Limits of Concern for the Risk Assessment of GMP EFSA, Parma 24 th - PowerPoint PPT Presentation

Argonaute-mediated silencing effects AGO POST-TRANSCRIPTIONAL “RNAi - like” “miRNA - like” mRNA degradation CCR4-NOT P-BODIES AGO2 GW182 AGO AAAAA AAAAA mRNA cleavage by AGO2 Inhibition of translation/deadenylation Eukaryots Eukaryots TRANSCRIPTIONAL RdDM other transcriptional silencing DNA heterochromatin methylation Plants e.g. S. pombe … poorly understood in animals

Argonaute structure and function AGO siRNA 3’ PAZ mRNA 5’ 3’ 5’ MID N PIWI • Argonaute structure explains principles of target recognition and repression

Argonaute structure and function AGO siRNA 3’ PAZ mRNA 5’ 3’ 5’ MID N PIWI • Dissociation constants for seed matching targets are in a pM range • low abundant miRNAs unlikely to have significant regulatory effects • seed match + abundance = siRNA off-targeting

Argonaute-mediated silencing effects AGO A small RNA seed sequence defines the minimal sequence complementarity required for silencing Parameters influencing silencing by small RNAs • small RNA abundance ( stoichiometry ) • target site accessibility • complementarity with the target • type of silencing (transcriptional/post-transcriptional)

OFF-TARGETING Perfect vs. imperfect basepairing

Argonaute – targeting & off-targeting AGO Ago RELOCATION TO P-BODIES AAAAA mRNA degradation Inhibition of translation Ago2 Ago AAAAA Cleavage of mRNA by Ago2 EXPRESSION VECTOR RISC Class 2 hairpin Class1 hairpin loading (miRNA-like) (shRNA) short RNAs (miRNAs and siRNAs) . TRANSFECTION siRNA nucleus cytoplasm

Argonaute – targeting & off-targeting AGO Jackson et al. (2003) Nature Biotech • off-targeting is siRNA-specific • any siRNA has off-targeting potential

Argonaute – targeting & off-targeting AGO Jackson et al. (2003) Nature Biotech • off-targeting is largely concentration-dependent • it is strongly reduced in sub-nanomolar range

Argonaute – targeting & off-targeting AGO transfected at 100nM siRNA2 siRNA4 siRNA3 siRNA1 pool Thermofisher/Dharmacom website • siRNA pooling is a way to reduce concentrations of individual siRNAs while keeping the constant siRNA amount in a transfection • natural siRNA pools produced from siRNAs are highly specific because of a highly diluted off-targeting effect

Argonaute – targeting & off-targeting AGO miRNA-like RNAi-like Inhibition Cleavage of Ago2 Ago of translation mRNA by Ago2 AAAAA AAAAA seed = nucleotides 2-7 seed = nucleotides 2-7 • off-targeting potential stems from seed sequence frequency • siRNA knock-downs - usually employ nM concentrations • hydrodynamic transfection (40 m g/mouse – Nature, 418, 38-39)

RNA silencing in different organisms

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer Dicer Arthropod set up AGO AGO inhibition mRNA of translation degradation defense gene control

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer Annelida set up (some Molluscs?) AGO inhibition mRNA of translation degradation defense gene control

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer PKR Vertebrate set up AGO interferon response defense inhibition mRNA of translation degradation defense gene control

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer RdRP Nematode set up (some Molluscs?) AGO inhibition mRNA of translation degradation defense gene control

