Functional RNAs; RNA catalysts, mirna,

Transcription

1 Functional RNAs; RNA catalysts, mirna, srna, RNAi... RNAs have many functions rrna (ribosomal RNA) trna (transfer RNA) mrna (Messenger RNA) snrna (including snorna) ) (Small nuclear RNA- splicing) Other ribozymes Telomerase RNA (telomerase maintenance) sirna (small interfering RNA RNA silencing) shrna (short hairpin RNA silencing) Pri-miRNA; mirna ( (primary) micro RNA - regulation) srna (small RNA bacteria) grna (guide RNA RNA editing) Genomic RNA (viruses) All of these RNAs have a function. Functions can be enzymatic, regulatory, or can encode information Small RNAs in Eukaryotes Micro RNAs (mirna( mirna) Similar to srnas in prokaryotes Small interfering RNA (sirna( sirna) Induces mrna degradation micrornas A novel class of ncrna gene Products are ~22 nt RNAs Precursors are nt hairpins Gene regulation by pairing to mrna Unknown before

2 Timeline mirna in PubMed Late 70 s lin-4 and let-7 regulate developmental timing in worm 1993 lin-4 codes for a ~22 nt RNA, complementary to 3 UTR of lin so does let-7 (strnas) 2000 let-7 is conserved in bilaterally symmetric animals 2001 ~100 mirnas discovered by cloning in worm, fly and human 2002 mirnas conserved in plants 2002 Science magazine s breakthrough of the year 2002 mirna Registry established 2003 mirnas may account for 1% of total gene count in animals 2003 a few targets of mirnas identified 2004 mirna Registry has 719 mirnas Number of publications Year mirna Genomically encoded Frequently are complimentary to mrna Usually imperfect complementarity Can be encoded in clusters on the genome, or individually Sequences are relatively conserved between species Can have multiple unlinked targets mirnas always interact with the 3 UTR Can alter mrna stability, and translational initiation mirna structures Originate as a precursor RNA (primary mirna; pri-mirna) of several hundred base pairs - Pri-miRNA contain Caps and poly A tail - Some contain introns while others do not - Can be encoded by introns of other genes - Transcribed by RNA Pol II - Contain ~ 80 bp imperfect stem loop 2

3 mirna Processing in human Drosha cleaves hairpin liberating a ~60 bp hairpin (pre- mirna) Pre-miRNA is exported to the cytoplasm In the cytoplasm, Dicer cleaves liberating a 22 nucleotide RNA duplex with 2 bp 3 overhangs. Drosha and Dicer Members of the RNase III family Cleave dsrna leaving 2 bp 3 overhangs mirna processing mirna biogenesis Adapted from DP Bartel, Cell 116: (2004) 3

4 mirna BIOGENESIS AND FUNCTIONS mirna function Mechanism of action is unknown Interacts with the 3 3 UTR of the target mrna Inhibits translation from a mrna without inducing mrna degradation Can form a ribonucleoprotein complex (mirnp (mirnp)) Can be complexed with ribosomes and target mrna Also important for silencing retrotransposons and endogenous retroviruses Important for development and differentiation (RNase III endonuclease) : DGCR8 Unwind Transcription al repression Drosha (RNase III endonucleas e) 7M GpppG AAAAA Perfect Complementari ty ORF Pol II Imperfect Complementar ity 5 UTR 7MGpp pg ORF 3 UTR 7MGpppG AAAAA Target AAAAA mrna cleavage mrna Translational repression mirnas targets PNAS 99: (2002) DP Bartel, Cell :

5 mirna Registry 3.0 Searchable database of published mirnas entries from human, mouse, rat, worm, fly, and plants Naming service Pre-publication Unique names for distinct mirnas Confidentiality for unpublished data Genomic context A model for the mirna biogenesis pathway and mechanism in mammals. 180 known mirnas in human 130 intergenic 50 intronic 60 polycistronic TRBP - Thyroid hormone receptorbinding protein 70 monocistronic 5

6 sirna... The Actions of Small Silencing RNAs(A) Messenger RNA cleavage specified by a mirna or sirna. Black arrowhead indicates site of cleavage.(b) Translational repression specified by mirnas or sirnas.(c) Transcriptional silencing, thought to be specified by heterochromatic sirnas. Originally characterized in plants Post transcriptional gene silencing Plays a large role in plant immune system Subsequently found in a wide variety of eukaryotes Triggered by dsrna sirna GFP silencing in transgenic C. elegans Similar to mirna.. BUT... Typically induces mrna degradation Requires exact base pairing Dicer processes double stranded RNAs RISC (or other complexes) carry out the effector function RISC cleaves RNA that is complementary to dsrna that activates pathway 6

7 RISC effector complex Contains a member of the argonaute family Between 130 kda and 500 kda Other components are being characterized Cleaves RNA complementary to the sirna,, in the middle of the sequence Assembling the RISC complex requires ATP, while RNA cleavage does not. Uses of sirna Gene knockdowns Look at function/phenotype of a gene Therapeutic techniques Anti viral Anti cancer Anti neurological diseases Others NOTE: sirna and other techniques are knockdowns and do NOT give 100% knockouts. Nor do they work on every gene. Protein/RNA genes DNA RNA X protein Bibliography Computational genomics of non-coding RNA genes. Sean R. Eddy, Cell 109: (2002) Non-coding RNAs: the architects of eukaryotic complexity. John S. Mattick,, EMBO Reports 2: (2001) MicroRNAs: : Genomics, biogenesis, mechanism and function. David P. Bartel,, Cell 116: (2004) Rfam: : An RNA family database. Sam Griffiths-Jones et al., Nucl. Acids Res. 31: (2003) 7

8 References Control of gene expression by a natural metabolite-responsive ribozyme ADE C. WINKLER 1,*, ALI NAHVI 2,*, ADAM ROTH 1,*, JENNIFER A. COLLINS 1,* & RONALD R. BREAKER 1 Nature 428,, Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo. Pennati M, Binda M, De Cesare M, Pratesi G, Folini M, Citti L, Daidone MG, Zunino F, Zaffaroni N. Carcinogenesis Jul;25(7): Epub 2004 Feb 04 Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. Lease RA, Cusick ME, Belfort M. Proc Natl Acad Sci U S A Oct 13;95(21): DsrA RNA regulates translation of RpoS message by an anti-antisenseantisense mechanism, independent of its action as an antisilencer of transcription Majdalani N, Cunning C, Sledjeski D, Elliott T, Gottesman S. Proc Natl Acad Sci U S A Oct 13;95(21): Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA. Lease RA, Woodson SA. J Mol Biol Dec 10;344(5): The small RNA regulators of Escherichia coli: roles and mechanisms*. Gottesman S Annu Rev Microbiol. 2004;58: Mechanisms of gene silencing by double-stranded RNA. Meister G, Tuschl T. Nature Sep 16;431(7006): A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate. William B. Lott, Brian W. Pontius *, and Peter H. von Hippel. PNAS. Vol. 95, Issue 2, , January 20, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. ANDREW FIRE*, SIQUN XU*, MARY K. MONTGOMERY*, STEVEN A. KOSTAS*, SAMUEL E. DRIVER & CRAIG C. MELLO Nature 391,, RNA interference: potential therapeutic targets. Jana S, Chakraborty C, Nandi S, Deb JK. Appl Microbiol Biotechnol Nov;65(6): Sam Griffiths-Jones The Wellcome Trust Sanger Institute [email protected] 8