The discovery of RNA interference

1. The discovery of RNA interference • An Unexpected Result… • petunias surprisingly developed areas of hypopigmentation when transduced with the gene encoding an enzyme required for pigment synthesis. • The phenomena was called Co-suppression • Similar effects seen in fungi. called “Quelling”

2. Later, in the C. elegans Camp... • Antisense RNA injection method for gene inactivation • Sense RNA injections gave same result! • Remained a mystery...“The basis for the sense effect is under investigation ...” [Guo and Kemphues, 1995]

3. Some Sharp Reasoning • Both sense and antisense RNAs sufficient for silencing • Silencing can persist, even though RNA is easily degraded • Could dsRNA be mediating a new silencing mechanism?

4. The RNAi revolution begins… • Fire and colleagues found that introducing long double-stranded RNA (dsRNA) into C. elegans led to the targeted degradation of homologous mRNA. • Coined the term “RNA interference” - RNAi

5. RNAi in C. elegans • Silencing of a green fluorescent protein (GFP) reporter in C. elegans occurs when animals feed on bacteria expressing GFP dsRNA (a) but not in animals that are defective for RNAi (b). • Note that silencing occurs throughout the body of the animal, with the exception of a few cells in the tail that express some residual GFP. • The lack of GFP-positive embryos in a (bracketed region) demonstrates the systemic spread and inheritance of silencing.

6. RNAi Timeline • 1990 • co-suppression of purple color in plants. • 1995 Guo S, and Kemphues KJ. • First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm C. elegans • 1998 Fire et al. • First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans which led to an efficient sequence-specific silencing and coined the term "RNA Interference". • 2000 Zamone et al. • Reported processing of long dsRNA by Rnase III (Dicer) into shorter fragments of 21-23-nt intervals in Drosophila extracts • 2001 Bernstein et al. • Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains. • 2002 Tuschl T and colleagues • First described RNAi in mammalian cells • 2003 Paddison et al. Sui et al. Paul et al. • Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. • 2003 Song et al. • First reported that siRNAs can be used therapeutically in whole animals • 2004 Kawasaki and Taira Morris et al. • First observed that siRNA silences gene at transcriptional level possibly through directing de novo DNA methylation.

7. RNAi Glossary • Dicer – • Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt. • Interferon – • A small and highly potent molecule that functions in an autocrine and paracrine manner, and that induces cells to resist viral replication. This term is related to RNAi because in mammals introduction of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response. • Micro-RNA – • Micro-RNAs (miRNA) are single-stranded RNAs of 22-nt that are processed from ~70-nt hairpin RNA precursors by Rnase III nuclease Dicer. Similar to siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA in plants or blocking its translation in plants and animals. • Post-Transcriptional Gene Silencing – • Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation system designed to act as an anti-viral defense mechanism. A form of PTGS triggered by transgenic DNA, called co-suppression, was initially described in plants and a related phenomenon, termed quelling, was later observed in the filamentous fungus Neurospora crassa • Ribozyme – • Ribozymes are RNA molecules that act as enzymes in the absence of proteins. • RNA Interference – • RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that small double-stranded RNA (referred as small interference RNA or siRNA) can induce efficient sequence-specific silence of gene expression. • RNA-Directed DNA Methylation – • RNA-directed DNA methylation (RdDM) is an RNA directed silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires a double-strand RNA that is cut into short 21-26-nt fragments. DNA sequences homologous to these short RNAs are then methylated and silenced. • RNA-Induced Silencing Complex – • RNA-induced silencing complex (RISC) is an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. • RNAi Trigger – • RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens strands hybridized to have 2 nt overhangs at both 3' ends. • Small Interfering RNA – • Small Interfering RNA (siRNA) is 21-23-nt double-strand RNA. It guides the cleavage and degradation of its cognate RNA. • Helicase – • Enzyme responsible for unwinding double stranded molecule

8. What is RNAi? • RNA interference (RNAi) is an evolutionally highly conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences. • It was first discovered in 1998 by Andrew Fire and Craig Mello in the nematode worm Caenorhabditis elegans and later found in a wide variety of organisms, including mammals. 

9. RNAi is a conserved mechanism • RNAi is a universal, omnipresent conserved mechanism in eukaryotic cells. • The cellular mechanism of RNAi Predates evolutionary divergence of plants and worms. • key proteins involved in RNAi in disparate organisms are highly conserved.

10. THE SILENCING MECHANISM • Two-step model to explain RNAi. • I. dsRNA is diced by an ATP-dependent ribonuclease (Dicer) into short interfering RNAs (siRNAs). • duplexes of 21 23 nucleotides bearing two-nucleotide 3' overhanging ends. • II. siRNAs are transferred to a second enzyme complex, designated RISC for RNAi-induced silencing complex.The siRNA guides RISC to the target mRNA, leading to its destruction. • the antisense strand of the siRNA is perfectly complementary

11. The classical RNA interference (RNAi) pathway in Drosophila • Long double-stranded RNAs (dsRNAs) are processed by the R2D2/Dicer heterodimer into small interfering RNAs (siRNAs). • The duplexed siRNA is unwound in an ATP-dependent manner*. • *starting at the 5' terminus that has the lowest relative free energy of base pairing. • This strand of the siRNA, the guide strand, is also preferentially taken up by the RNA-induced silencing complex (RISC). • The single-stranded siRNA guides the endonuclease activity of the activated RISC ("holoRISC") to the homologous site on the mRNA, cleaving the mRNA.

12. RNAi Amplification • A small amount of dsRNA can silence a vast amount of target mRNA in C. elegans. • Mechanistic explanations for this observations: • Each siRNA fragment can target the homologous mRNA • Catalytic mechanism: each siRNA fragment can be used several times. • RNA directed RNA synthesis

13. RNA Dependent RNA Polymerase (RdRP) • RdRP activity found in plants and C. elegans • Required for RNAi? • Not found in mammals or drosophila • RdRP deficient plants and worms... Results not decisive • Proposed mechanism: Random degenerative PCR [Lipardi et al., 2001] • siRNA acts as primer for elongation on target mRNA

14. Immunity via RNAi • RNAi is used as a form of primitive immunity to protect the genome from invasion by exogenous nucleic acids introduced by mobile genetic elements, such as viruses and transposons.

15. Putting a stop to the promiscuous Duplication Fodder… Richard Dawkins • Cellular machinery is extremely good at copying DNA. • The cell nucleus is a paradise for DNA, humming with sophisticated, fast, and accurate duplicating machinery. • Cellular machinery is so friendly towards DNA duplication that it is small wonder cells play host to DNA parasites --- viruses, viroids, plasmids and a riff-raff of other genetic fellow travelers.

16. The plot thickens… The Discovery of Endogenous Effectors for RNAi • Discovery of the first naturally occurring small RNA specie , lin-4 • Non-coding, 22nt RNA • Identified in screen for defects in timing of larval development • lin-4 partially complementary to conserved sites in lin-14 3’UTR [Lee et al., 1993] • lin-4 binds these sites • lin-4 negatively regulates lin-14 translation • The naturally occurring small RNA designated microRNAs (miRNAs) (only later) • No other miRNAs found for 7 years! • Second miRNA – let-7 [Reinhart et al., 2000] • Non coding, 21nt RNA • Regulates lin-14 in same way as lin-4 • Note: Homologues of lin-4 escaped bioinformaticsLet-7 Homologs were easily detected [Pasquinelli et al., 2000] • Drosophila, sea urchins, mice, humans...

17. Endogenous RNAi: miRNA in the Genome • Characteristic Properties • Highly conserved, particularly 5’ end • All from hairpin precursors • Genome Wide miRNA Identification • Most has been done experimentally (Cloning and sequencing) • Over 100 novel miRNAs identified from C. elegans, Drosophila, and mammals • Expected to represent ~1% of predicted genes [Lim et al., 2003] • Same as other gene families with regulatory roles • 200-255 miRNAs in humans • >175 have now been experimentally confirmed [Griffiths-Jones, 2004] • Functional Characterization • Lewis et al., (2003) estimate average of five mRNA targets per miRNA • Many targets are transcription factors - miRNAs regulate the regulators • Suggests major role in highly regulated processes • Thousands of proteins may be regulated by miRNA

18. miRNA vs. siRNA • miRNA: microRNA. • Encoded by endogenous genes. • Hairpin precursors - pre-miRNAs • The pre-miRNAs are hairpins with imperfect complementarity in their stems and frequent bulges, mismatches and G:U wobble base pairings. • Recognize multiple targets. • siRNA: short-interfering RNA. • Mostly exogenous origin. • dsRNA precursors • May be target specific • Discovered in different ways • Similar biogenesis • Share common pathway components and outcomes • Understanding of miRNA comes from research on siRNA and vice versa • Maybe current understanding does not allow us to distinguish them

19. MULTIPLE MECHANISMS OF SMALL-RNA-MEDIATED GENE SILENCING • The endogenous RNAi pathway contributes significantly to regulating cellular gene expression. • Silencing of endogenous genes regulates basic biological processes, including the transition from one developmental stage to the next. • the archetype miRNAs, let-7 and lin-4, regulate C. elegans larval development • miRNAs are expressed in a specific spatial and temporal pattern during development in D. melanogaster or differentiation of mouse embryonic stem cells • The function of most miRNAs remains unknown…

20. miRNA Biogenesis • Transcribed from endogenous gene as pri-miRNA • Primary miRNA: long with multiple hairpins • Imperfect internal sequence complementarity • It is processed into 70-nt hairpins by the RNase III family member Drosha to become the pre-miRNA. • Note: How does it identify pri-miRNA? • Hairpin terminal loop size • Stem structure • Hairpin flanking sequences • The pre-miRNA is exported to the cytoplasm by Exportin 5. • It is cleaved by the R2D2/Dicer heterodimer into the mature miRNA. • Symmetric 2nt 3’ overhangs, 5’ phosphate groups

21. The miRNA pathway • pri-miRNA • processed by Drosha to become the pre-miRNA. • exported to the cytoplasm by Exportin 5. • cleaved by the R2D2/Dicer heterodimer into the mature miRNA. • The miRNA is loaded into RISC and guides it to sites on the mRNA that have only partial sequence complementarity to the miRNA, leading to repression of translation.