Nematodes antiviral miRNA exoRNAi endoRNAi defense injection pri-miRNA replication feeding dsRNA dsRNA dsRNA soaking DRH-1 DRH-1 Microprocessor RDE-4 RDE-4 RDE-4 Dicer complex Dicer Dicer ERI pre-miRNA 1 o siRNA 1 o siRNA 1 o siRNA nucleus 22-23 nt 22-23 nt 26G RNA cytoplasm Dicer RDE-1 ERGO-1 RDE-1 1 o siRNA 1 o siRNA 1 o siRNA AAAAA AAAAA AAAAA RDE-8 RDE-8 miRNA mRNA cleavage mRNA cleavage mRNA cleavage 22-23 nt ALG-1/2 RdRP RdRP RdRP DRH-3 DRH-3 DRH-3 AIN-1 ERI AGO WAGOs AAAAA WAGOs SAGO-2 2 o siRNA 2 o siRNA 2 o siRNA inhibition of translation 22G RNA 22G RNA 22G RNA gene control RNA clearance gene control immunity

Nematodes exoRNAi injection feeding dsRNA soaking DRH-1 RDE-4 Dicer 1 o siRNA 22-23 nt RDE-1 1 o siRNA Tabara et al., 1998 AAAAA RDE-8 mRNA cleavage RdRP 0.5 - 1.0x10 6 dsRNA molecules per each gonad arm DRH-3 WAGOs 2 o siRNA 22G RNA RNA clearance

Plants tasiRNA transgene & viral silencing sense RNA dsRNA TAS loci viral SDE3 long hairpin RDR6 sense RNA SGS3 AGO miRNA DCL4/2 DCL3 SDE3 DRB4 HEN1 DRB3 HEN1 RDR6 RDR6 21/22 nt siRNA 24 nt siRNA AGO AGO4/6 DCL4 DRB HEN1 AGO1/7 AGO RdDM 21nt tasiRNA RNA clearance DNA methylation Gene regulation during (post-transcriptional) (transcriptional) development

miRNA pathway in plants & animals Plants Mammals Arthropods pri-miRNA pri-miRNA pri-miRNA DCL1 DGCR8 DGCR8 Drosha Drosha DGCR8 DGCR8 HYL1 SE DCL1 pre-miRNA pre-miRNA pre-miRNA SE HYL1 nucleus nucleus cytoplasm cytoplasm HEN1 HEN1 TARBP2 LOQS Dicer-1 Dicer-1 nucleus cytoplasm miRNA miRNA 21-23 nt 21-23 nt AGO1-4 AGO1 miRNA AGO1 21 nt SUO GW182 GW182 AGO1 AGO1 AGO1-4 AAAAA AAAAA AAAAA mRNA cleavage inhibition of translation inhibition of translation inhibition of translation gene control gene control gene control

Plants MAIN miRNA PATHWAY ALTERNATIVE miRNA PATHWAY long inverted repeats (evolving miRNAs) DCL1 DCL2 DCL4 DCL3 AGO1 AGO2 AGO7 AGO10 AGO4/6/9 A A U U miR-390 miR-156/166 21 nt 24 nt • highly complex RNA silencing system 4x Dicer, 10-20 Argonautes • a number of small RNAs, TGS & PTGS effects

Plants – transcriptional silencing Canonical RdDM Non-canonical RdDM • highly complex RNA silencing – crosstalks & redundancy

small RNA mobility

RNAi mobility - systemic RNAi dsRNA delivery RNAi effect Example dsRNA Cell autonomous mammals RNAi dsRNA C. elegans Systemic some Arthropods ( Tribolium ) RNAi plants dsRNA C. elegans Environmental insects RNAi dsRNA 0.5 - 1.0x10 6 dsRNA molecules per each gonad arm

Plants – spreading of RNA silencing short distance long distance

Plants – > Animals environmental & systemic RNAi  ? Huang et al., 2006 ? ? ?  ? circulating miRNAs environmental & systemic RNAi Baum et al., 2007 Mao et al. 2007

Plants – > Animals Unclear/controversial issues: Mechanism of transport • Mechanism of transport across membranes not explained • Unclear if free or bound to a protein • Survival in digestive tract? Effector complex structure • Would require binding of methylated single stranded RNAs by AGO Targeting stoichiometry • Concentrations estimated 68-250 fM – too low • Authors calculate ~850 molecules per cell, cannot be verified – data not released

