The Silence of the Genes

Transcription

1 The Silence of the Genes Initial Observation: Plant geneticists aim to turn pink petunia to purple by over-expression of chalcone synthetase wt wt + Chalcone synthetase cdna driven by cauliflower mosaic virus promoter inaccurate petunia rendering expected result ~80% of transgenics observed result ~ 20% of transgenics At reasonably high frequency, a transgene silences the endogenous copy of the gene

2 This observation eventually lead to the development of a powerful experimental tool GFP expressing C. elegans Fly S2 cells eating bacteria not expressing double strand GFP RNA eating bacteria expressing double Strand GFP RNA grown in supplemented with double RNA Mice injected with tumour cells GFP expressing fly brains with tissue specific expression of long double stranded RNAs to brain specific genes -/+ short RNA that forms a hairpin, RNA sequence from a tumour promoting gene

3 Genetic, molecular, and biochemical analyses uncover multiple pathways for RNA directed gene silencing (RNA interference - RNAi) How did we get from white petunias to RNAi?

4 Which stage of gene expression is blocked? Northern analysis of chalcone synthetase mrna wt chalcone synthetase mrna wt wt + transgene wt + transgene (pink) (purple) (white) cdna transgene mrna Vander Krol et al, 1990 Napoli et al, 1990 Addition of transgene = decreased steady state mrna levels

5 Silencing of endogenous and transgenic copy is posttranscriptional, demonstrated by nuclear run-on analysis wt pink tg purple tg white isolate nuclei * * * * * * * * * incubate nuclei with 32P-UTP (*), to allow transcription to proceed (elongation, not initiation) isolate mrna * * * * * * * * * * * * * * * * * * * * * * * * * * * Hybridize hot mrna to filters containing sequences of interest Exon 1 sense antisense Intron 1 Exon 2 Silencing accompanied by appearance of antisense transcripts

6 Is this effect unique to petunias? Genotype of plant wt tg 35S pro GFP nos pro GUS (Nicotiana Benthamania) Infecting Bacterium (Agrobacterium Tumefaciens) tg GFP tg GUS no tg tg GFP GFP+ GUS+ tg GUS no tg to assay GUS remove leaf inside hybridize for mrna Resulting plant GFP+ GUS+ -1- GFP-/ GUS+ GFP+/ GUS- GFP+/ GUS+ -silencing is sequence specific -spreads beyond site of innoculation (bacteria doesn t) -spreads through phloem and plasmodesmata Ability of silencing to spread suggests amplification step

7 Is antisense RNA sufficient for silencing? wild type wild type C. elegans mutant 1. ds RNA must be directed at exon, not inton 2. ~85% homology over 200 bp required 3. Targeted mrna lost - appears to be a decrease in abundance of cytoplasmic but not nuclear RNA 4. Non-stoichometric effects - small amount of ds RNA can destroy excess mrna 5. Transmissible between generations Effective silencing requires double strand RNA

8 Small Molecular Weight RNAs are associated with silencing no tgs GFP tg GFP tg +GFP Agrobacterium wt GFP tg GFPtg + GFP bacterium Probe: GFP high mw northern low mw northerns GFP wt tg sense strand GFPtg + GFP bacterium loss of transgenic GFP mrna upon infection GFP tg Hamilton & Balcombe, 1999 Silencing correlates with production of small dsrna wt GFPtg + GFP bacterium 25 nt 25 nt antisense strand

9 What we have so far: +

10 Steps inferred: uptake of long dsrna cleavage of long dsrna Movement of small dsrnas amplification of small dsrna small dsrna recognition of mrna degradation of mrna

11 Genetic screen to ID RNAi defective (rde) mutants: mutagenize F2 selected on E. coli expressing pos-1 dsrna Bag of dead embryos = pos-1 mutant phenotype Viable progeny = RNAi deficient mutants (rde) Tabara et al, 1999 Grishok et al, 2000 rde: homologs found in plants, animals, flies, fungi Conservation suggests fundamentally important mechanism in most eukaryotes

