1. Chapter 16 Other RNA Processing Events
Trans-splicing, Editing, RNAi, miRNAs

2. Trans-splicing section 16.3
First seen in a parasitic protozoa
Trypanosomes, protozoan that causes African sleeping sickness
trans-splicing used to generate changing surface coat proteins that help outwit the immune system
Trans-splicing also occurs in C. elegans.

3. trans-splicing
Figure 16.12
200 copies of a 35 n leader encodes in a different place in the genome.

4. Editing protozoa = U-insertion protozoa = U-deletion
mammals, insects & plants = nucleotide deaminiation
16.4
Focus on this one

5. RNA editing by deamination
ADAR = Adenosine deaminase acting on RNA
adenosine -> inosine
inosine bp with cytidine
So codons change
ACG codon (threonine) changes to an ICG codon which is read as GCG (alanine)
pg 493 4th ed.

6. Results in major changes in properties of the protein
Example
Glutamate receptor ion channel
GluR-B changes glutamine->arginine
Reduces Ca2+-permeability.

7. How?
Usually codons to be changed are near introns. A guide RNA molecule base pairs to an intron and then points ADAR at the correct codon.

8. So what? Not a trivial change.
It is extremely important for the normal development and function of the nervous system.
In mammals, it appears to be part of the way that the nervous system generates diversity and complexity (ADAR 3 unique to brain).

9. Cytidine deaminaton
CDAR cytidine deaminase acting on RNA
C-->U

10. Discovery of post-transcriptinal gene silencing (PTGS) or post-transcriptional control of gene expression
• Involved attempts to manipulate pigment synthesis genes in petunia
• Genes were enzymes of the flavonoid/anthocyanin pathway:
CHS: chalcone synthase
DFR: dihydroflavonol reductase
When these genes were introduced into petunia using a strong viral promoter, mRNA levels dropped and so did pigment levels in many transgenics.

11. Discovery of PTGS First observed in plants (R. Jorgensen, 1990)
Introduction of a transgene homologous to an endogenous gene resulted in both genes being suppressed!
Also called Co-suppression
involved enhanced degradation of the endogenous and transgene mRNAs

12. DFR construct introduced into petunia
CaMV - 35S promoter from Cauliflower Mosaic Virus
DFR cDNA – cDNA copy of the DFR mRNA (intronless DFR gene)
T Nos - 3’ processing signal from the Nopaline synthase gene
Flowers from 3 different transgenic petunia plants carrying copies of the chimeric DFR gene above. The flowers had low DFR mRNA levels in the non-pigmented areas, but gene was still being transcribed.
These results resembled more what you get with antisense DFR genes, which suppress the endogenous DFR gene.

13. RNAi RNA interferance Discovered in a control experiment
pg 501 Weaver 4th edition
Su Guy and Kenneth Kenphues 1995

14. 2006 Nobel Prize in Physiology & Medicine
RNAi
RNAi discovered in C. elegans (first animal) while attempting to use antisense RNA in vivo
Control “sense” RNAs also produced suppression of target gene!
sense (and antisense) RNAs were contaminated with dsRNA.
dsRNA was the suppressing agent.
Su Guy and Kenneth Kenphues 1995
Craig Mello
Andrew Fire
2006 Nobel Prize in Physiology & Medicine

15. 2. The experiment.
unc22 gene nonessential myofilament protein. Mutations in unc-22 cause a twitching phenotype. dbstded unc-22 RNA phenocopies.
In 2. actually required high dose to see an effect.
Used GFP and LacZ reporters to test specificity.

16. Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode C. elegans.
Fig Weaver 4th Ed.
Inject antisense RNA (c) or dsRNA (d) for the mex-3 (mRNA) into C. elegans ovaries.
mex-3 mRNA was detected in embryos by in situ hybridization with a mex-3 probe.
negative control
positive control
no probe
mex-3 antisense
mex-3 dsRNA
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense mRNA. (2) the suppressing signal moved from cell to cell.

