were performed using the BLASTN non-
redundant database (http://www.ncbi.nlm.nih.gov/BLAST).
Alignment analysis was carried out using the Gene stream
Align tool (http://www2.igh.cnrs.fr/bin/align-guess.cgi) and
ClustalW tool available at www.ebi.ac.uk/clustalw [19].
TFBIND (http://tfbind.hgc.jp/), TFSEARCH (http://www.
cbrc.jp/research/db/TFSEARCH.html) and WWW signal
scan (http://www-bimas.cit.nih.gov/molbio/signal/) were
used to study the transcription factors binding to the
upstream region of the exons. Some of the properties of the
amino acid sequences coded by the new exons were analyzed
using ExPASy tools (http://ca.expasy.org/).
Results and Discussion
Three New and Differentially Expressed Transcripts
of gria1
AMPARs are expressed in various subunit combinations.
The four subunits (GluR1–GluR4) of the receptor assemble
in various stoichiometries to form functional ion channels
[20]. The different subunit composition determines channel
gating kinetics, conductance, vesicular trafficking and tar-
geting to specific synapse [21–23]. In mouse, gria1 gene is
located on chromosome 11 and a large 50UTR of almost
20 kb is present, the 1st intron is 3 kb and the 2nd intron is
150 kb in size as depicted in Fig 1a. The second intron is
unusually large. Mouse database searches revealed that this
subunit is coded by a single gene consisting of 16 exons
and 15 introns. cDNA corresponding to gria1 is present in
the database with Accession number NM001113325 that
encodes for a protein of 907 amino acids. In order to
investigate the presence of possible new splice variants
50RACE was performed. Total RNA isolated from cere-
bellum and cortex was amplified using a 50RACE kit using
the primers spanning exon 5 and exon 6. We were able to
amplify multiple transcripts from the RNA preparations of
mouse brain as shown in Fig 2. Following 50RACE,
sequencing of the products was done as described in
‘‘Materials and Methods’’ section. The cDNA sequences
containing the reverse primer sequence corresponding to
the part of 5th and 6th exon was further analyzed for their
50 ends. We obtained several clones containing new 50 end
sequences. The analysis of the sequencing data revealed the
identification of three new cDNA of gria1 differing at the
50 ends which were not reported earlier. The alignment of
these sequences with the genomic sequence confirmed that
these cDNA were transcribed from gria1 gene and the new
exons were derived from 50UTR, 1st intron and 2nd intron
of the gene. Three new sequences differing in their
N-terminus were produced from the usage of four novel
exons. These exons were designated as N1, N2, N3 and N4
and the resulting transcripts as T1, T2 and T3.
N1 exon derived from 50UTR splices with the 1st
reported exon (E1) producing a variant (T1) which encodes
for an additional 19 amino acid residues to the published
N-terminus of GluR1. N2 exon was found to be non-coding
and splices with the 2nd reported exon (E2) to produce a
variant (T2) which is truncated at the N-terminal by 69
amino acids. N3 exon splices with a novel N4 exon which
then splices with the 3rd reported exon (E3) producing a
different variant (T3) which is 40 amino acid residues
shorter as compared to the reported sequence. N4 exon is
also located in the 2nd intron and is 42,173 bp downstream
of exon N3. These transcripts variants produced through
alternative splicing are depicted in Fig 1b. The presence of
Neurochem Res
123
N2 variant was further supported through Expressed
Sequence Tag (EST). EST with accession number
BY719132 from adult male olfactory brain encodes for a
sequence identical to N2 variant. These analyses were
further confirmed by RT-PCR using upstream and down-
stream primer (given in ‘‘Materials and Methods’’) from
the 1st newly identified exons and from the junction of 5th
and 6th exon (MREV1GRIA1) respectively. The RT-PCR
results revealed bands of expected sizes as predicted from
50RACE sequence data. These results were further con-
firmed through semi-nested PCR using the downstream
primer MREV2GRIA1 (from exon 5th) which was
designed from a sequence internal to the first reverse pri-
mer (MREV1GRIA1). The agarose gel electrophoresis of
the semi-nested PCR revealed bands of expected sizes for
T1 (752 bp), T2 (684 bp) and T3 (573 bp) in both cere-
bellum and cortex of mouse brain (Fig 3). These semi-
nested RT-PCR products were also cloned and sequenced.
