CLONING, FUNCTIONAL AND

MOLECULAR CHARACTERIZATION

CHAPTER 1

Introduction

In early 1990s, the serendipitous discovery of few genes like Xist (Brown CJ,

Willard HF. 1989; Brown SD et al. 1991) H19 (Bartolomei MS et al. 1991) and Air

(Sleutels F et al. 2002, Sotomaru Y et al. 2002), which were found to be functional as

RNAs led to a new era in the history of cellular regulation. Around the same time, two

small RNAs - let-4 and lin-7 were discovered in C. elegans as regulators of

developmental timing by post transcriptional gene regulation (PTGS) mechanism (Lee

RC et al. 1993; Wightman B et al. 1993; Reinhart BJ et al. 2000; Slack FJ et al. 2000).

Earlier, PTGS was established to be functional only in plants and was called as gene co-

suppression (Napoli C et al. 1990). This new class of functional RNAs which possess no

specific long ORFs and do not encode for proteins are known as noncoding RNAs

(ncRNAs), The human and mouse genome sequencing and annotation has revealed

that 98% of the transcriptional output consists of ncRNAs (Lander ES et al. 2001; Venter

CJ et al. 2001; Waterston RH et al. 2002). The ncRNAs besides being omnipresent and

omnipotent are known to fulfill a wide range of normal functions, which includes control

of chromosome dynamics, splicing, RNA editing, translational inhibition and mRNA

destruction. Earlier, ncRNAs were thought to be an exception in the cellular mechanism,

however now they are considered as a new tier of gene regulation in eukaryotic biology

(Costa FF. 2005; Mattick JS, Makunin IV. 2006).

Noncoding RNAs are implicated in many cancers and various other diseases.

Genome-wide profiling has revealed that ultra conserved regions (UCRs), which encode

a particular set of ncRNAs, have distinct signatures in human leukemia’s and

carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in

cancers (Calin GA et al. 2007). HEPT3, a novel ncRNA is over expressed in 87%

(20/23) of HCC (hepatocellular carcinoma) patients and in 4/5 of HCC cell lines. It is an

un-spliced RNA and lacks extensive ORF. It contains an Alu sequence near the 5'

terminus. Reduction in HEPT3 expression leads to reduced cell proliferation. Hence, it

may be proposed that it’s over expression may play a role in hepato-carcinogenesis

(Moh MC et al. 2007). HULC (highly up-regulated in liver cancer) is mRNA like ncRNA

that is spliced and polyadenlyated and resembles the mammalian LTR transposon 1A.

HULC is higly expressed in HCC. HULC is present in the cytoplasm, where it copurifies

with ribosomes. siRNA mediated knockdown of HULC RNA in two HCC cell lines

affected HCC (hepatocellular carcinoma) (Panzitt K et al. 2007). Similarly, H19

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(Bartolomei MS et al. 1991), MALAT1 (Ji P et al. 2003), DD3 (Bussemakers MJG et al.

1999) play a role in carcinogenesis. Several miRNAs whose expression is increased in

tumors act as oncogenes. These oncogenic miRNAs are called “oncomirs”, These

oncomirs usually promote tumor development by negatively inhibiting tumor suppressor

genes and/or genes that control cell differentiation or apoptosis (Cho WC. 2007;

Esquela-Kerscher A, Slack FJ. 2006).

The genome as well as transcriptome annotation of the eukaryotic genome has

revealed a surprising abundance of antisense transcripts. Around 20% of human and

mouse genes overlap resulting in a potential pair of sense- antisense transcripts (Werner

A. 2005). These natural antisense transcripts (NATs) are transcribed from opposite

strands of their sense partners and regulate sense genes at multiple levels by

complimentary base pairing. In recent years, NATs have been implicated in many

aspects of eukaryotic gene expression including genomic imprinting, RNA interference,

translational regulation, alternative splicing, X-inactivation and RNA editing (Yin Y et al.

2007). Therefore, it is possible that antisense gene expression might be a common

mechanism of regulation in higher organisms like mouse and humans. There are two

types of NATs present, the one transcribed from opposing DNA strands at the same

genomic locus known as cis-NATs and the other transcribed from separate genomic loci

known as trans-NATs (Werner A, Berdal A. 2005; Lavorgna G et al. 2004). Several long

ncRNAs exist as cis-NATs like Xist and Tsix which act antagonistically in X-chromosome

inactivation in higher mammals (Lee et al. 1999; Stavropoulos et al. 2001). Mouse Igf2r

imprinting depends on the expression of Air RNA. The truncated product of AIR, with

deletion of antisense region to Igf2r shows complete loss in silencing of the Igf2r gene

cluster, indicating that ncRNAs have an active role in genomic imprinting (Sleutels F et

al. 2002).

This chapter includes the cloning of human M3TR and sequence analysis using

bioinformatics. M3TR functional analysis was assessed using stable ectopic expression

followed by in vitro and in vivo transformation assays. Molecular characterization of

M3TR i.e., transcript analysis, sense/antisense expression, subcellular localization etc.,

was investigated using techniques like RT-PCR, northern hybridization, ribonuclease

protection assay, rapid amplification of cDNA ends, strand specific RT assay and real

time PCR etc.

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Results

1.1 Cloning and sequence analysis of human M3TR (hM3TR). 1.1.1 Expression in human tissues and cell lines.

Several human tissues and cell lines were screened for presence of M3TR by

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis using M3TR

specific internal primers. Fig. 1.1 shows the analyses of the RT-PCR products on an

agarose gel, wherein the upper panel represents M3TR gene expression and the lower

panel is for β-actin used as a loading control. The human fetal brain, testis and cell-lines

SK-N-MC (neuroepithelioma), HeLa (cervical carcinoma), Raji and Jurkat (hematopoetic)

expressed M3TR. Weak expression was detected in WI38 and MRC5 (fetal lung

fibroblast) cell lines and the expression was absent in the adult brain and U373-MG (Fig.