17. Hammond et al Nature 404: An RNA-directed nuclease is purified from Drosophila cells that seems to specifically degrade mRNAs.
S2 cells
extract destroys cognate RNAs
As others have seen, notice the accumulation of a 25 nt RNA which can bp to the target mRNA.
dsRNA T7
RNAi introduced by calcium phosphate co-precipitation or with Superfect lipid reagents. Same results seen.
Destruction of 25 nt RNA with micrococcal nuclease blocks reaction.
Hammond et al An RNA-directed nuclease mediates post-trancriptional gene silencing in Drosophila cells. Nature 404:
Figure is not in Weaver 4th but is mentioned on pg

18. Short interfering RNAs -siRNAs

19. Drosophila embryo lysate system simplifies step by step analysis.
Processes the trigger to the 21-23nt fragments. Both strands of the trigger are cut. - show by radiolabelling one strand and then the other strand (sense, antisense). Processing of trigger is not dependent on mRNA.
dsRNA p p
Zamore et al Cell 101:25-33
Zamore et al Cell 101:25-33
Fig th ed

20. The dsRNA that is added dictates where the destabilized mRNA is cleaved.
The dsRNAs A, B, or C were added to the Drosophila extract together with a Rr-luc mRNA that is 32P-labeled at the 5’ end. The RNA was then analyzed on a polyacrylamide gel and autoradiographed.
Results: the products of Rr-luc mRNA degradation triggered by dsRNA B are ~100nt longer than those triggered by dsRNA C (and ~100 nt longer again for dsRNA A-induced degradation).
Fig 16.31

21. Enzyme cleaves at ~23-nt intervals & after U.
High resolution gel analysis of the products of Rr-luc mRNA degradation from the previous slide.
Fig
Results: the cleavages occur mainly at nt intervals; 14 of 16 cleavage sites were at a U.There is an exceptional cleavage only 9 nt away from the adjacent site (induced by dsRNA C); this site had a stretch of 7 Us.
Enzyme cleaves at ~23-nt intervals & after U.
In 2001 Hammond et al purify the enzyme and name it DICER.

22. dsRNA DICER - RNase III family member
Weaver 4th edition pg
DICER - RNase III family member
RISC - one of the proteins is SLICER. In Drosophila SLICER is the product of the Argonaute gene.
Argonaute has a PAZ and a PIWI domain.
PIWI domain forms a shape like an RNase H.
In mice there are 4 Ago genes but only Ago2 appears to be SLICER.
Dicer participates in selecting the guide RNA that is passed on to Argonaute.
Roles of R2D2 and Armitrage are not clear.
ATP
Dicer
ADP+Pi p p
Dicer leaves 2nt 3’ overhangs & phosphorylated 5’ ends
21-23 nt siRNP p
PIWI PAZ
The 2 domains of Argonaute
-Dicer
-R2D2
-Armitrage p
RISC loading complex
Dicer member RNase III family of dsRNA-specific endonucleases.
The RNase in RISC has not yet been identified as of 2002.
Hutvagner & Zamore Current Opinion in Genetics & Development 12:
First enzyme activity named SLICER. A gene was independently named Argonaute. Then it is shown that the Argonaute protein is SLICER.
Very efficient process because many small interfering RNAs (siRNAs) generated from a larger dsRNA.
RISC=RNA-induced silencing complex.
ATP
ADP+Pi
RISC p
Argonaute
mRNA
Target recognition p p
Target cleavage
mRNA p p

23. Bizarre figure see next one for explanation.
Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.
Bizarre figure see next one for explanation.

24. Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.
RNA transcript made
siRNA1 could bp about 140n from 5’ end of transcript
siRNA2 could bp about 180n 3’ end of transcript
Argo2 that has been produced in bacteria
lane1
lane2
lane 1 transcript + siRNA2 + Argonaute + MgCl2
lane 2 transcript + siRNA1 + Argonaute + MgCl2

25. Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.

26. Ago2 knock out in mice embryonic lethal with severe defects
important for RNAi & miRNA

27. Function of RNAi
Antiviral - Double stranded RNA is an intermediate in the replication of some RNAi viruses.
Suppress transposon activity
Great research tool because it provides a way to experimentally eliminate a gene product
Might be a useful therapy for cancer, etc.

28. How to evoke RNAi Inject double stranded RNA
Express or inject antisense RNA inside a cell
Express a gene which has an inverted repeat.
Two promoters which point at one other.
Expression of 2 different genes whose mRNAs can base-pair over a short region.

29. But wait there’s (too much )more
Amplification of siRNA
Role of RNAi machinery in the formation of heterochromatin
miRNAs - inhibition of translation
miRNAs - stimulation of translation
Feed C. elegans bacteria expressing RNAi and they develop RNAi response.

