chapter 16 other rna processing events

Chapter 16 Other RNA Processing Events

Trans-splicing, Editing, RNAi, miRNAs

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

trans-splicing

Figure 16.12

200 copies of a 35 n leader encodes in a different place in the genome.

Editing

protozoa = U-insertionprotozoa = U-deletion

mammals, insects & plants = nucleotide deaminiation

16.4

Focus on this oneFocus on this one

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.

Results in major changes in properties of the protein

Example

Glutamate receptor ion channel

GluR-B changes glutamine->arginine

Reduces Ca2+-permeability.

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.

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).

Cytidine deaminaton

CDAR cytidine deaminase acting on RNA

C-->U

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.

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

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.

RNAi

Discovered in a control experiment

pg 501 Weaver 4th edition

RNA interferance

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

unc22 gene nonessential myofilament

protein. Mutations in unc-

22 cause a twitching

phenotype. dbstded unc-22

RNA phenocopies.

2. The experiment.

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.

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.

Short interfering RNAs -siRNAs

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.

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).

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

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

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

Bizarre figure see

next one for explanation.

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

lane 1 transcript + siRNA2 + Argonaute + MgCl2

lane1

lane 2 transcript + siRNA1 + Argonaute + MgCl2lane2

Ago2 knock out in mice

embryonic lethal with severe defects

important for RNAi & miRNA

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.

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.

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


Amplification of siRNATiny amounts of a trigger can have a very large and long lasting effect. Occurs in Plants, Drosophila and C. elegans.


miRNAs - inhibition of translation


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

Potential for exon spreading

Reference: Nishikura 2001 Cell 107:415-418.




miRNAs - degradation of mRNA or inhibition of translation



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).

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.




miRNAs - degradation of mRNA or inhibition of translation


Comparison of Mechanisms of MiRNA Biogenesis and Action

Better complementarity of MiRNAs and targets in plants.

40

Fig. 16.45

RNAIchannel

Source of miRNA’s

45

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:

45

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.

chapter 16 other rna processing events

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