1.1). Sequence analyses of M3TR PCR products confirmed complete homology of

human derived M3TR with mouse M3TR sequence. The expression of M3TR in fetal

brain, testis (germinal tissues) and its presence in few of the cancerous cell lines like

SK-N-MC, HeLa, Raji and Jurkat suggests a plausible role for M3TR in embryonic

development and malignancy.

Figure 1.1 M3TR expression in human tissues and cell lines by RT-PCR analysis.

5 µg of DNased total RNA was used to synthesize cDNA with oligo dT15 primer from human

cell lines- SK-N-MC, U373-MG, Hela, Raji, Jurkat, WI38, and MRC5. The cDNAs from

human tissues- adult brain, fetal brain, fetal lung and testis were commercially procured (BD

clontech). M3TR was amplified from 100 ng of cDNA using GSPs for M3TR i.e., AS89

(sense) and AS90 (reverse). Positivity of expression is indicated by the presence of a ~500

bp amplicon. β-actin is used as internal control that produced a ~500 bp product.

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1.1.2 Cloning and sequencing of hM3TR from SK-N-MC cell line. Human M3TR (hM3TR) was cloned from SK-N-MC (ATCC no- HTB-10), a cell

line developed from neuro-epithelioma of adult brain. This cell-line was chosen for its

high M3TR expression compared to other cell-lines. Moreover, the accumulating

evidences suggest that development and function of the nervous system is heavily

dependent on RNA editing and the intricate spatiotemporal expression of a wide

repertoire of non-coding RNAs, including micro RNAs, small nucleolar RNAs and longer

ncRNAs (Mehler MF, Mattick JS. 2006; Cao X et al. 2006). Total RNA from SK-N-MC

was used to synthesize cDNA using oligo dT primer. hM3TR was PCR amplified from

the cDNA employing M3TR specific 5’ and 3’ end primers (AS 216 and AS 218). The

agarose gel analysis revealed a 557bp PCR product of M3TR (Fig. 1.2.A) that was gel

eluted and cloned into pTarget mammalian expression vector using TA cloning strategy

(Fig. 1.2.B). The clones with hM3TR in sense and anti-sense orientations were screened

by PCR amplification using vector specific T7 primer and either of the insert specific

primers- AS89 or AS90. The clone that showed amplification of the insert with T7/AS90

was designated as hM3TR (sense) whereas the clone with insert that got amplified with

T7/AS89 was hM3TR-as (anti-sense) (Fig. 1.2.C). Sequencing with T7 and M3TR

specific primers served as an additional criteria for the confirmation of directionality of

cloning. Further, the insert size ~600bp was confirmed by restriction digestion analysis

with EcoRI (Fig. 1.2.D).

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Figure 1.2 Cloning and confirmation of hM3TR in pTarget expression vector.

A. Map of mammalian expression vector- pTargetT (Promega, USA). The vector has CMV

early promoter to drive the expression of cloned insert and neomycin resistance gene for

G418 stable selection in mammalian cells. B. SK-N-MC cDNA was synthesized using 5 µg

of RNA using AMV-RT and Oligo dT15. 100 ng of cDNA was used for PCR amplification of

hM3TR using M3TR specific 5’ and 3’ end primers, AS216 and AS218 respectively. A 557 bp

amplicon was seen with SK-N-NC cDNA but not in water control. C. Directionality of hM3TR

clones was determined by PCR amplification using the vector based T7 primer and either of

the gene specific primers (AS89-sense /AS90-reverse). pTarget Empty vector shows no

amplification with AS89/AS90 (lane 1). pT-hM3TR (sense) shows amplification with AS89/90

and T7/AS90 (lanes 1&3). pT-hM3TR-as (reverse) shows amplification with AS89/AS90 and

T7/AS89 (lanes 1&2). D. An overnight digestion of 5 µg of hM3TR sense and reverse

plasmid DNAs with EcoRI restriction digestion yielded a 600 bp product.

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The directionality of pT-hM3TR (sense) and pT-hM3TR-as (anti-sense) clones

was confirmed by sequencing using vector (pTarget) specific T7 primer. The hM3TR

sequence was found to be 557 bp as shown in Fig. 1.3.

Figure 1.3 hM3TR sequence (557bp). The hM3TR sequence was determined by automated sequencing using T7 primer and found

to be 557 bp in length. The sequencing was hM3TR was affirmed by sequencing of at least 5

cDNA clones from SK-N-MC. The representative human M3TR sequence in sense

orientation is shown.

1.1.3 In silico analyses. The computational analysis of hM3TR sequence was done using the BLAST

programs available at www.ncbi.nlm.nih.gov and www.ensembl.org. The BLAST (Basic

Local Alignment and Sorting Tool) analysis of hM3TR sequence showed 100%

homology to a genomic contig (XNT_039706.6) located on mouse X-chromosome

indicating that the M3TR sequence was conserved in mouse and human (Fig. 1.4.A).

The NBLAST analyses of M3TR sequence with nonredundant (nr) human genome

sequence database resulted in a single major hit. A genomic contig (NW_928032.1)

from Celera genomics showed 71% homology with hM3TR sequence (Fig. 1.4.B, red

line). Further, BLAST analysis with human HTGS database (high throughput genomic

sequences) showed a high homology of >80% on human chromosomes 8, 17 and 2.

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However, the M3TR sequence did not show any specific long ORFs and contained

several stop codons indicating the noncoding RNA nature of M3TR.

Figure 1.4 BLAST analyses of hM3TR sequence.

A. hM3TR (557bp) sequence was aligned with mouse M3TR sequence using BLAST2

program. The human M3TR shows 100% identity to mouse M3TR, as shown pictorially. B.

hM3TR sequence was analyzed using N-BLAST for homologies in the non-redundant human

genome sequence database at www.ncbi.nlm.nih.gov. The pictorial representation of hM3TR

homology shows a single major hit. An unidentified genomic contig from Celera genomics

shows 71% homology to hM3TR (red line).

