13 Functional RNAsThomas Schmitt-John

Functional and Biomedical Aspects of Genome Research

20 11 35 Vorlesung SS 04 Bartsch, Jockusch & Schmitt-JohnMi. 9:15 - 10:00, in W7-135

• micro RNAs• small interfering RNAs• RNA interference

rRNAs (ribosomal RNAs): Function of Ribosomes

tRNAs (transfer RNAs): AA transfer for protein synthesis

Ribozymes: RNAs with enzymatic function

miRNAs (micro RNAs): Putative translational regulatory RNAs

stRNAs (small temporally regulated RNAs): let-7 and lin-4 heterochromatic miRNAs (control developmental programs).

siRNAs (small interfering RNAs): Generated out of cleavage of long dsRNAs; antiviral, defense mechanism

snRNAs (small nulear RNAs): Splicing of pre-mRNAs

snoRNAs (small nucloelar RNAs): modification of other RNAs.

Non protein-coding RNAs

ribonucleic acid with catalytic characteristics (cleavage of other ribonucleic acids);

RNase P hammerhead ribozyme

Ribozymes

http://biochemie.web.med.uni-muenchen.de/biotutor/ribozym.htm

snRNAs

snRNAs are incorperated in multi protein complexes(snRNP) and play a crucial role in splicing of primarymRNA transcripts

MPI Göttingen

snoRNAs

snoRNAs are also incorperated in multi protein complexes (snoRNP);most frequently located in the nucleolus; two functional classes of snoRNA

C/D box containin snoRNPs are associated with methylation of mRNA (stability), snRNA(structure), DNA (imprinting)

HACA box containin snoRNPs are associated to pseudourilation of e.g. tRNA

Hüttenhofer et al. 2002

stRNAs

miRNAs

miRNA biogenesis

miRNA genes are typically found in intergenic areas but canalso be found in sense or antisense orientation within intronsof known genes; some miRNAs are cluster and transcribed as polycystronic genes the RNA polymerase(s) or regulatory elements that control miRNA transcription are unknown

the primary transcript of miRNAs are named pri-miRNAs

pri-miRNA is processed to pre-miRNA by DROSHA;pre-miRNAs always occur as hairpin intermediates (60–80 nt)

export of pre-miRNA (but not pri-miRNA) is mediated byExportin 5 which is Ran-GTP dependent and recognizes the hairpin structure; sequence-motif independent procedure

Nelson et al. 2003

miRNA biogenesis Dicer and miRNP

in the cytosol pre-miRNAs are processed by DICER to form the mature miRNA

the miRNA is located in a Protein complex called miRNP or RISC (RNA-induced silencing complex)

miRNPs regulate translation of target mRNAs and RISC induces degradation

recently discoverd new class of endogenous miRNA based posttranscriptional gene regulation was awarded by Science in 2002 as the „Breakthrough of the Year“

pA

endonucleolytic cleavage

CAP

degradation

21-23 nt miRNA

pACAPpACAP

inhibit translation

imperfect binding of miRNAs to specific sites in the 3´UTR of the target mRNA leads to repression of the translation

perfect binding of siRNAs to the coding regionof the target mRNA leads to gene silencing bydegradation of the mRNA

miRNAs and siRNAs (small interfering RNAs)

imperfect binding of miRNAs to specific sites in the 3´UTR of the target mRNA leads to gene silencing by repression of translation

perfect binding of siRNAs to the coding region of the target mRNA leadsto gene silencing by degradation of themRNA

What is the origin of miRNAs and siRNAs and what do they have in common?