Plants – > Animals RNA, advanced online, Jan 6., 2017 doi: 10.1261/rna.059725.116 • meta-study of xenomiRs of 824 datasets from human tissues and body fluids • xenomiRs commonly present in tissues (17%) and body fluids (69%), • low abundance, 0.001% of host human miRNA counts • no significant enrichment in sequencing data from tissues and body fluids exposed to dietary intake (e.g. liver). • no significant depletion in tissues and body fluids that are relatively separated from the main bloodstream (e.g, brain and cerebro-spinal fluids) • the majority (81%) of body fluid xenomiRs stem from rodents, which are rare human dietary contributions, but common laboratory animals. • body fluid samples from the same studies are clustered by xenomiR compositions - suggesting technical batch effects. • feeding studies - no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood.

Key points • a targeting repertoire of a small RNA is largely determined by its seed – nucleotides 2-8. • not absolute rule (non-canonical binding) • allows some predictability, especially for conserved targets • RNAi-like cleavage or miRNA-like target repression silencing effects are primarily defined by AGO isoform and basepairing • targeting efficiency is determined by: • small RNA abundance ( stoichiometry ) • target site accessibility • complementarity with the target • vertebrates have lack systemic RNAi, an RdRP amplification system, and highly processive Dicer -> inefficient RNAi • plant small RNA pathways use 3’ end 2 -O-methyl modification of all small RNAs. In mammals, such modification is found only in piRNAs bound to PIWI AGO cladein the germline

END CREDITS: Miloslav Nic Tomas Novotny Jan Paces

Rana 2007

Mammalian microRNA pathway GW182 AGO relocation AAAAA to P-bodies miRISC Inhibition of translation mRNA degradation targeting GW182 GW182 AGO AGO2 AAAAA RISC Cleavage of loading mRNA by Ago2 Microprocessor miRNA complex duplex DGCR8 Drosha Exportin 5-mediated DGCR8 Dicer transport Dicer cleavage pri-miRNA pre-miRNA pre-miRNA nucleus cytoplasm

Task I Mode-of-action of dsRNA and miRNA pathways RNA silencing in selected taxonomic groups

Animal Dicer evolution Dicer “miRNA” Dicer • RNAi-dedicated Dicer-2 in Arthropods is a derived character • the mammalian “miRNA” Dicer is related to miRNA-producing Dicer-1 in Arthropods • Dicer in C. elegans produces efficiently miRNAs and siRNAs

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer Dicer AGO AGO inhibition mRNA of translation degradation defense gene control

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer AGO inhibition mRNA of translation degradation defense gene control

Co-existence of miRNA & RNAi pathways RNAi miRNA Dicer PKR AGO interferon response defense inhibition mRNA of translation degradation defense gene control

Interferon response induced by long dsRNA (>30bp) TLR3 MDA5 sensing OAS PKR RIG-I specific common responses response 2’,5’ -OA INTERFERON RESPONSE eIF2a P RNaseL ISG interferon-stimulated genes global inhibition global mRNA of translation degradation • The interferon response can be detected/monitored

Small RNA pathways in animals mammals birds Chordata fish Trilobita † Arthropoda Chelicerata ECDYSOZOA Myriapoda Nematoda Crustacea Hexapoda Annelida LOPHOTROCHOZOA Mollusca Cnidaria Porifera

Mammals (and vertebrates in general) Interferon RNA silencing response miRNA RNAi dsRNA common sensors pre-miRNA dsRNA Dicer translational PKR repression TARBP2 PACT OAS OAS RNAse L siRNA RIG-I AGO1-4 IFN signaling MDA5 miRNA TLR3 GW182 AGO2 AGO1-4 interferons & AAAAA AAAAA interferon stimulated genes mRNA cleavage inhibition of translation gene control antiviral defense • miRNA pathway is the main RNA silencing pathway • main dsRNA response = sequence-independent interferon response