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1.2 Functional characterization of hM3TR. The biological significance of hM3TR was determined by ectopic expression

studies wherein hM3TR was stably transfected by lipofection into NIH3T3 (ATCC-CRL-

1658), a mouse embryonic fibroblast cell line. NIH3T3 is one of the most commonly used

recipient cell line in DNA transfection studies.

1.2.1 Stable ectopic expression of hM3TR.

The pT-EV (empty vector), pT-hM3TR and pT-hM3TR-as plasmids were stably

transfected into NIH 3T3 cells by G418 selection. Both, hM3TR and hM3TR-as yielded

20-25 G418 resistant colonies per 1 µg of DNA used. The colonies were pooled and

developed into stable cell lines and named as NIH-pT-EV, NIH-hM3TR and NIH-hM3TR-

as respectively. Fig. 1.5 shows the phase contrast micrographs of the transfected cells.

The recipient NIH 3T3 cells appeared non-refractile and had a contact inhibited nature.

Similarly, the NIH-pT-EV and NIH-hM3TR-as cells grew in a contact-inhibited manner.

Interestingly, the NIH-hM3TR cells appeared morphologically distinct and were highly

refractile. The cells possessed high proliferative potential, were not contact inhibited and

had the ability to grow in a piled up manner (Fig. 1.5). The stable transfections were

performed at least 3 times using independent batches of NIH 3T3 cells and plasmid

DNA’s to ensure the reproducibility in data.

Figure 1.5 Micrographs of stably expressing hM3TR, hM3TR-as cell lines.

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NIH 3T3 cells (1 X 105) were transfected with 5 µg of plasmid DNA using 10 µl of

lipofectamine 2000. Stable clones were obtained by selection on G418 (500 µg/ml) for 15

days. The resistant colonies were pooled and developed into cell lines. The NIH 3T3, pT-EV

and hM3TR-as cells grow as flat sheets and showed contact inhibition, In contrast, the

hM3TR cells grew in a piled up manner. The phase contrast micrographs of the stable cell

lines were shown at 10X magnification.

The ectopic expression of M3TR in stable cell lines pT-EV, hM3TR and hM3TR-

as was confirmed by RT-PCR analysis. The hM3TR and hM3TR-as cell lines showed a

marked over-expression of M3TR over the pT-control cell line (Fig. 1.6).

Figure 1.6 Ectopic expression of M3TR in stable transfections.

Total RNA from pT-control, hM3TR and hM3TR-as stable cell lines was used for cDNA

synthesis followed by PCR amplification using M3TR internal primers (AS89 and AS90).

Stable cell lines- hM3TR and hM3TR-as on RT-PCR analysis showed specific over-

expression of M3TR compared to pT-EV, seen as a 557 bp product on a 1% agarose gel.

1.2.2 Biological function analysis by in vitro clonogenecity assay.

The transformed cells are characterized by loss of contact inhibition and potential

for colony formation in soft agar. This attribute is useful for studying transformation

potential in vitro (Clark SS et al. 1995). The M3TR transfected cells revealed high

clonogenecity within 15 days of seeding with development of colonies seen as early as 5

days of seeding, indicating the transforming nature of hM3TR- sense transcript. The pT-

EV and hM3TR-as cells did not show any colony formation even after 15 days of

seeding (Fig. 1.7).

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Figure 1.7 hM3TR stable cell line shows colony formation in soft agar assay.

The anchorage independent growth potential was assayed by soft agar colony formation

assay, wherein 5X103 cells were seeded in complete medium with 0.3% agarose, overlaid

upon a basal layer of 0.6% agarose in complete medium. The colony formation was

observed for 15 days. The hM3TR cells show high clonogenecity in soft agar. The pT-control

and hM3TR-as cells did not develop colonies in agarose. Three independent experiments

were performed to validate the reproducibility in results. The pictures shown are the

representative pictures at 4X magnification.

1.2.3 Biological function analysis by in vivo tumorigenecity assay.

The transformed cells generate tumors in the nude mice, a property of highly

transformed cells. Therefore, the transforming potential exhibited by M3TR in vitro was

validated in vivo by tumorigenecity assays in nude mice. The pT-control cells, hM3TR

and hM3TR-as stable cell lines were injected subcutaneously into the right flanks of

athymic nude mice and observed for tumorigenecity at the site of injection. hM3TR cells

showed development of large sized tumors of 1.5 - 2 cm3 in volume, in nude mice within

4 weeks. However, hM3TR-as and pT-control cell lines were non-tumorigenic even after

8-12 weeks of injections (Fig. 1.8).

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Figure 1.8 hM3TR drives tumor formation in nude mice.

The in vivo tumorigenecity assays in athymic nude mice (6-8 weeks old) were performed by

subcutaneous injection of 1X106 cells/ mouse into the right flanks of the nude mice and

observed for tumor development for 8 weeks. The mice injected with vector control and

hM3TR-as cells did not show any tumor formation even after 8 weeks. The mice injected with

hM3TR cells showed tumors as early as 7-10 days with tumor growth progressing to 1.5 - 2

cm3 (4/3.πr3) in size within 4-6 weeks. The experiments were performed thrice by injecting

into a set of three mice per cell line. The mice pictures shown are representative pictures

from one of the experiments.

The above results of transformation assay were similar to the results obtained using

mouse M3TR. Earlier, our lab has demonstrated the transforming nature of mouse

M3TR sense transcript and non-transforming property of the reverse transcript.

Moreover, the bioinformatics analyses have proven the 100% homology of M3TR

sequence in mouse and human. Therefore, ‘M3TR’ notation would only be used for

human M3TR in future. The study reported here can be considered as a collective work

for the M3TR- noncoding RNA in human and mouse.

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1.3 ORF analysis of M3TR sequence and functional studies. 1.3.1 ORF analysis of M3TR sequence.

