Regulation of gene expression at the mRNA level: RNA interference and microRNAs • Overview: • The silencing effect of double-stranded RNA • The mechanism: Dicer, RISC, Argonaute and siRNAs • microRNAs: Nobel Prize in the next few years?
The breakthrough paper that started The RNAi revolution Cited 10026 times* in 5393 days (as of Nov 25, 2012) *Source: Google Scholar
Only 8 years later… ? David Baulcombe
Injection of double-stranded RNA triggers RNA interference The dsRNA represents the unc-22 gene. unc-22 mRNA is highly expressed Injection of unc-22 dsRNA results in a phenotype similar to that of unc-22 mutants. Neither injection of sense or antisense unc-22 RNA has any effect.
Injection of dsRNA results in depletion of the corresponding mRNA • injection of single- or double-stranded mex-3 RNA into the gonad of C. elegans • mex-3 mRNA is normally abundant in the gonads and early embryos. • mex-3 mRNA is lost after injection of double-stranded RNA • injection of antisense RNA only moderately reduced mex-3 mRNA
Summary of key findings • dsRNA was highly effective, single-stranded RNAs were not • only a few dsRNA molecules required per cell • dsRNA triggers mRNA degradation • dsRNA corresponding to introns and promoters • does not work • dsRNA effect crossed cellular boundaries • offspring often still displays dsRNA effect • …suggesting a catalytic or amplification mechanism
RNA interference generates small interfering RNAs Dicer: Ribonuclease III. Cuts double stranded RNA RISC: RNA-Induced Silencing Complex, mediates degradation of target mRNA siRNA: small interfering RNAs, ~20-25 nt long RNA end products of dicer action
~20-30 nt RNA mRNA degradation (siRNAs & microRNAs) Germ cell development (piwiRNAs) Translational repression (microRNAs)
Significance and Impact of RNAi • RNAi protects against viral infections • RNAi is essential for maintaining genome stability through • repressing mobile genetic elements • RNAi machinery represses gene expression through • post-transcriptional gene silencing • RNAi also suppresses transcription through heterochromatin • formation • RNAi offers a new tool to interfere with gene function • RNAi might provide a future tool for gene therapy
Species comparison: Amplification of dsRNA RdRP: RNA-dependent RNA polymerase Amplification: new dsRNA is synthesized by RdRP using antisense RNA as a primer
microRNAs: from worms to cancer • What are microRNAs? • How microRNAs were discovered • The first microRNA: lin-4 and developmental timing • microRNA structure and function
microRNA versus siRNA: what is the difference? • microRNAs are derived from genes, usually trigger inhibition of translation • siRNA is derived from double-stranded RNA, usually mediates RNA degradation • The structure of the primary microRNA transcript determines the • sequence and structure of the mature microRNA. • siRNAs are heterogenous because they are more randomly generated • from a long dsRNA • However, both share pathway components such as Dicer and similar • RISC complexes containing Argonaute proteins.
A short history of microRNAs • 1993: C. eleganslin-4 (abnormal cell lineage-4) is the first identified microRNA • discovered in a genetic screen • lin-4 mutants affect developmental timing (heterochronic phenotypes) • The term “small temporal RNA” (stRNA) is coined • lin-4 works in the same pathway as lin-14, a transcription factor • lin-4 displays sequence complementary to the 3’ UTR of lin-14 mRNA • 2000: A second stRNA is found: let-7 (for lethal-7) • let-7 mutants also affect developmental timing • let-7 homologs are discovered in all metazoans, including humans • 2001/2: hundreds of microRNAs are discovered in humans, worms and flies
Heterochronic phenotypes in C. elegans lin-4 and let-7: micro-RNAs (formerly stRNAs)
Temporal control functions of miRNA during C. elegans development
Let-7 is conserved in all animals and associated with late stages
Eukaryotic Initiation Factors and Loop Formation • eIF4E binds the cap structure • (7-methyl guanine) • PABP: Poly-A-Binding Protein • eIF4G acts as a bridge between eIF4E and PABP
Argonaute proteins display homology to Cap-binding protein eIF4E
Three groups of primary microRNAs (pri-miRNAs) examples of exonic pri-miRNAs in non-coding RNAs intronic pri-miRNAs in non-coding transcripts intronic pri-miRNAs in coding transcripts
RNA Pol II miRNA biogenesis: overview