Annelids RNA silencing dsRNA response miRNA RNAi dsRNA common sensors pre-miRNA dsRNA Dicer ? TARBP2 ? OAS RNAse L siRNA AGO RIG-I ? signaling miRNA MDA5 ? ? AGO AGO AAAAA AAAAA innate immunity? mRNA cleavage inhibition of translation • almost no functional data, set-up seems similar to mammals

Molluscs Interferon RNA silencing response miRNA RNAi dsRNA common sensors pre-miRNA dsRNA Dicer translational PKR repression TARBP2 OAS RNAse L RdRP siRNA AGO RIG-I ? MDA5 miRNA IFN signaling MX GW182 AGO AGO interferons & AAAAA AAAAA interferon stimulated mRNA cleavage genes inhibition of translation gene control & antiviral defense? antiviral defense • almost no functional data, set-up seems similar to mammals • possible RdRP loop – would make it similar to nematodes

Arthropods Interferon miRNA RNAi response pri-miRNA Microprocessor dsRNA dsRNA pre-miRNA R2D2 nucleus Dicer-2 common sensors cytoplasm LOQS Dicer-1 siRNA PKR miRNA 21-23 nt RIG-I AGO2 AGO1 signaling MDA5 TLR3? GW182 AGO2 AGO1 AAAAA AAAAA mRNA cleavage inhibition of translation innate immunity? defense gene control gene control • separated miRNA & RNAi • sensors of the interferon response present

Nematodes C. elegans is an outstanding model for analyzing RNA silencing • highly complex RNA silencing system • one Dicer but 26 Argonautes and 3 RdRPs • four pathways can be recognized • miRNA • exoRNAi • endoRNAi • antiviral defense • primary and secondary RNAs (amplification of the response) • cytoplasmic and nuclear Argonautes • systemic RNAi, sensitive, cheap, temperate areas worldwide Tabara et al., 1998 0.5 - 1.0x10 6 dsRNA molecules per each gonad arm

Nematodes antiviral miRNA exoRNAi endoRNAi defense injection pri-miRNA replication feeding dsRNA dsRNA dsRNA soaking DRH-1 DRH-1 Microprocessor RDE-4 RDE-4 RDE-4 Dicer Dicer Dicer complex ERI pre-miRNA 1 o siRNA 1 o siRNA 1 o siRNA nucleus 22-23 nt 22-23 nt 26G RNA cytoplasm Dicer RDE-1 ERGO-1 RDE-1 1 o siRNA 1 o siRNA 1 o siRNA AAAAA AAAAA AAAAA RDE-8 RDE-8 miRNA mRNA cleavage mRNA cleavage mRNA cleavage 22-23 nt ALG-1/2 RdRP RdRP RdRP DRH-3 DRH-3 DRH-3 ERI AIN-1 AGO WAGOs WAGOs SAGO-2 AAAAA 2 o siRNA 2 o siRNA 2 o siRNA inhibition of translation 22G RNA 22G RNA 22G RNA gene control RNA clearance gene control immunity

Plants MAIN miRNA PATHWAY ALTERNATIVE miRNA PATHWAY long inverted repeats (evolving miRNAs) DCL1 DCL2 DCL4 DCL3 AGO1 AGO2 AGO7 AGO10 AGO4/6/9 A A U U miR-390 miR-156/166 21 nt 24 nt • highly complex RNA silencing system 4x Dicer, 10-20 Argonautes • a number of small RNAs, TGS & PTGS effects

miRNA pathway in plants & animals Plants Mammals Arthropods pri-miRNA pri-miRNA pri-miRNA DCL1 DGCR8 DGCR8 Drosha Drosha DGCR8 DGCR8 HYL1 SE DCL1 pre-miRNA pre-miRNA pre-miRNA SE HYL1 nucleus nucleus cytoplasm cytoplasm HEN1 HEN1 TARBP2 LOQS Dicer-1 Dicer-1 nucleus cytoplasm miRNA miRNA 21-23 nt 21-23 nt AGO1-4 AGO1 miRNA AGO1 21 nt SUO GW182 GW182 AGO1 AGO1 AGO1-4 AAAAA AAAAA AAAAA mRNA cleavage inhibition of translation inhibition of translation inhibition of translation gene control gene control gene control