The ORF analysis of M3TR sequence done using ‘ORF finder’ program at NCBI

website revealed three small putative ORFs: ORF1, ORF2, and ORF3 of 138 bp, 156 bp

and 312 bp respectively (Fig. 1.9.A). The three ORFs are highlighted in the 557bp M3TR

sequence (Fig. 1.9.B). The possibility of these ORFs contributing towards the

transformation induced by M3TR was assayed experimentally by cloning the ORFs

individually into pEGFP-N1 vector followed by in vivo functional studies.

Figure 1.9 ORF analysis of M3TR sequence.

A. The ORF analysis of M3TR sequence reveals three putative ORFs, namely ORF1, ORF2

and ORF3, from top to bottom respectively (www.ncbi.nlm.nih.gov/orffinder). B. The three

putative ORFs are represented in M3TR sequence. The arrow represents start codon. The

ORF1 (red, long dotted line) and ORF2 (green, dotted line) have ATG as start codon. ORF3

(blue, line) has CAG as start codon. The ORF1 has a stop codon TAA.

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1.3.2 Cloning of putative ORFs as fusion products to EGFP. An upstream Kozak consensus sequence was added to the three putative ORFs

in M3TR sequence and they were cloned and expressed in pEGFP-N1 vector as fusion

products with GFP, at the N-termini of EGFP (Fig. 1.10.A). The M3TR in sense and

reverse orientations were also cloned and expressed in pEGFP-N1 vector. For cloning,

the three ORFs as well as M3TR and M3TR-as cDNAs were PCR amplified with

respective sense and reverse primers using BglII and HindIII adapters respectively, at

the 5’ ends. They were cloned into pEGFP-N1 vector by directional cloning strategy into

BglII and HindIII restriction sites in the multiple cloning site (MCS). The positive clones

named as pEGFP-M3TR, pEGFP-M3TR-as, pEGFP-ORF1, pEGFP-ORF2 and pEGFP-

ORF3 were confirmed for the presence of specific insert by PCR analyses using

respective internal primers (Fig. 1.10.B). The identity of the clones was confirmed by

sequencing.

Figure 1.10 Cloning and confirmation of the three putative ORFs in pEGFP-N1

expression vector. A. The vector map of pEGFP-N1. It has CMV early promoter and a neomycin selection

marker. The three putative ORFs, M3TR and M3TR-as were PCR amplified by incorporating

5’ BglII and 3’ HindIII adapters, and cloned in frame with GFP by directional cloning strategy

into BglII and HindIII sites in multiple cloning site (MCS) of pEGFP-N1 vector. B. The positive

clones were confirmed by PCR amplification with insert specific primers. ORF1, ORF2 and

ORF3 show 138bp, 156bp, 312bp products respectively. M3TR and M3TR-as show 557 bp

products. However, the EGFP-N1 shows no amplification. (The primers used for cloning

were provided in materials section).

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1.3.3 Stable ectopic expression of putative ORFs. The EGFP plasmid constructs- M3TR-EGFP, M3TR-as–EGFP, ORF1-EGFP,

ORF2-EGFP, ORF3-EGFP were stably transfected into NIH 3T3 cells and selected on

G418 (500 µg/ml) containing medium for ~15 days. The resistant colonies obtained were

pooled and sub-cultured to develop into stable cell lines. pEGFP-N1 stable cell line

served as control. The ectopic expression of the cloned inserts was confirmed by RT-

PCR analysis using the respective insert specific primers. The agarose gel analysis

showed specific overexpression of M3TR in M3TR-EGFP and M3TR-as-EGFP, shown

as ~500bp product. ORF1-EGFP, ORF2-EGFP and ORF3-EGFP showed specific

amplification of 138 bp, 156 bp and 312 bp respectively. EGFP-NIH 3T3 cell line showed

a weak expression of M3TR (Fig. 1.11). Figure 1.11 Confirmation of ectopic expression of ORFs by RT-PCR analysis.

The ectopic expression of specific inserts in EGFP-N1 constructs confirmed by RT-PCR

analysis. Total RNA from EGFP-N1, M3TR-EGFP, M3TR-as-EGFP, ORF1-EGFP, ORF2-

EGFP, and ORF3-EGFP stable cell lines was used to synthesize cDNAs, followed by PCR

amplification with the insert specific primers (shown in materials). The agarose gel analysis

shows over-expression of specific ORFs over EGFP control cells. β-actin was used as an

internal control.

1.3.4 ORFs functionality by in vivo tumorigenecity assays.

The transforming potential of three putative ORFs was assessed by in vivo

tumorigenecity assays in nude mice as explained earlier. The individual cell lines were

injected in a set of 5 mice. None of the three ORFs induced tumor formation in nude

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mice even after 8 weeks of inoculation indicating that the ORF encoded proteins are

non-transforming. Consistent with our earlier results, EGFP-M3TR cells showed high

tumorigenecity in all the five mice that were inoculated, within a short span of 4 weeks.

EGFP-M3TR-as and EGFP control cells were non-tumorigenic (Table. 1). Taken

together, these studies suggests that the possible protein products from M3TR

sequence were not responsible for tumorigenecity caused by M3TR, but the entire

M3TR noncoding RNA was responsible for the transforming function.

Table.1. ORFs show no tumorigenecity in nude mice.

The nude mice tumorigenecity assays were performed as explained above, using stable cell

lines developed by transfection of EGFP-N1 constructs into NIH 3T3. A set of 5 mice were

injected with each of EGFP-N1, EGFP-M3TR, EGFP-M3TR-as, ORF1-EGFP, ORF2-EGFP

and ORF3-EGFP. While none of the 3 ORFs induced tumors, as expected M3TR in sense

orientation is tumorigenic.

1.3.5 ORFs expression as GFP fusion proteins. Expression of ORFs as fusion products with GFP was studied by Western

blotting by using anti-GFP antibody. The three putative ORF-GFP constructs were

transiently transfected into NIH 3T3 cells and analyzed for formation of GFP- fusion

products. The Western blot analyses revealed the expression of ORF1 and ORF2 as

fusion products with EGFP, evident by the 34 kDa and 38 kDa size bands detected on

the blot compared to 27 kDa of native GFP (Fig. 1.12). However, ORF3 did not form any

fusion protein with EGFP and manifested by a 27 kDa band as of GFP (Fig. 1.12). The

expected protein products of ORF1, ORF2 and ORF3 were 5.3 kDa, 6.0 kDa and 11.5

kDa respectively.