Nelson et al. 2003

miRNAs and siRNAs (small interfering RNAs)

siRNAmiRNA

incorporated inmiRNP RISC

imperfect binding to 3´UTR of the target leads to inhibition of translaition

Function ? Gene Regulation

perfect binding to coding regionleads to cleavage of the mRNA at the RNA: RNA hybrid location

Function ? Virus Defence

miRNP: ribonucleoprotein complex containing as known an Argonaute protein (eIF2C2), Gemin 3 (helicase), Gemin 4

RISC: RNA-induced silencing complex, multiprotein complex also containing the Argonauteproteins eIF2C1 and eIF2C2, further proteins like a multisubunit nuclease

up to now ~900 miRNAs have been found in different species

http://www.sanger.ac.uk/cgi-bin/Rfam/mirna/browse.pl

207 miRNAs147 intergenic55 intronic (+)9 intronic (-)

32 cluster2miRNA-cluster 213miRNA-cluster 96miRNA-cluster 2

miRNA targets

leaf morphogeneseMYB33Ath-159

leaf morphogeneseTCPAth-JAW

flower developmentAPATALA2Ath-172

meristem init. and leaf morphogenesePhabulosa/PhavolutaAth-165/166

oncogenesis?Hsa-mir-15/16/142/155

hematopoetic differentiation?Hsa-181

notch signaling pathwayE(spl);BRD,hairyDme-mir-7

apoptoseReaper/grim/sickleDme-mir2

asymmetry chemor. expr.hid pro-apopt.Dme-batam

neural developmental timinglin 41cel-lin-6

larval developmental timinglin 28cel-lin-7

larval developmental timinglin-14cel-lin-4

Function of TargetTargetmiRNA

Validation of predicted of miRNA targets

Luciferase

constitutive promotor 3´ UTR

predicted miRNA target site

Luciferase

Luciferase

Luciferin

Biomedical aspects of the miRNA system

Dicer-KO in mice is lethal during early embryonic development

Let7 and lin4 regulate the timing of C. elegans development

Fragile X-Syndrome is caused by mutations in FMR1, the Drosophila homolog of which is a component of the RISC complex

SMN1 (survival of motoneuron 1) mutated in SMA patients is associatedwith Gemin 3 and might be associated with the miRNP

pre-miRNA

dsRNA

Dicer complex

miRNA + miRNP gene-silencing by inhibition of translation

siRNA duplex + RISC genesilencing by mRNAdegradation

siRNAduplex

miRNA

Long double-stranded RNAs (dsRNAs; typically >200nt) can be used to silence the expression of targetgenes in a variety of organisms and cell types (e.g.,worms, fruit flies, and plants). Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi)pathway. First, the dsRNAs get processed into 20-25nucleotide (nt) small interfering RNAs (siRNAs) by anRNase III-like enzyme called Dicer (initiation step).Then, the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step).Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. In mammalian cells, introduction of long dsRNA(>30 nt) initiates a potent antiviral response,exemplified by nonspecific inhibition of protein synthesis and RNA degradation. The mammalian antiviral response can be bypassed, however, by the introduction or expression of siRNAs.

RNA-interference (RNAi)

RNA-interference (RNAi)as used for functional Genomicsin C. elegans and Drosophila

PCR a cDNA flanked by T7 and Sp6 Promoters

in vitro transcription of both strands

anneal transcripts to dsRNA

microinject dsRNA into C. elegans embryos

analyze the phenotype of the developing worm

http://www.wormbase.org

Effects of GAPDH siRNA on GAPDH mRNA and Protein Levels. HeLa cells were plated at 200,000 cells/well into a 6 well culture plate. 24 hours later they were transfected with either achemically synthesized siRNA at a final concentration of 100 nM or with pSilencer™ 2.0-GAPDH;both target human GAPDH. Samples were harvested 48 hours after transfection, and both RNA andprotein were isolated using the PARIS™ Kit. mRNA knockdown was evaluated by Northern blot using an antisense radiolabeled RNA probe transcribed from pTRI-GAPDH human (Ambion Cat#7430). For the Western blot, anti-GAPDH antibody (Ambion Cat #4300) was used.

siRNA for functional gene analyis in mammalian cells

Vector based siRNA

insertion of chemically synthezised siRNA-target sequence

transfection of the expression vector in target cells

transcription in situ by RNA pol III

spontaneous folding of the transcript

processing of the loop RNA by endogenous DICER and RISCrevealing a knock-down of the target gene

Biomedical perspectives siRNA

Functional analysis of the mammalian genome

Analysis of disease genes

Therapeutic knock-down of disease genes

Antiviral therapy launched for HIV and Hepatitis Virus