Download - 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-insertionprotozoa = U-deletion
mammals, insects & plants = nucleotide deaminiation
16.4
Focus on this oneFocus on this one
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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.
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Results in major changes in properties of the protein
Example
Glutamate receptor ion channel
GluR-B changes glutamine->arginine
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-->U
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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 petuniaCaMV - 35S promoter from Cauliflower Mosaic VirusDFR 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
Discovered in a control experiment
pg 501 Weaver 4th edition
RNA interferance
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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 Fire2006 Nobel Prize in Physiology & Medicine
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unc22 gene nonessential myofilament
protein. Mutations in unc-
22 cause a twitching
phenotype. dbstded unc-22
RNA phenocopies.
2. The experiment.
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Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode C. elegans.
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
mex-3 antisense mex-3 dsRNA
no probe
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense mRNA. (2) the suppressing signal moved from cell to cell.
Fig. 16.29Weaver 4th Ed.
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Hammond et al. 2000. An RNA-directed nuclease mediates post-trancriptional gene silencing in Drosophila cells. Nature 404:293-296Figure is not in Weaver 4th but is mentioned on pg 501-502.
Hammond et al. 2000. Nature 404:293-296.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.
dsRNAT7
T7
Destruction of 25 nt RNA with micrococcal nuclease blocks reaction.
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Short interfering RNAs -siRNAs
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dsRNA
p
p
Fig 16.30 4th ed
p
p
Zamore et al. 2000. Cell 101:25-33
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.
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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.
Fig 16.31
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).
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High resolution gel analysis of the products of Rr-luc mRNA degradation from the previous slide.
Enzyme cleaves at ~23-nt intervals & after U.
In 2001 Hammond et al purify the enzyme and name it DICER.
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.
Fig. 16.32
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Target cleavage
dsRNAATP
ADP+PiDicer
p
p
p
21-23 nt siRNP
p
p
p
ATPADP+Pi
mRNA Target recognition
p
mRNA
p
p
p
p
RISC loading complex
Dicer leaves 2nt 3’ overhangs &
phosphorylated 5’ ends
RISC=RNA-induced silencing complex.
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.
RISC
PIWI PAZ
The 2 domains of Argonaute
Argonaute
p
p
-Dicer
-R2D2
-Armitrage
Weaver 4th edition pg 501-507
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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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RNA transcript made
siRNA1 could bp about 140n from 5’ end of transcript
siRNA2 could bp about 180n 3’ end of transcript
Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.
Argo2 that has been produced in bacteria
lane 1 transcript + siRNA2 + Argonaute + MgCl2
lane1
lane 2 transcript + siRNA1 + Argonaute + MgCl2lane2
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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 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
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But wait there’s (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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p
mRNA
p
p
p
p
p
p
p
Amplification (pg508 4th ed)
NTPsPPi
RdRp (RNA directed RNA polymerase)
ATPADP+Pi
Dicer
Target cleavage
dsRNAATP
ADP+PiDicer
p
p
p
21-23 nt siRNP
p
p
p
ATPADP+Pi
mRNA Target recognition
p
mRNA
p
p
p
p
RISC loading complex
Dicer leaves 2nt 3’ overhangs &
phosphorylated 5’ ends
RISC=RNA-induced silencing complex.
RISC
Argonaute
p
p
-Dicer
-R2D2
-Armitrage
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Potential for exon spreading
Reference: Nishikura 2001 Cell 107:415-418.
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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
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Role of RNAi machinery in the formation of heterochromatin
Heterochromatin - condensed chromatin, silenced chromatin Centromeres - include much heterochromatinCentromeres - 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 genesmeH3lys9 - 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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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.
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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
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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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RNAIchannel
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Stop
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Source of miRNA’s
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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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