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Figure 1.12 Western hybridization using EGFP antibody confirms the fusion proteins

by ORF1 and ORF2. NIH 3T3 cells were transiently transfected with EGFP-ORF1, ORF2 and ORF3 constructs for

48 hours. The protein lysate was prepared from the transfectants using RIPA buffer with 1X

protease inhibitor cocktail. Protein estimation was performed using Lowry method.

Approximately 60 µg of protein was separated on 10% SDS-PAGE, blotted onto PVDF

membrane using wet transfer in neutral buffer. The blots were hybridized with anti-GFP 1°

antibody (1:1000) followed by anti-IgG HRP (1:2500), and bands were visualized using ECL

chemiluminescent kit. The expression of ORF1 and ORF2 as GFP-fusion products of

molecular weights 34 kDa and 38 kDa respectively is shown. ORF3 did not form a fusion

protein with EGFP.

1.3.6 Sub-cellular localization of ORF-GFP constructs.

The intra-cellular localization of ORF1, ORF2 and ORF3 was analyzed by

transient transfection studies in NIH 3T3 cells by tracking the GFP reporter. The ORF1

was localized exclusively to the nuclei in NIH3T3 cells and ORF2 was targeted to

mitochondria. However, ORF3 showed no specific localization and was expressed all

over the cell similar to the native GFP protein (Fig 1.13.A). The nuclear localization of

ORF1 was confirmed by co-localization studies using DAPI (stains nuclei). The

mitochondrial localization of ORF2 was determined with mitotracker dye that specifically

targets to the mitochondria (Fig 1.13.B). The amino acid sequence analyses revealed

that ORF1 possessed a nuclear localization signal (NLS) whereas ORF2 had a

mitochondrial targeting signal (MTS) at their N-termini (Fig 1.13.C). The PSORTII

program at http://psort.nibb.ac.jp showed 82.6% possibility of ORF1 to be localized into

nuclei. The NLS is known to constitute positively charged aminoacids i.e., Lysine or

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Arginine at the amino termini of the peptide sequence. MTS is characterized by

presence of basic amino acids i.e., Serine and Leucine at the N-terminal region with less

number of acidic amino acids.

Figure 1.13 Subcellular localization studies show ORF1 targeting to nuclei and ORF2

to mitochondria.

A. NIH 3T3 cells, on coverslips were transfected with GFP-ORF constructs and 36 hours

post transfection cells were fixed using 4% paraformaldehyde, mounted using mounting

medium with 1% DABCO and observed under fluorescent microscope (Olympus BX51) with

green filter. ORF1 shows nuclear localization, ORF2 shows mitochondrial localization. ORF3

shows no specific localization, similar to EGFP. B. ORF1 cells were fixed and counterstained

with DAPI. ORF2 cells were incubated with mitotracker dye (100 ng/ml) in culture medium for

15 min and fixed. ORF1 shows co-localization with DAPI. ORF2 co-localizes with

mitotracker dye. C. The nuclear localization signal (NLS) in ORF1 and mitochondrial

targeting signal (MTS) in ORF2 amino acid sequences are shown.

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1.4 Molecular characterization of M3TR. Noncoding RNAs are reported to possess unusual characters compared with the

coding genes. In brief, the ncRNAs may or may not possess polyadenylation signals at

3’ end, may have several transcripts of different sizes and may be expressed in both

sense and antisense orientations. A detailed molecular characterization of M3TR RNA

was performed using a myriad of cellular and molecular biology techniques.

1.4.1 Transcript analysis by Northern hybridization.

The transcript size of M3TR-RNA was determined with strand specific northern

hybridization. The SK-N-MC mRNA was separated on denaturing agarose gel and

blotted onto nylon membranes and hybridized with M3TR sense and reverse riboprobes,

independently. A 1.9 kb band was consistently observed in the SK-N-MC blots

hybridized with M3TR sense riboprobe, indicating its transcript length to be 1.9 kb.

(Fig.1.14). However, the blots probed with M3TR antisense riboprobe did not show any

hybridization. The consistent hybridization seen only with M3TR sense riboprobe

confirms that M3TR is expressed as an antisense transcript. The hybridization seen with

SK-N-MC mRNA suggests that the M3TR transcript is polyadenylated at its 3’ end.

Figure 1.14 M3TR transcript size analyses by strand specific northern hybridization. 1 µg of polyA+ RNA from SK-N-MC and RNA ladder was separated on 1.2% denaturing

formaldehyde agarose gel (1.2%) electrophoresis, blotted onto Nylon+ membrane and UV

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crosslinked. The blots were hybridized overnight with M3TR sense and reverse riboprobes

(4.5X 106 CPM/ml) at 68°C followed by stringent washing steps (0.1X SSC, 0.1% SDS at

72°C). The blots were exposed to phosphor screen and scanned using molecular imager FX

(Biorad). A 1.9 kb band is detected in the blot hybridized with M3TR sense riboprobe. The

M3TR reverse riboprobe shows no hybridization.

1.4.2 Transcript analysis by RPA.

The antisense expression of M3TR was further investigated by ribonuclease

protection assay. The DNased total RNA from SK-N-MC cells was hybridized with M3TR

sense and reverse riboprobes, separately, later digested with RNaseA/T1 mix and

analyzed using denaturing urea-PAGE. The gel analysis showed the protection of

M3TR-sense riboprobe (~600bp) by SK-N-MC RNA, confirming the anti-sense

expression of M3TR (Fig. 1.15). However, the M3TR riboprobe did not yield any

protection (Fig. 1.15). This confirms the natural anti-sense expression of M3TR

transcript.

