Brief introduction to non- protein-coding RNAs Mihaela Zavolan - PowerPoint PPT Presentation

Brief introduction to non- protein-coding RNAs Mihaela Zavolan Biozentrum, Basel Swiss Institute of Bioinformatics

Classes of functional RNAs • rRNA - protein synthesis • tRNA - transport of amino acids • snRNA - spliceosome • snoRNA - rRNA methylation and pseudouridylation • RNase P - removal of 5’ sequence from tRNAs • SRP RNA - protein secretion pathway • miRNA,siRNA - translation inhibition / mRNA degradation • antisense RNAs (Xist) - X chromosome inactivation • bacterial noncoding RNAs

From genes to proteins Start Start Stop Stop ) ) ( )) ( ) ( genome 5’ splice site ) Transcription 3’ splice site ( Exon 1 Exon 2 Exon 3 Exon 4 ) ( ) ) ( ) ( ) pre-mRNA Splicing, capping, polyadenylation m3G A A mature mRNA A Translation A Capture, cDNA synthesis A cDNA protein

snRNA

tRNA

rRNA Prokaryotic Eukaryotic 70 S 80 S 2.5x10 6 Da 4.2x10 6 Da 50 S 60 S 30 S 40 S 1.6x10 6 Da 2.8x10 6 Da 0.9x10 6 Da 1.4x10 6 Da 5 S rRNA 23 S rRNA 16 S rRNA 5 S rRNA 28 S rRNA 5.8 S rRNA 18 S rRNA 120 nc 2900 nc 1540 nc 120 nc 4700 nc 160 nc 1900 nc 21 proteins 34 proteins ~33 proteins ~49 proteins

rRNA RNA - gray Peptide - gold backbone Large ribosomal subunit of Haloarcula mortui - Ban et al. Science 289:905-920 (2000)

snoRNA Weinstein & Steitz, 1999

snoRNA Cavaille et al. 2002 Cavaille et al. 2000

RNAi: why so much excitement? • Unsuspected, intricate regulatory pathways • Involving many new enzymes and RNA-binding proteins • Regulating a large fraction of the genes • Extremely versatile tool to knock down gene expression

Short history of RNAi

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.)

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.) • 1993 - The group of V. Ambros discovers the first microRNA (lin-4) in the worm

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.) • 1993 - The group of V. Ambros discovers the first microRNA (lin-4) in the worm • 1995 - Guo & Kemphues discover RNA interference

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.) • 1993 - The group of V. Ambros discovers the first microRNA (lin-4) in the worm • 1995 - Guo & Kemphues discover RNA interference • 2000 - The group of G. Ruvkun discovers the first evolutionarily conserved microRNA (let-7) in the worm

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.) • 1993 - The group of V. Ambros discovers the first microRNA (lin-4) in the worm • 1995 - Guo & Kemphues discover RNA interference • 2000 - The group of G. Ruvkun discovers the first evolutionarily conserved microRNA (let-7) in the worm • 2001 - The group of T. Tuschl shows that RNAi is mediated by small (21-22 nc) RNAs

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.) • 1993 - The group of V. Ambros discovers the first microRNA (lin-4) in the worm • 1995 - Guo & Kemphues discover RNA interference • 2000 - The group of G. Ruvkun discovers the first evolutionarily conserved microRNA (let-7) in the worm • 2001 - The group of T. Tuschl shows that RNAi is mediated by small (21-22 nc) RNAs • 2002 - Hutvagner & Zamore show that miRNAs and siRNAs share components of the effector pathway

Short history of RNAi • 1990 - multiple copies of a pigment transgene result in colorless petunias (Napoli et al.) • 1993 - The group of V. Ambros discovers the first microRNA (lin-4) in the worm • 1995 - Guo & Kemphues discover RNA interference • 2000 - The group of G. Ruvkun discovers the first evolutionarily conserved microRNA (let-7) in the worm • 2001 - The group of T. Tuschl shows that RNAi is mediated by small (21-22 nc) RNAs • 2002 - Hutvagner & Zamore show that miRNAs and siRNAs share components of the effector pathway • 2004 - RITS complex purified by the group of D. Moazed • 2005 - hundreds on miRNA genes in human (321), mouse (245), rat (194), chicken (122), fly (78), worm (114); hundreds more predicted.

miRNA, siRNA, rasiRNA biogenesis RNA interference pathway MicroRNA pathway Exogenous dsRNA Endogenous dsRNA MicroRNA precursors • Viral infection • Repetitive elements encoded in the genome • Transfection • Antisense transcripts Drosha dsRNA DICER DICER siRNA rasiRNA miRNA RISC RITS RISC-like miRNP mRNA AAAA 3’-UTR Me Me Me Me mRNA cleavage Translational repression Chromatin silencing

Biological functions of RNAi/miRNAs • Defense mechanisms against foreign nucleic acids - Viral infection - Active transposons • Regulation of gene expression - globally (formation of heterochromatin, centromers) - locally ( stellate locus silencing) - translational regulation by miRNAs - transcriptional regulation by miRNAs (plants) • Regulation of genomic rearrangement ( Tetrahymena, Paramecium )

Proteins involved in RNAi function Components of RISC and miRNPs Paz (siRNA-) Piwi (Dicer-binding) domain family (Ago, Hiwi) Fragile X Mental Retardation Protein (FMRP) and related proteins VIG, Gemins, Tudor-SN, R2D2, Dicer Other proteins RNA-dependent RNA polymerases (RdRPs) RNA helicases Proteins modulating RNAi (eri-1) Viral supressors of RNAi Various genes of unknown function identified by screening

Main problems with RNAi technology Off-target effects siRNAs RISC RISC-like miRNP mRNA AAAA Cleavage 3’-UTR Delivery of siRNAs to the appropriate tissue Sustainability of the inhibitory effect

Specificity of RNAi/miRNAs RISC Ago2 mRNA mRNA cleavage Ago2 is a cleaving endonuclease. 5’ end of the miRNA is critical for miRNA Joshua-Tor & Hannon 2004 function. Doench & Sharp 2004.

Applications of RNAi High-throughput RNAi screens for elucidating gene function

Applications of RNAi Development of RNA- based anti-viral (HIV1, Influenza, Coxsackie, HCV) and anti-cancer (targets: angiogenesis, DNA repair, STAT3, bcl-2) therapeutics

Functions of miRNAs Let-7 miRNA regulates developmental timing in C.elegans. Reinhart et al. 2000

Functions of miRNAs Ras is regulated by let-7 miRNA. Johnson et al. 2005

Functions of miRNAs Pancreatic-specific miR-375 inhibits insulin secretion. Poy et al. 2004

Functions of miRNAs miRNAs regulate brain morphogenesis in zebrafish. Giraldez et al. 2005

rasiRNAs in epigenetic regulation Heterochromatic RITS-associated siRNAs in fission Not demonstrated yeast originate from diverse DNA elements. yet in mammals. Cam et al. 2005.

Some ncRNA databases • general: ncRNA database http://noncode.bioinfo.org.cn/ • microRNA repository http://microrna.sanger.ac.uk/sequences/ • tRNA databases http://lowelab.ucsc.edu/GtRNAdb/ http://medstat.med.utah.edu/RNAmods/trnabase/ http://www.staff.uni-bayreuth.de/~btc914/search/ • rRNA database http://www.psb.ugent.be/rRNA/