Sequencing confirmed that these cDNAs were produced
through alternative splicing of gria1 gene and the tran-
scripts were formed through the use of N1, N2 and N3
exons in combination with the internal exons as discussed
above. The sequences were submitted to the GenBank and
following accession numbers JN180918, JN180919 and
JN180920 were obtained for T1, T2 and T3 respectively.
The study of the structure of these exons showed that the
donor–acceptor residues associated with these new exons
are found to be conserved. The donor ‘‘GT’’ and the
Fig. 1 a Genomic organization of gria1 gene in mouse. The gene is
located on chromosome 11 with a large 50UTR of almost 20 kb, the
1st and 2nd intron are 3 and 150 kb in size, respectively. The
positions of reported exons (E1–E6, E16) as well as new exons (N1,
N2 and N3 and N4) are shown. The positions of the primers are
shown by arrows above the exons. CtrF. N1F, N2F, N3F are exon
specific forward primers and R1 and R2 are reverse primers. b The
transcript variants produced through alternative splicing. Only exons
are shown in mature transcripts. Each rectangle represents an exon,
dark filled rectangles represent newly identified exons while light
filled rectangles are published exons. (i) The published sequence
containing all the exons. (ii) T1 variant produced through the splicing
of a new exon (N1) to the preexisting first exon (E1). The rest of the
sequence is identical to the reported sequence. (iii) A new exon (N2)
replaces E1 and splices with E2 to produce a new transcript variant
T2. (iv) Two new exons N3 and N4 replace E1 and E2 of the reported
sequence and splices with E3 to produce T3 transcript. All these
transcript variants differ in their 50 ends. Sizes of exons are not to
scale
Fig. 2 Agarose gel electrophoresis of 50 Rapid Amplification of
cDNA ends (RACE). a Amplified 50RACE product from cerebellum.
b Amplified 50RACE product from cortex. These amplified products
were electrophoresed on 1.2% gel and stained with ethidium bromide.
In both (a) and (b) Lanes M, -RT, ?RT are 100 bp DNA ladder,
RACE products in absence of reverse transcriptase and RACE
products in presence of reverse transcriptase respectively. Products
from ?RT lane were excised and cloned. Sequencing data confirmed
the presence of transcripts published earlier in addition to the newly
identified transcripts
Neurochem Res
123
acceptor ‘‘AG’’ residues are conserved in all these new
exons (Fig 4). In order to understand the differential
expression of these variants we analyzed the 50 upstream of
these new exons. In silico analysis employing programs
like TFBIND, TFSEARCH and SIGSCAN of the upstream
region of these newly predicted exons showed that a
number of transcription factor (TF) binding sites are
available as depicted in Fig 5. The 50 upstream region of
N1, N2 and N3 exons contain TATA Binding Protein
(TBP) sites. TBP is a key component of eukaryotic tran-
scription initiation machinery [24]. The locations of other
general TFs are also shown in the Fig 5. SP-1 TF binding
site is present in the upstream of all these exons. This TF is
involved in the gene expression in early development of an
organism [25]. Multiple Nuclear Factor-1 (NF-1) binding
sites are present preferably this is associated with the
upstream region of the genes that are highly expressed [26].
Upstream of E1, N1 and N2 exons report Myc-associated
zinc-finger protein (MAZ) TF binding site. MAZ and SP-1
are often found regulating the same gene and may even
bind to the same sites [27]. Activating protein-2 (AP-2) TF
binding site is located upstream of N2 and AP-2 play
important regulatory roles in vertebrate development,
apoptosis and cell-cycle control [28]. Pit-1 TF is a pitui-
tary-specific TF while serum response factor (SRF) is
ubiquitous nuclear protein that stimulates both cell prolif-
eration and differentiation. A number of TFs bind to
CCAAT box such as NF-Y, CP-1, CDP, GATA-1, and
NFE3. Some TFs are tissue specific while others are con-
stitutively expressed. These analyses suggest that these
transcripts may involve various TF activities which might
be responsible for their differential activity in different
regions of the mouse brain.
