Puzzles of the RNAi Both sense and anti-sense ssRNA effective Catalytic – very few copies of dsRNA could silence abundant mRNA Therefore not conventional antisense Only dsRNA targeting mature mRNA are effective, not to introns or promoters RNAi can cross cellular boundaries
ClassLength (nt)Function Micro RNA (miRNA)19-25Translational repression Small interfering RNA (siRNA)19-21Target mRNA cleavage Piwi-interacting RNA (piRNA)26-31Transposon control in germ cells Classes of small RNAs ClassLength (nt)FunctionOrganism Trans-acting siRNA (tasiRNA) 21-22mRNA cleavagePlants Small-scan RNA (scnRNA)~28Histone methylation and DNA elimination Tetrahymena Repeat-associated siRNA (rasiRNA) 24-27Transposon control/transcripti onal silencing Yeast, plants, flies dsRNA
RNAi Dicing and slicing All RNA silencing pathways are triggered by 21-27 nt long small RNAs –Small interfering RNAs – siRNA –Micro RNAs – miRNA –Piwi RNA RNAi induction using long dsRNA only operates in plants and invertebrates In mammals, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNaseL, degrades mRNA
siRNA and shRNA siRNA (short interfering RNA) –typically synthesized chemically then introduced into target cells shRNA (short hairpin RNA) –typically introduced as a plasmid or viral vector –endogenous production, can be long term –enters the RNAi pathway upstream of siRNA
Dicer Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity
RISC RISC has helicase, endonucelase “slicer”,S and homology searching domains. Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense strand.
Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 1. Only mammalian Ago2 can form cleavage-competent RISC Identification of Ago2 as “Slicer” in the RISC
Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 2. Argonaute2 is essential for mouse development
Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 3. Argonaute2 is essential for RNAi in MEFs
The ago1 mutant Arabidopsis develops abnormally because it does not produce an effector of silencing. The Argonaute genes were so named because the mutant plants look like an argonaute squid. The Sainsbury Laboratory John Innes Centre Colney Lane Norwich, NR4 7UH, UK
miRNA Abundant ssRNA from a few thousand to 40,000 molecules /cell Found in all metazoans 0.5-1% of genes siRNA targets genes from which it is derived in a sequence specific manner miRNA regulates separate genes and has imperfect complementarity. May be 100’s/miRNA. Usually have many binding sites in each 3’ UTR, and several different miRNA can target same 3’ region. Combinatorial control 30 – 50 % of genes regulated by miRNA
miRNA Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes. miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.
Plasterk RHA Cell 124:877 2006 Tissue-specific expression of miRNA
Du, T. et al. Development 2005;132:4645-4652 The structure of human pri-miRNAs
Processing of miRNA Long primary Pol II transcript (pri-miRNA) Cleaved by Drosha, nuclear RNase III endonuclease to establish one end of the miRNA (pre-miRNA) –Also need dsRNA binding protein Pasha (flies) DGCR8 (humans) The pre-miRNA exported from the nucleus by Exportin 5 Cut by Dicer→ miRNA Strand with the less-tightly base pared 5’ end becomes mature miRNA, other strand becomes miRNA* and degraded Worms and mammals only one Dicer and it makes miRNA and siRNA. Flies have one for each.
Imperfect homology between miRNA and 3’ UTR of target mRNA Seed sequence has perfect homology
Players in miRNA biogenesis Drosha –Nuclear RNase III enzyme. Initiates miRAN biogenesis by cleaving pri-miRNA into pre- miRNA Pasha –Partner of drosha is a dsRNA binding protein. Human DGCR8 Exportin-5 –Nuclear transmembrane protein that transports pre-miRNA form nucleus to cytoplasm. Works in conjunction with GTP-Ran
Players in miRNA and siRNA Argonaute (AGO) –PAZ domain binds the characteristic two-base 3' overhangs of siRNAs –PIWI domain: dsRNA guided hydrolysis of ssRNA –Slicer in RISC Dicer (DCR) –Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA TRBP –Cofactor for Dicer RISC –RNA induced silencing complex
Mechanism of miRNA suppression of gene expression Transcription mRNA degradation Translational repression –1 Initiation –2 Post-initiation step Co-translational degradation of the nascent peptide
miRNA miRNA in disease –Loss of function mutation of miRNA –Gain of function mutation of miRNA, e.g overexpression –Mutation of target site, no longer binds miRNA –Mutation of target site, now binds miRNA –Tumor suppressors –Oncogenes “oncomirs”
In vivo applications of RNAi Highly specific –Silence a single nucleotide difference in a dominant negative allele Resistance not (less) a problem –Can design new RNAi if a mutation arises and original targeted sequence is changed Problems –Stability –Delivery –Toxicity
Couzin Science 312:1121 2006Grimm, et al. Nature 441:537-541 2006 Liver damage in mice expressing shRNA long-term
Off Target Effects Global, due to induction of innate immune responses Cross reactive, due to sequence homology with other mRNA sequences Not easy to recognize unless global gene expression studies performed. Good to have multiple target sequences
http://www.glocalbeer.dk http://tolweb.org/tree?group=Ascomycota&contgroup=Fungi Taphrina S. pombe S. cerevisiae Morel Penicillium Swahili word for beer (Pombe) Schizosaccharomyces pombe has DCR and AGO but not in Saccharomyces cerevisiae