Plants tasiRNA transgene & viral silencing sense RNA dsRNA TAS loci viral SDE3 long hairpin RDR6 sense RNA SGS3 AGO miRNA DCL4/2 DCL3 SDE3 DRB4 HEN1 DRB3 HEN1 RDR6 RDR6 21/22 nt siRNA 24 nt siRNA AGO AGO4/6 DCL4 DRB HEN1 AGO1/7 AGO RdDM 21nt tasiRNA RNA clearance DNA methylation Gene regulation during (post-transcriptional) (transcriptional) development

Plants – transcriptional silencing Canonical RdDM Non-canonical RdDM • highly complex RNA silencing – crosstalks & redundancy

Plants – spreading of RNA silencing

Timeline Pubmed: RNAi OR RNA interference OR miRNA OR microRNA OR dsRNA • extremely large volume of literature • majority not related to the review purpose (RNAi technology, miRNA biology, innate immunity …)

Timeline RNAi mechanism solved discovery Single- co-suppression (AGO2 crystalized) of RNAi molecule Dicer Dicer analysis discovered crystalized GW182:AGO2 of AGO Let-7 binding 1 st miRNA & siRNA 1990 2000 2015 2010 miRNA research pre-RNAi era Plant co-suppression, PTGS, VIGS, TIGS etc. mechanisms - mainly plant PTGS research - initial miRNA research RNAi research RNA silencing core molecular mechanism deciphering - mutation screens - biochemical approach

Literature review process 1. Searches in bibliographic databases n = 641 975 2. Citation pearl growing using publications known n = 682 911 to be landmark publications in the field. (Annex C) 3. Removal of duplicates (compilation of a n = 239 987 comprehensive set of scientific and grey literature). 4. Exclusion of references published since 2000 n = 190 734 without DOI 5. Filtering for relevance to individual ELS questions 6. Screening of titles and abstracts 7. Study selection based on full-text reports

Literature review process 1. Searches in bibliographic databases n = 641 975 2. Citation pearl growing using publications known n = 682 911 to be landmark publications in the field. (Annex C) 3. Removal of duplicates (compilation of a n = 239 987 comprehensive set of scientific and grey literature). 4. Exclusion of references published since 2000 n = 190 734 without DOI 5. Filtering for relevance to individual ELS questions 6. Screening of titles and abstracts 7. Study selection based on full-text reports

Literature review process keyword search double strand* rna, dsrna rna interference, rnai, gene silenc*, ptgs Dicer, rnase III, argonau*, ago1, ago2, Piwi, wago, rde1 or rde-1, r2d2 tarbp2 or trbp2 mirna or microrna, sirna, 21u rna oligoadenylate, Pkr Pubmed WoS Scopus ProQuest reference database citations of 47 landmark papers covers highly-cited pioneering papers from the pioneering times when nomenclature was not established and uniformly adopted across the field

Literature review process 1. Searches in bibliographic databases n = 641 975 2. Citation pearl growing using publications known n = 682 911 to be landmark publications in the field. (Annex C) 3. Removal of duplicates (compilation of a n = 239 987 comprehensive set of scientific and grey literature). 4. Exclusion of references published since 2000 n = 190 734 without DOI 5. Filtering for relevance to individual ELS questions - described in detail in 6. Screening of titles and abstracts 2. Data & Methodologies 7. Study selection based on full-text reports

Literature review process SPECIFIC SET-UP FOR EACH TASK/ELS QUESTION OR TAXONOMIC GROUP: MAMMALS BIRDS FISH MOLLUSCS ANNELIDS ARTHROPODS NEMATODES PLANTS

Literature review process include choice of keywords for reference inspection exclude chosen filtering keywords relevant/irrelevant choice references with abstracts with highlighted relevant keywords