Comparative Analysis of the New Variants
In order to understand the structure and functions of these
variants we analyzed their conceptually translated amino
Fig. 3 Agarose gel electrophoresis of the semi-nested PCR products
from a cerebellum and b cortex of mouse brain. 10 ll of the semi-
nested PCR product was electrophoresed on 1.2% agarose gel
electrophoresis. The lanes are labeled as M, ctrl, T1, T2 and T3 which
correspond to DNA marker 100 bp ladder and RT-PCR products
obtained using exon specific primers. Ctrl lane corresponds to 679 bp
and is the amplified product obtained from primers designed from
published sequence, T1 corresponds to the first new transcript having
anticipated size 752 bp, T2 is second new transcript with an
anticipated size of 684 bp and T3 is the third new transcript variant
where the band corresponds to the anticipated 573 bp. The expression
levels of these transcripts are different as seen by the intensity of the
band
Fig. 4 Splice donor–acceptor sites of first few exons of the transcripts. The junction residues are conserved in all the exons. The donor ‘‘GT’’
and the acceptor ‘‘AG’’ residues are conserved in all these new exons and are shown by capital italicized alphabets
Neurochem Res
123
Fig. 5 Analysis of the upstream
region of these exons using
web-based programs TFBIND,
TFSEARCH and SIGSCAN.
The upstream region of the
exons is represented by
rectangular boxes and the
binding positions of various
transcription factors (TFs) are
shown. 600 bp upstream
sequences are analyzed to locate
the position of TFs. Analysis
revealed that many brain
specific TFs can potentially bind
to upstream regions of the
newly identified exons
Fig. 6 Multiple sequence
alignment (ClustalW) of the
deduced amino acid sequences
of the published transcripts (E1)
and the newly identified
transcripts (N1 N2 and N3). (*),
colon (:) and dash (–) represents
identical, similar and no residue
respectively. The accession
numbers are provided at the end
of the sequences. All sequences
are identical from third exon
onwards. Accession numbers
are given at the end of each
sequence
Neurochem Res
123
Ta
ble
1A
com
par
ativ
ean
aly
sis
of
the
amin
oac
idse
qu
ence
of
the
tran
slat
edtr
ansc
rip
ts
Pro
per
ties
E1
?E
2N
1?
E1
E2
?E
3N
3?
N4
Am
ino
acid
seq
uen
ceM
PY
IFA
FF
CT
GF
LG
AV
VG
AN
FP
NN
IQIG
GL
FP
NQ
QS
QE
HA
AF
RF
AL
SQ
LT
EP
PK
LL
PQ
IDIV
NIS
DS
FE
MT
YR
ML
SS
GR
LG
KG
GR
AC
ILL
LQ
MP
YIF
AF
FC
T
GF
LG
AV
VG
AN
FP
NN
IQI
MT
YR
FC
SQ
FS
KG
VY
AIF
GF
YE
RR
TV
NM
LT
SF
CG
AL
HV
CF
ITP
SF
P
VD
TS
NQ
FV
LQ
LR
PE
LQ
EA
LIS
IID
HY
KW
QT
FV
YIY
DA
DR
MQ
LIG
LS
ME
MG
YD
AL
Q
ET
KA
NR
EN
VQ
NM
AL
GL
AV
No
of
aa7
34
68
43
4
pI
4.9
09
.50
6.6
94
.41
Mw
8,1
70
.37
4,9
07
.89
9,9
36
.43
3,7
40
.34
Sig
nal
pep
tid
eY
es
VV
G-A
N
Sig
nal
anch
or
No
n-s
ecre
tory
pro
tein
No
n-s
ecre
tory
pro
tein
Mit
och
on
dri
alta
rget
ing
seq
uen
ceN
oN
oY
escl
eav
age
site
at2
4
Cle
aved
seq
uen
ceM
TY
RF
CS
QF
SK
GV
YA
IFG
FY
ER
R
No
Net
Og
lyc
No
No
No
No
Net
Ng
lyc
MP
YIF
AF
FC
TG
FL
GA
VV
G
AN
FP
NN
IQIG
GL
FP
NQ
QS
Q
EH
AA
FR
FA
LS
QL
TE
PP
KL
L
PQ
IDIV
NIS
DS
FE
MT
YR
No
No
No
Net
Ph
os
S36,
S67,
T71
Ser
:2T
hr:
1T
yr:
0
No
Yes
S66,T
24,
T48,
Y14
Ser
:1T
hr:
2T
yr:
1
Yes
,S
7
Net
acet
No
Yes
3S
Yes
2T
No
Net
Ph
osK
PK
CT
71
PK
CS
4P
KC
T2
No
My
rist
oy
lati
on
No
No
No
No
Th
en
um
ber
assu
per
scri
pt
ind
icat
esth
ep
osi
tio
no
fth
eam
ino
acid
resi
du
eu
nd
erg
oin
gm
od
ifica
tio
n.