Figure 1.15 M3TR transcript analyses by Ribonuclease Protection Assay. The expression of M3TR as anti-sense transcript was assessed by ribonuclease protection

assay. 50 µg of DNased total RNA from SK-N-MC cells was hybridized with M3TR and

M3TR-as riboprobes (4.5 X 106 CPM/ml), digested with 10 units of RNase A/T1 mix,

separated on 6% denaturing urea PAGE, exposed onto phosphor screen and scanned using

molecular imager FX. The gel analysis shows the protection of a ~600 bp M3TR sense

riboprobe by SK-N-MC RNA. M3TR reverse riboprobe is not protected. The yeast total RNA

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(50 µg) was used for control reactions. The positive control (without RNaseA/T1) shows the

undigested riboprobe and negative control (with RNaseA/T1) shows no protection.

1.4.3 Transcript analysis by strand specific RT assay.

Our results with Northern and RPA indicated the expression of M3TR as a

natural antisense transcript (NAT). NATs are endogenous RNA molecules that exhibit

partial or complete complementarity to other transcripts, thereby regulating the molecular

expression at various levels. 30% of the transcripts in human are known to have NATs.

The cis-encoded anti-sense NATs (both the strands are coded from the same genomic

locus) are called Sense-Antisense transcript pairs (SAST). The transcripts arising from a

different genomic locus are referred to as trans. cis-NATs are usually related in a one to-

one fashion to the sense transcript, whereas a single trans-NAT may target several

sense transcripts (Werner A. 2005; Lavorgna G et al. 2004; Werner A, Berdal A. 2005).

1.4.3.1 SAST in human cell lines. The existence of M3TR as a sense antisense transcript pair was studied using a

more sensitive strand specific Reverse Transcriptase assay. Human cell lines with high

M3TR expression i.e., SK-N-MC, HeLa, Raji and Jurkat were used in the study. The total

RNA from these cell lines was used for synthesis of strand specific cDNAs. The M3TR

sense and reverse primers were used to reverse transcribe the cellular M3TR-antisense

and sense transcripts, respectively. These cDNAs were used for assessing M3TR

expression by semi-quantitative RT-PCR. The agarose gel analysis of RT-PCR products

showed presence of M3TR mainly as an antisense transcript. There was however a very

weak expression of sense transcript seen in most cell-lines studied with Jurkat showing

a distinct sense transcript. (Fig.1.16.A). The densitometric analysis wherein, the sense

over antisense transcript levels was compared in the above mentioned cell lines, the

levels obtained for Jurkat, Hela, SK-N-MC and Raji were in the ratio of 0.418 fold, 0.158

fold, 0.052 fold and 0.023 fold respectively (Fig. 1.16.B). These results indicated towards

the expression of M3TR as cis-SAST pairs.

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0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

SK-N-MC HeLa Raji Jurkat

Rel

ativ

e ex

pres

sion

M3TR-asM3TR

Figure 1.16 Strand specific RT assay using human cell lines.

A. 5 µg of DNased total RNA from human cell lines- SK-N-MC, Hela, Raji and Jurkat were

used to synthesize orientation specific cDNAs with M3TR specific AS216 (sense) and AS218

(reverse) primers, along with C2, the β-actin reverse primer. β-actin expression was used for

normalization. The RNA and primers were denatured at 70°C for 2 min, followed by RT

reaction at 48°C for 1 hour. RT was inactivated by incubation at 90°C for 5 min. Semi-

quantitative RT-PCR was performed with 40 cycles to amplify M3TR (~500 bp) and β-actin

(~500 bp). The agarose gel analysis of PCR products is shown. The upper panel shows the

expression of M3TR transcript and lower panel shows the respective β-actin controls. B. The

quantitation of transcript expression was performed by densitometric analysis of the bands

using GeneTools software (Syngene, USA). The M3TR expression was normalized with the

respective β-actin expression and represented graphically.

1.4.3.2 SAST in M3TR stable cell lines. The SAST mechanism was assessed in M3TR and M3TR-as stable cell lines by strand

specific semi-quantitative RT assay, as described above. The NIH 3T3 cell line was

used as a control. Consistent with the human cell line data, the NIH 3T3 cells showed

high expression of M3TR antisense transcript and a low expression of M3TR sense

transcript (~50% of antisense transcript). This indicated that M3TR existed as a SAST

pair in NIH3T3 cells. The stable cell lines generated by ectopic expression of M3TR

possessed high expression of the sense transcript whereas the M3TR-as cells showed a

strong M3TR antisense transcript. Interestingly, both the stably transfected cell lines

expressing either the sense or antisense transcripts had an elevated expression of the

complementary M3TR transcript that was not introduced (Fig. 1.17.A). A ~2.5 fold

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upregulation of M3TR antisense transcript in M3TR cell line and a ~3 fold increase in

M3TR sense transcript in M3TR-as cell line was obtained by densitomeric analyses of

the PCR products. Their levels are calculated in accordance with the respective

transcript levels in NIH 3T3 cells (Fig. 1.17.B). An important data that emerges from this

experiment is about the co-regulation of M3TR sense antisense pair i.e. ectopic

expression of one transcript effects the expression of its complementary transcript.

Figure 1.17 SAST analyses in M3TR stable cell lines. A. 5 µg of RNA from NIH 3T3, M3TR and M3TR-as stable cell lines was used to prepare

orientation specific cDNAs followed by semi-quantitative RT-PCR, as described above. The

agarose gel analysis of PCR products is shown. It is a representative picture of 3

independent experiments performed. B. The densitometric analyses of three independent

experiments are represented graphically. The error bars represents standard deviation with

p<0.001. The densitometric analysis was done using GeneTools software (Syngene).