30. But wait there’s (too much )more
Amplification of siRNA Tiny amounts of a trigger can have a very large and long lasting effect. Occurs in Plants, Drosophila and C. elegans.
Role of RNAi machinery in the formation of heterochromatin
miRNAs - inhibition of translation
miRNAs - stimulation of translation
Feed C. elegans bacteria expressing RNAi and they develop RNAi response.

31. dsRNA Amplification (pg508 4th ed) mRNA ATP Dicer NTPs ADP+Pi
RdRp (RNA directed RNA polymerase)
PPi p p
Dicer leaves 2nt 3’ overhangs & phosphorylated 5’ ends
21-23 nt siRNP p p
ATP
Dicer
ADP+Pi
-Dicer
-R2D2
-Armitrage p p p p
RISC loading complex
Dicer member RNase III family of dsRNA-specific endonucleases.
The RNase in RISC has not yet been identified as of 2002.
Hutvagner & Zamore Current Opinion in Genetics & Development 12:
First enzyme activity named SLICER. A gene was independently named Argonaute. Then it is shown that the Argonaute protein is SLICER.
RISC=RNA-induced silencing complex.
ATP
ADP+Pi
RISC p
Argonaute
mRNA
Target recognition p p
Target cleavage
mRNA p p

32. Potential for exon spreading
Reference: Nishikura 2001 Cell 107:

33. But wait there’s (too much )more
Amplification of siRNA
Role of RNAi machinery in the formation of heterochromatin
miRNAs - degradation of mRNA or inhibition of translation
miRNAs - stimulation of translation
Feed C. elegans bacteria expressing RNAi and they develop RNAi response.

34. Role of RNAi machinery in the formation of heterochromatin
Heterochromatin - condensed chromatin, silenced chromatin Centromeres - include much heterochromatin
Centromeres - One does not observe transcription from material adjacent to the centromeres.
In yeast, mutations in Dicer, Argonaute and RdRp cause such transcripts to appear.
pg 510 Weaver 4th edition
meH3lys4 - associated with active genes
meH3lys9 - associated with inactive genes.
Normally centromeres would have low meH3lys4 and high meH3lys9.
Mutants have the opposite.
RdRP found associated with centromere (but called RDRC there).

35. But mammals appear to lack RdRp.
RITS - RNA-induced initiator of transcriptional gene silencing contains Ago1 + siRNA
RDRC - RNA-directed RNA polymerase complex contains RdRp
Supposed to indicate that the RDRC copies (amplifies) the siRNA
Swi6 is required to form heterochromatin. It is attracted to meH3lys9
outer edge of a centromere
Histone methyl transferase bound by RITS.
Forward transcripts only accumulate in mutants because the strong RNAi destroys them.
See in chickens too.
But mammals appear to lack RdRp.
Mammals also have methylation of CpG sequences. But RNAi machinery appears to be inf=volved.

36. But wait there’s (too much )more
Amplification of siRNA
Role of RNAi machinery in the formation of heterochromatin
miRNAs - degradation of mRNA or inhibition of translation
miRNAs - stimulation of translation
Feed C. elegans bacteria expressing RNAi and they develop RNAi response.
MicroRNAs are nt RNAs produced naturally in plant and animal cells by cleavage from 75-nt stem-loop precursor RNA

38. Comparison of Mechanisms of MiRNA Biogenesis and Action
DCL– dicer-like nuclease in plants; Drosha-nuclease that process pre-MiRNAs in animals.
The MicroRNA can inhibit binding of an initiation factor (in animals).
Better complementarity of MiRNAs and targets in plants.

39. Fig

40. RNAI
channel
Plants - plasmodesmata

41. Stop

42. Source of miRNA’s

43. Why RNA silencing?
Original view is that RNAi evolved to protect the genome from viruses, and perhaps transposons or mobile DNAs.
Some viruses have proteins that suppress silencing:
HCPro - Vicki Vance, P19 – D. Baulcombe (?)HCPro in plant potyviruses (first example)P19 in tomato bushy stunt virus binds to siRNAs, and prevents them from being recruited into the RISC.Tat protein in HIV 45

44. References
Baulcombe, D. (2004) RNA silencing in plants. Nature 431:
Millar, A.A. and P.M. Waterhouse (2005) Plant and animal microRNAs: similarities and differences. Functional & Integrative Genomics 5: