1. Presenting: Ofir Cohen Supervisors: Tal Pupko Benny Chor
RNA Interference:
Ancient Mechanism
With A Promising Future
Presenting: Ofir Cohen Supervisors: Tal Pupko Benny Chor

2. 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”

3. 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]

4. 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?

5. 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

6. 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.

7. RNAi Timeline 1990 1995 Guo S, and Kemphues KJ. 1998 Fire et al.
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 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.

8. RNAi Glossary Dicer – Interferon – Micro-RNA –
Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Dicer processes long dsRNA into siRNA of 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 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 nt sense and antisens strands hybridized to have 2 nt overhangs at both 3' ends.
Small Interfering RNA –
Small Interfering RNA (siRNA) is nt double-strand RNA. It guides the cleavage and degradation of its cognate RNA.
Helicase –
Enzyme responsible for unwinding double stranded molecule

9. 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. 

10. 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.

11. 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 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

12. 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.

13. 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

14. 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

15. 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.

16. 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.

17. 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., ]
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., ]
Non coding, 21nt RNA
Regulates lin-14 in same way as lin-4
Note: Homologues of lin-4 escaped bioinformatics Let-7 Homologs were easily detected [Pasquinelli et al., ]
Drosophila, sea urchins, mice, humans...

18. 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., ]
Same as other gene families with regulatory roles
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

19. miRNA vs. siRNA miRNA: microRNA. siRNA: short-interfering RNA.
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

20. 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…

21. 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

22. 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.

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

24. 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.

25. 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.

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

27. Presenting the cast:

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

33. 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).

34. 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., ]
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., ]
Discovered in mice
Conserved in vertebrates
Interacts with regulatory protein
Turns transcriptional repressor into activator

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

36. 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.