12 A story of one gene product ID d as RNAi defective: rde-1 = Argonaute (Ago) conserved across eukaryotes, unknown function insight from biochemical assays dsrna B-gal cyce cyca B-gal cyce cyca in fly S2 cells: Northern blot: cyclin E probe cyclin A probe Transfect cells with dsrna Make whole cell extracts Incubate with radiolabeled RNA with extract hot RNA: cyc E lac Z cyc E lac Z incubation time: cyc E lac Z dsrna transfected: cyc E lac Z lac Z + RNAse Fractionated extract, find discrete peak of activity, ~ 500 kda complex + pre-treatment fly Ago present, associated with ~24-mer RNA, termed RNA interference specific complex RISC Hammond et al, 2000

13 A story of one gene product ID d as RNAi defective: rde-1 = Argonaute (Ago) conserved across eukaryotes, RISC component functional insight from crystal structure Mid contains MC motif = the cap structure motif found in eif4e. MC motif is involved in binding cap structure and translation control PAZ = RNA binding domain PIWI = RNAse H domain Structure suggests Ago may be an RNA-directed RNAse involved in turnover or translational suppression of target mrna

14 A story of one gene product ID d as RNAi defective: rde-1 = defective in RNAi, but accumulate small dsrna uptake of long dsrna cleavage of long dsrna block in rde-1 occurs beyond this step Movement of small dsrnas amplification of small dsrna small dsrna recognition of mrna degradation of mrna A second RNAse activity is required for small ds RNA production

15 ID of the second RNAse: Identified using genomics approach scan Drosophila genome for ds-specific RNase encoding genes (RNase III family) 3 RNase III family members in flies introduce myc tag onto cdnas, transfect into S2 cells find one yields IP able Rnase activity that cleaves into nt pieces: Dicer use RNAi to disrupt Dicer, loose RNAi in cells mutation/rnai of worm Dicer also disrupts RNAi (Bernstein et al, 2001) 2 RNAse III domains, one positioned to cleave each strand ruler helix correct size and position to determine size of resulting small dsrna PAZ = RNA binding domain

16 ID of the second RNAse: Dicer = defective in RNAi, does not accumulate small dsrna Movement of small dsrnas amplification of small dsrna uptake of long dsrna cleavage of long dsrna small dsrna recognition of mrna degradation of mrna block in Dicer and rde-4 mutants occurs beyond this step RNAi in Dicer mutant rescued by microinjection of small dsrna How does the small dsrna move from Dicer to Ago?

17 How does small dsrna move from Dicer to Ago? 1. two RNase III domains to cleave both strands 3. dsrna-binding protein (here, TRBP) senses the asymmetry and positions the RNA in an orientation 2. short dsrna dissociates from and reassociates with Dicer at a different position 4. dsrna is transferred to Argonaute kept in an open conformation by heat shock protein 90 (HSP90) 4b. HSP90 dimer + co-chaperone binds unloaded Argonaute holding it in an open conformation 5.After small RNA binding, the passenger strand is removed, HSP90 hydrolyses ATP, and the AGO protein loaded with the ssrna transitions into a closed conformation

18 How does small dsrna move from Dicer to Ago? C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing sirna passenger strand cleavage products

19 RNA dependent RNA polymerases (RdRPs) ID d as important for RNAi in worms and plants, organisms in which RNAi activity moves between cells uptake of long dsrna this activity absent in mammals cleavage of long dsrna small dsrna recognition of mrna Movement of small dsrnas this module absent in flies and mammals amplification of small dsrna degradation of mrna Are RdRPs necessary for amplification?

20 Model for amplification target RNA 5 3 guide RNA use target as a template to direct production of long dsrna Cleavage of ds RNA to produce more dsrna (2 sirna) Prediction: 2 sirna would exclusively contain sequences arising from region of the target RNA 5 to the guide RNA

21 target mrna dsrna Test of prediction endogenous worm transcript injected into worms isolate cytoplasmic RNA Do RNAase protection with probes 3 & 5 of injected dsrna dsrna target mrna 5 3 probe 1 2 ~ 21 nt Probe alone 2. Probe +RNAse 3. Probe + RNA (RdRP mutant) 4. Probe + RNA (wild type RdRP) probe 1 2 Only 2 sirnas 5 of original guide dsrna are detected

