Chapter 16 Other RNA Processing Events

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Chapter 16 Other RNA Processing Events

• Trans-splicing, Editing, RNAi, miRNAs

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Trans-splicingsection 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

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

• protozoa U-insertion
• protozoa U-deletion
• mammals, insects plants nucleotide
  deaminiation
• 16.4

Focus on this one
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RNA editing by deamination

• ADAR Adenosine deaminase acting on RNA
• adenosine -gt 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.
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Results in major changes in properties of the
protein

• Example
• Glutamate receptor ion channel
• GluR-B changes glutamine-gtarginine
• Reduces Ca2-permeability.

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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.

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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).

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Cytidine deaminaton

• CDAR cytidine deaminase acting on RNA
• C--gtU

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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.

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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

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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.
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RNAi
RNA interferance

• Discovered in a control experiment
• pg 501 Weaver 4th edition

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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.

Craig Mello
Andrew Fire
2006 Nobel Prize in Physiology Medicine
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2. The experiment.
unc22 gene nonessential myofilament protein.
Mutations in unc-22 cause a twitching phenotype.
dbstded unc-22 RNA phenocopies.
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Double-stranded RNA (dsRNA) induced interference
of the Mex-3 mRNA in the nematode C. elegans.
Fig. 16.29Weaver 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.
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Hammond et al. 2000. Nature 404293-296.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.

• Destruction of 25 nt RNA with micrococcal
  nuclease blocks reaction.

Hammond et al. 2000. An RNA-directed nuclease
mediates post-trancriptional gene silencing in
Drosophila cells. Nature 404293-296 Figure is
not in Weaver 4th but is mentioned on pg 501-502.
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Short interfering RNAs -siRNAs
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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
Zamore et al. 2000. Cell 10125-33
Fig 16.30 4th ed
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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
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High resolution gel analysis of the products of
Rr-luc mRNA degradation from the previous slide.
Fig. 16.32
Results the cleavages occur mainly at 21-23 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.
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dsRNA
Weaver 4th edition pg 501-507
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
ADPPi
Dicer leaves 2nt 3 overhangs phosphorylated 5
ends
RISC loading complex
RISCRNA-induced silencing complex.
ATP
ADPPi
RISC
p
mRNA
Target recognition
p
p
Target cleavage
mRNA
p
p
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Argo2 is Sliceris shown by building highly
specfic siRNA complexes in vitro using
bacterially expressed Argo2.
Bizarre figure see next one for explanation.
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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
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Argo2 is Sliceris shown by building highly
specfic siRNA complexes in vitro using
bacterially expressed Argo2.
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Ago2 knock out in mice

• embryonic lethal with severe defects
• important for RNAi miRNA

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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.

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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.

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But wait theres (too much )more

• Amplification of siRNA
• Role of RNAi machinery in the formation of
  heterochromatin
• miRNAs - inhibition of translation
• miRNAs - stimulation of translation

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But wait theres (too much )more

• Amplification of siRNATiny 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

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dsRNA
Amplification (pg508 4th ed)
mRNA
ATP
Dicer
NTPs
ADPPi
RdRp (RNA directed RNA polymerase)
PPi
Dicer leaves 2nt 3 overhangs phosphorylated 5
ends
ATP
Dicer
ADPPi
RISC loading complex
RISCRNA-induced silencing complex.
ATP
ADPPi
RISC
p
mRNA
Target recognition
p
p
Target cleavage
mRNA
p
p
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Potential for exon spreading
Reference Nishikura 2001 Cell 107415-418.
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In plants, C. elegans Drosophila, a
RNA-dependent RNA polymerase (RdRp) is involved
in initiation or amplification of silencing.
CBP and PABP block access for RDR.
RdRp
PABP missing.
D. Baulcombe 2004 Nature 431356
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But wait theres (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

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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.
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).
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But wait theres (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

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Comparison of Mechanisms of MiRNA Biogenesis and
Action
Better complementarity of MiRNAs and targets in
plants.
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Fig. 16.45
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Are the final mi and si complexes very different?
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RNAI channel
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Stop
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• Source of miRNAs

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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

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References

• Baulcombe, D. (2004) RNA silencing in plants.
  Nature 431 356-363.
• Millar, A.A. and P.M. Waterhouse (2005) Plant and
  animal microRNAs similarities and differences.
  Functional Integrative Genomics 5 129-135.

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