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1.4.4 Sub-cellular localization of M3TR and M3TR-as transcripts. Several ncRNAs were reported to elicit their function specific to their sub cellular

localization. Like, small nucleolar RNAs are specifically identified from nucleoli that are

involved in post-transcriptional processing (Kiss T. 2001). The XIST RNA is reported to

localize in nuclei and coats one of the two X chromosomes in human females leading to

inactivation (Herzing LB et al. 1997; Plath K et al. 2002). Most of the ncRNAs were

known to exhibit greater nuclear localization than cytoplasmic localization and exhibit

functions like imprinting and antisense transcription (Furuno M et al. 2006).

The subcellular localization of M3TR sense and antisense transcripts was

examined by transfecting NIH 3T3 cells with Atto520 labeled M3TR and M3TR-as

(nascent) RNA transcripts, which are synthesized by in vitro transcription. 36 hours post-

transfection, cells were counterstained with DAPI and observed under fluorescent

microscope. The M3TR sense transcript was found intensely localized into nuclei than

M3TR antisense transcript (Fig. 1.18), which implies an abundance of natural M3TR

antisense transcript in nuclei rather than cytoplasm that hybridizes with the ectopically

introduced Fl-labeled M3TR sense transcript. The cytoplasm showed very less staining

with both the transcripts, indicating a weak expression of M3TR transcripts in the

cytoplasm.

Figure 1.18 Sub-cellular localization of Atto520 labeled M3TR and M3TR-as transcripts. The linearised M3TR and M3TR-as plasmid DNA was used to synthesize Atto-520 labeled

(amino-allyl atto-520 CTP, 5mM + 10mM other NTPs) M3TR and M3TR-as RNA transcripts

by in vitro transcription using T7 RNA polymerase. 0.5 µg of purified transcripts were

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transfected into 1 X 104 NIH 3T3 cells on coverslips for 36 hours. The cells were methanol

fixed, counterstained with DAPI and observed under Olympus BX51 with green and blue

filters. The Flourescently labeled M3TR transcript shows an intense localization into nuclei;

and Fl. labeled M3TR-as transcript is weakly localized into nuclei.

1.4.5 Nuclear and cytoplasmic expression of M3TR transcripts. The above results were validated by M3TR expression analysis specifically from

isolated nuclei and cytoplasm using semi-quantitative RT-PCR. The total RNA from

nuclei and cytoplasm of NIH 3T3 cells was isolated and used for preparing cDNAs with

oligo dT and orientation specific cDNAs using M3TR specific primers as explained

earlier. The oligo dT primed cDNAs were used to analyze the expression of M3TR and

β-actin. The agarose gel analysis of PCR products revealed a high expression (~10 fold)

of M3TR in nucleus compared to cytoplasm (Fig. 1.19.A), indicative of the high

expression of M3TR in nuclei rather than in cytoplasm. Next, the abundance of specific

M3TR sense and reverse transcripts in the nucleus and cytoplasm was investigated

using strand specific cDNAs. The agarose gel analysis of semi-quantitative RT-PCR

products showed a high expression of M3TR-antisense transcript in nuclei compared to

cytoplasm. However, the M3TR sense transcript was nearly absent in both nuclei and

cytoplasm (Fig. 1.19.B) corroborating the earlier sub-cellular localization studies using

fluorescently labeled M3TR transcripts.

M3TR

Figure 1.19 M3TR and M3TR-as transcript analyses in nucleus and cytoplasm. NIH 3T3 cells were trypsinized and washed twice with 1X PBS. The cell pellet was

resuspended in TKM buffer to dissolve the cell membrane. The undamaged free nuclei were

isolated by centrifugation at 400Xg. The supernatant was aspired and RNA extraction was

performed (cytoplasmic RNA). The above procedure was repeated once more and RNA

extraction was performed from the pelleted nuclei (nuclear RNA). The strand specific RT-

PCR was performed as explained earlier. The gel analysis shows a high expression of

M3TR-as transcript in the nucleus.

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1.4.6 M3TR transcripts localization on condensed chromosomes. Further, the localization of M3TR transcripts on the condensed chromosomes

was examined by transfection of Atto-520 labeled M3TR sense and reverse transcripts

into NIH3T3 cells for 36 hours followed by chromosomal preparation by standard

cytogenetic procedures. Cells were treated with Calyculin A (50 nM) for 30 min to

improve the chromosomal condensation prior to proceeding for chromosomal

preparation. Calyculin A is an inhibitor of protein phosphatases (type1 and type2A

serine/ threonine). Inactivation of these phosphatases leads to premature chromosome

condensation (PCC) in all phases of the cell cycle (Gotoh A et al. 1995). Consistent with

earlier results, the Fl- labeled M3TR transcript was intensely seen in nuclei than the Fl-

labeled M3TR-as transcript. The examination of chromosomal spreads showed a

uniform and intense staining in cells transfected with Fl- labeled M3TR sense transcript.

This indicated of the presence of M3TR natural antisense transcripts on the condensed

chromosomes, which showed hybridization with fluorescently labeled M3TR sense

transcript. The fluorescently labeled M3TR anti-sense transcript showed a weak

localization onto the condensed chromosomes (Fig. 1.20). These results implicated of a

possible role of M3TR NATs in chromatin organization.

Figure 1.20 Chromosomal localization of flourescently labeled M3TR transcripts. Atto-520 tagged M3TR (5 µg) and M3TR-as (5 µg) transcripts were transfected into NIH 3T3

(5 X 106 cells) for 36 hours, treated with calyculin A (50 ng/ml) for 30 min and processed for

chromosomal preparation using standard cytogenetics procedures that include hypotonic

treatment, fixing, and washing. The nuclei were dropped onto slides, counter stained with

DAPI and observed under Olympus BX51 with green and blue filter. The M3TR sense

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transcript is found to be equally painted on all chromosomes. M3TR-as shows a weak

localization onto chromosomes.

1.4.7 M3TR expression under stress. The ncRNAs are implicated in diverse cellular events including cellular stress.

The first stress inducible ncRNA gene reported was hsrω (heat shock RNA omega) in

D.melanogaster, which was shown to be one of the highly activated genes after heat

exposure along with the other heat shock genes (Lakhotia SC et al. 1996). The exposure

of cells to external stress activates the cellular mechanisms against the deleterious

effects of stress. Therefore, the M3TR expression was assessed for its response in

presence of external physical stress. The NIH 3T3 cells were subjected to physical

treatments like UV light (30 mJ/cm2) and hyperthermia (42°C for 45 min). The untreated

NIH 3T3 cells served as experimental control. With 4 hours post- stress period, the cells

were lysed and estimated for M3TR expression using qRT-PCR. The analyses revealed

a downregulation of M3TR upon induction of external stresses like UV and

Hyperthermia. The UV treatment resulted in a ~10 fold down regulation, and

hyperthermia in ~ 4 fold down regulation of M3TTR transcripts (Fig. 1.21).

Figure 1.21 M3TR transcript analyses upon UV and hyperthermia.

NIH 3T3 cells were treated with UV (30 mJ/cm2) in a UV crosslinker or subjected to

hyperthermia at 42°C for 45 min in an incubator, followed by 4 hours of recovery period. The

untreated NIH 3T3 cells were used as control. Total RNA was isolated and cDNA synthesis

was performed with oligo dT. M3TR expression was assayed by qRT-PCR using Taqman

probes. Gapdh was used as internal control. The relative expression was calculated by 2-ΔΔCt

method. The assay showed down regulation in M3TR level upon UV treatment and

Hyperthermia.

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1.4.8 M3TR sequence extension using RACE. Experiments using Northern hybridization showed M3TR transcript to be 1.9kb in

size. Efforts were made to extend the existing M3TR sequence (557bp) towards 5’ and

3’ ends and to obtain the full length sequence by using rapid amplification of cDNA ends

(RACE). The RACE reaction was performed by Marathon cDNA amplification kit (BD

clontech). The RACE double strand cDNAs with ligated adapters were prepared using

mRNA from SK-N-MC cell line. These RACE cDNAs were used for primary and nested

PCR using M3TR specific primers and adapter primers. Fig. 1.22.A shows no

amplification in the primary PCR in either of 5’ or 3’ RACE, but a prominent band of

~600bp was seen in nested 3’ RACE. The genuinity of RACE products was confirmed by

southern hybridization using M3TR DNA probe, where the nested 3’ RACE product

showed hybridization. Similarly, in a separate experiment using different RACE cDNAs,

3’ nested RACE PCR showed a single prominent band; however 5’ nested RACE PCR

showed multiple bands (Fig. 1.22.C). The southern hybridization showed hybridization of

~600bp product in 3’ RACE and ~ 500 bp products in 5’ RACE (Fig. 1.22.B & D). The

hybridized bands were gel eluted and cloned into pGEM-T vector and sequenced using

vector specific T7 and SP6 primers. The RACE experiments have extended M3TR

sequence by 36 bases at 5’ end and 23 bases at 3’ end (by considering the M3TR

Natural antisense transcript), extending the M3TR sequence to 612bp.

igure 1.22 Cloning of 5’ and 3’ end M3TR sequence using RACE. F

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A. 1µg of polyA+ RNA from SK-N-MC was used to prepare ds-cDNA using Marathon cDNA

mplification kit, BD Clontech. The Marathon adaptors were ligated using T4 DNA ligase.

aq polymerase (BD

the

sults obtained by RACE to amplify the 612 bp M3TR product. SK-N-MC cDNA was

e agarose gel analysis showed a

Figure 1.23 Cloning and confirmation of RACE extended M3TR.

a

RACE PCRs, (primary and nested) were performed using Advantage2 T

clontech) with adapter primers (AP1, AP2) and M3TR specific primers (AS89, AS165 for 3’

RACE and AS90, AS164 for 5’ RACE). For nested PCR, 1µl of the primary PCR product was

used as a template. The 3’ RACE nested PCR shows an amplicon of ~550 bp. B. The PCR

products were transferred to Hybond N+ (GE Healthcare) using neutral transfer. Southern

hybridization was performed using p32 labeled M3TR DNA probe using RapidHyb buffer (GE

healthcare) overnight at 60°C. The blot shows signal from 3’ RACE nested PCR product. C.

Another independent experiment was performed as explained above. The gel analysis of 3’

and 5’ RACE nested PCR products show amplicons of various sizes. D. Southern

hybridization show signals form 3’ and 5’ RACE products of sizes ~550bp and ~500bp.

1.4.9 Cloning and sequencing of extended M3TR. The 5’ and 3’ end oligos (AS217 and AS 224) were designed according to

re

synthesized using oligo dT to amplify M3TR-612bp. Th

~612bp PCR product (Fig. 1.23.A), which was gel eluted and cloned in sense and anti-

sense orientations into pTarget mammalian expression vector by TA cloning strategy as

explained earlier. The directionality of the positive clones was confirmed by directional

PCR using vector specific T7 with M3TR specific AS89 or AS90 primers as explained

earlier (Fig. 1.23.B). The EcoRI restriction digestion analyses of positive clones showed

an insert size of 612bp, confirming the authenticity of the clones (Fig.1.23.C).

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A. The gel analysis shows 612bp PCR product. It was cloned into pTarget mammalian

ategy. B. The agarose

d reverse clones were confirmed by sequencing with

(as explained earlier) and analyzed using BLAST program. The sequence of M3TR

Figure 1.24 The extended M3TR sequence (612bp). (Acc. No. EF649772).

he sequence above shows the extended M3TR sequence using RACE. 5’ RACE added 23

e M3TR

expression vector in sense and reverse orientations by TA cloning str

gel picture shows the confirmation of M3TR-612bp sense and antisense clones (as

explained in hM3TR cloning) C. The EcoRI restriction digestion analysis of M3TR-612bp

clones shows an insert of ~612bp.

The M3TR-612bp sense an

T7

using RACE extended M3TR sequence to 612bp as shown in Fig. 1.24.

T

bases and 3’ RACE added 36 bases, extending the M3TR sequence to 612bp. Th

sense sequence is represented in 5’ to 3’ direction.

85