22. Intermediate Summary: • miRNA vs. siRNA • mRNA cleavage vs. Translational Repression Initiation Execution

23. An additional mechanism: Heterochromatin formation • The repeat-associated siRNA (rasiRNA) pathway • Transcription from opposing promoters found in repetitive DNA elements, such as centromeric repeats and satellite DNA, leads to the formation of long dsRNAs. • These long dsRNAs are cleaved by Dicer, presumably the R2D2/Dicer heterodimer, into siRNAs. • These are unwound and taken up by the RNA-induced transcriptional silencing complex (RITS) • RITS directs the establishment of silenced chromatin over the region of DNA homologous to the siRNAs. • This silenced chromatin is characterized by sequence-specific DNA methylation and histone methylation and by recruiting heterochromatin-associated proteins.

24. A model for the mechanism of RNAi • Silencing triggers in the form of double-stranded RNA may be presented in the cell as synthetic RNAs, replicating viruses or may be transcribed from nuclear genes. • These are recognized and processed into small interfering RNAs by Dicer. • The duplex siRNAs are passed to RISC (RNA-induced silencing complex) • The complex becomes activated by unwinding of the duplex. • Activated RISC complexes can regulate gene expression at many levels: • promoting RNA degradation • translational inhibition • chromatin remodelling • Amplification of the silencing signal in plants may be accomplished by siRNAs priming RNA-directed RNA polymerase (RdRP)-dependent synthesis of new dsRNA.

25. RNAi movie - http://www.nature.com//focus/rnai/animations/index.html

26. Presenting the cast:

31. RNAi applications • Genome-wide RNAi screening • Done in C. elegans • 19 757 protein coding genes (predicted) • 16 757 inactivated using RNAi • New standard for systematic genome wide functional studies • RNAi as a solution for mammalian genetics • Potential therapeutic use

32. Double-stranded RNA can be introduced experimentally to silence target genes of interest • silencing is systemic and spreads throughout the organism. • a, A silencing signal moves from the veins into leaf tissue. red is chlorophyll fluorescence that is seen upon silencing of the GFP transgene. • b, C. elegans engineered to express GFP in nuclei. Animals on the right have been treated with a control dsRNA, whereas those on the left have been exposed to GFP dsRNA. • Some neuronal nuclei remain florescent, correlating with low expression of a protein required for systemic RNAi59. • c, HeLa cells treated with an ORC6 siRNA and stained for tubulin (green) and DNA (red). Depletion of ORC6 results in accumulation of multinucleated cells. • Stable silencing can also be induced by expression of dsRNA as hairpins or snap-back RNAs. • d, Adult Drosophila express a hairpin homologous to the white gene (left), which results in unpigmented eyes compared with wild type (right).

33. New Frontiers for RNA… • Small RNAs likely to have bigger impact on gene and protein regulation • New classes of small RNAs: • Tiny non-coding RNA [Ambros et al., 2003] • tncRNA – 20-22nt • Discovered in C. elegans • Not likely generated from hairpin loops • Not conserved among species • Many complementary to mRNAs • Function unknown • RNA as a Molecular Switch: Small Modulatory RNA – smRNA [Kuwabara et al., 2004] • Discovered in mice • Conserved in vertebrates • Interacts with regulatory protein • Turns transcriptional repressor into activator

34. Just Scratching the Surface… • New roles for RNA added to our current paradigm for gene and protein regulation. • A new buzz word? “Regulomics”

35. The difficulties in STP research fundamentally owe their complexity to the designer – natural selection. The reason lies in the profound difference between systems “designed” by natural selection and those designed by intelligent engineers (Langton 1989). For instance, human designers being farsighted but blinkered, tend to find their designs thwarted by unforeseen side effects and interactions, so they try to guard against them by giving each element a single function insulating it from other elements and allowing the minimally required interconnections. In contrast natural selection is ultimately myopic and having no foresight the process tries out designs in which many side effects occur; most such designs are terrible, but every now and then there is a serendipitous side effect: few unrelated functional systems interact to produce a bonus. This “design” process ultimately produced the biological systems we investigate; system which operate with an outrageously complex weave of interconnections.