22 RDE-1, RDE-4, and CSR-1 (AGO protein) also required for 2 sirna production and activity Are 2 sirnas competent for silencing? Transgenic worms: NLS-GFP-LACZ MITO-GFP MITO-GFP + NLS-GFP-LACZ MITO-GFP + NLS-LACZ-GFP GFP GFP dsrna + UNC--2A dsrna LACZ dsrna

23 Test other RNAi proteins for role in amplification uptake of long dsrna cleavage of long dsrna small dsrna recognition of mrna Movement of small dsrnas amplification of small dsrna degradation of mrna What is role of RDE-1 in 2 sirna production?

24 2 sirna pathway (C. elegans version) dsrna is processed into 23 nt 1 sirnas by Dicer (DCR-1) and the ds RNA binding protein RDE-4 (R2D2 in flies). 1 sirnas are double-stranded, with a 5 monophosphate and a 3 OH which overhangs the duplex by two nucleotides. The Argonaute protein RDE-1 escorts one strand of the primary sirna to its target mrna. The RdRP, RRF-1, is recruited to the target message where it synthesizes 2 sirnas that are antisense to the mrna. The sirnas are generated by de novo synthesis, are nucleotides in length, and contain a 5 triphosphate (ppp). The Argonaute protein CSR-1 promotes cleavage of mrnas that are base-paired to the 2 sirna. How does the 2 RNA load onto CSR-1?

25 Highly unlikely RNAi pathways evolved to make our lives as experimental biologists simpler Plant RdRP (SGS-2) mutants are defective in resistance to many plant viruses X amount of viral mrna potyvirus or tubamovirus wt 5X amount of viral mrna Sgs-2- Mourrain et al, 2000 Suggests that RNAi a mechanism for viral resistance in plants In worms deficient in RNAi there is activation of transposases and transposition Hypothesis: natural function of RNAi silencing of multicopy sequences (viruses, transposons), which give themselves away by producing transcripts of each strand?

26 Is the sole use of the RNAi pathway to mediate transposon silencing and viral defense? Lee, Feinbaum and Ambros, 1993 RNAi involved in normal developmental regulation in worms, flies, and mammals

27 Are there other endogenous small regulatory RNAs? Clone RNAs from C. elegans with the following features expected of Dicer products: small 5 -terminal monophosphate 3 -hydroxyl 28 nt 16 nt gel purify appropriately sized RNA P 5 -adaptor OH pre-adenylated 3 -adaptor clone and sequence, >300 new small RNAs, termed micrornas (mirnas) develop bioinformatic approaches to predict targets by homology (this remains an inexact procedure) In humans there are mirnas and each may target up to 400 mrnaa Lau, Lim and Bartel, 2003

28 Outcomes differ if small RNA is a perfect or imperfect match for the target mrna Why?

29 Where do mirnas come from? long RNA nuclear intermediate RNA cytoplasmic 22 nt RNA cytoplasmic primary mirna (pri-mirna) pre-mirna (60 nt) mirna What cleaves pri-mirna?

30 ID enzymes for pri-mirna cleavage with biochemistry Which RNase III cleaves pri-mirna? 3 candidates: dicer - already know no L44 - mitochondrial Drosha - predominantly nuclear Test Drosha-FLAG - in an in vitro processing assay marker FLAG IP Drosha IP Dicer IP Drosha + Dicer IP 220 nt 60 nt 22 nt Lee et al, 2003 confirm with genetics: RNAi of mammalian Drosha or mutation of worm Drosha blocks pre-mirna accumulation How does Drosha hand the pre-mirna to Dicer?

31 Does Drosha hand of the pre-mirna to Dicer? How does one pre-mirna know which Dicer to load to (many organisms have >1)?

32 Do sirnas function in the nucleus? In Schizosaccharomyces pombe, the RNA-induced transcriptional silencing complex establishes a physical connection between sirnas and heterochromatin by targeting a nascent transcript, and forms the basis of a self-sustaining feedback mechanism that couples sirna production to chromatin modification.