Th
e1
sttw
oex
on
so
fth
en
ewtr
ansc
rip
tsar
ean
aly
zed
ino
rder
tost
ud
yth
ed
iffe
ren
ce
asso
ciat
edw
ith
the
N-t
erm
ini
var
iati
on
of
thes
etr
ansc
rip
ts.
Th
ese
qu
ence
‘NIS
’in
bo
ldle
tter
defi
nes
the
gly
cosy
lati
on
site
Neurochem Res
123
acid sequences. Multiple sequence alignment employing
web-based ClustalW of these translated sequences revealed
that they differed only in their N-terminal (Fig 6). A
number of properties of these variants were studied and
compared using ExPASy tool. Table 1 compares some of
the properties of these translated exon sequences and pro-
vides a number of difference in the properties of these new
exons. The phosphorylation and acetylation pattern of these
sequences are very different. N1 and N2 contain 3S and 2T
respectively which can be potentially acetylated. However
no such residues are present in E1 and N3 variants.
Although a majority of eukaryotic proteins, almost 50% of
yeast proteins and 80% of the human proteins are N-ter-
minally acetylated (Nt-acetylated), the function of this
modification is largely unknown [29, 30]. For some pro-
teins such as actin, Nt-acetylation has been reported to
affect protein functionality, e.g. non-acetylated actin is less
efficient at assembling microfilaments [31]. Recently the
other role suggested for acetylation is that Nt-acetylation of
a protein can function as a degradation signal (degron)
playing a role in protein turnover and homeostasis [29]. N1
and N2 variants have potential PKC dependent phosphor-
ylation sites. Protein phosphorylation of neuronal gluta-
mate receptors by cyclic AMP-dependent protein kinase
(PKA) and protein kinase C (PKC) may regulate their
function and play a role in some forms of synaptic plas-
ticity [32]. E1 contains a signal peptide while N1 contains
signal anchor. Many integral proteins contain signal non-
cleavable transmembrane signal sequence called signal
anchor sequence which anchors the protein to the mem-
brane [33–35]. N2 contains a mitochondrial targeting
sequence. Glutamate excitotoxicity often leads to mito-
chondrial dysfunctioning associated with apoptotic and
necrotic neuronal cell death [36]. N3 does not contain such
signal sequence. The detailed study of this variant might
provide new pathways to study the regulation in neuronal
cell death. The diverse molecular properties of the variants
might be responsible their differential expression in cere-
bellum and cortex of the mouse brain as shown by the
RT-PCR result in our study.
Our studies provide the 1st experimental evidence for
the presence of multiple alternatively spliced transcripts
variants of the gria1 gene encoding different N-termini.
The new exons are produced from the large intronic
regions of the gene. The large introns are frequently
associated with the evolution of a gene. These transcripts
being differentially expressed in cerebellum and cortex of
mouse brain suggest that the N-termini may be associated
with differential targeting of the receptor to different
regions of the brain. The properties of these variants are
highly heterogeneous and might play a role in providing
functional, structural and molecular diversity to the AMPA
receptors. A detailed study of these variants might throw
light on the mechanism of synaptic plasticity, neuronal
development and various neuropathological conditions
involving AMPARs.
Acknowledgments The authors are thankful to CSIR, New Delhi,
India for providing senior research fellowship to SA. Partial funding
from the Department of Biotechnology (DBT), Ministry of Science
and Technology, New Delhi and necessary facilities provided by
AMU, Aligarh, India is thankfully acknowledged.
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