RNA PROCESSING

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RNA PROCESSING BCH 6415 Dr. Yang Spring
2006 Part I
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Transcription termination, mRNA Stability, and
Turnover 1) mRNA Decay 2) Transcription
termination decay 3) Nonsense-mediated decay
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Messenger RNA decay pathways in eukaryotic cells.
The left panel depicts deadenylation-dependent
mRNA decay. Deadenylases remove the poly(A) tail
which then allows decapping and 5'-3'
degradation. Additionally following
deadenylation, the exosome can execute 3'-5'
decay of the mRNA body. In mammalian cells,
3'-5' decay can occur without prior decapping. In
the nonstop decay pathway, removal of the poly(A)
tail and 3'-5' degradation of the mRNA body
requires only the exosome and associated factors
the major deadenylases are not involved. The
right panel depicts deadenylation-independent
decay pathways. Following endonucleolytic
cleavage of a mRNA, the two products can undergo
both 5'-3' and 3'-5' degradation to complete
destruction of the mRNA. In the NMD pathway of S.
cerevisiae, decapping ensues without prior
deadenylation, followed by 5'-3' degradation.
The NMD pathway in mammalian cells is still
unclear.
Brewer, G. Aging Res. Rev. 1607 2002
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Switch from PAN2-PAN3 deadenylation to CCR4-CAF1
adenylation may be mediated by PABP PAN2-PAN3
stimulated by PABP
Fr. Muhlmann 2005
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Close physical functioanal interaction bwtn 5
cap and 3 poly(A) tail complex, as well as
translation initiation and mRNA
decay Poly(A)-binding protein (Pab1/PAB1) bound
to poly(A) tail inhibits decapping ? interaction
btwn 5 and 3 ends of mRNA (circularization?) 2
potential deadenylases poly(A)-binding protein
(Pab)1-dependent Poly(A) nuclease (Pan2/pan3)
Ccr4/Caf1 In mammals, deadenylase may be PARN
(poly(A) ribonuclease) Cap complex bound by
eIF4F eIF4E eIF4G (binds PAB1) eiF-4A
Deadenylation inhibits translation initiation ?
interaction btwn 3 poly(A) tail and 5 cap
complex Inhibition of translation (e.g.,
mutations in eIF4F) increases mRNA deadenylation
and degradation ? mRNA turnover may be
regulated by translation initiation
complex Deadenylation removes PAB1 binding to
poly(A) tail ? mRNA decay Yeast decapping enz,
Dcp1, interacts w/ eIF4F complex Pab1 AREs
(adenylate- uridylate-rich instability elements)
in mRNAs enhance deadenylation and decay rates
(but some AREs can stabilize mRNAs) AREs are
bound by factors (e.g., HuR/HuA, AUF1/hnRNP D)
that modulate stability of ARE-containing mRNAs
mechanism of action not clear
(Dcp1/Dcp2)
(Cap removal occurs when poly(A) tail 10 nts.)
(Xrn1)
Deadenylation-dependent mRNA decay (in yeast)
Minor decay pathway Endoribonucleases recognized
specific sequences in RNA transcript and
cleave RNA at internal site Endonuc. cleavage
followed by exonuc. digestion from exposed 5
3 ends Can regulate process w/ proteins that
bind and protect cleavage sites
Endoribonucleolytic decay
Wilusz et al. Nat. Rev. Molec Cell Biol. 2237
2001
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ARE bound by destabilizing factor AUF1 reduces
affinity of PABP for poly(A) tail ? allows access
of PARN
HuR binding to ARE enhances binding of PABP to
poly(A) tail ? blocks deadenylation stabilizes
mRNA
Model for how the AU-rich element
mediates stability and instability
ARE AU-rich element Found in 3 UTRs of some
mRNAs Several classes of AREs w/ slightly
different sequences Can mediate mRNA decay or
stabilization Many ARE-binding proteins
identified Binding of these proteins can either
promote stability or decay Not clear exactly how
AREs their binding prots. regulate mRNA decay
Two potential models 1) ARE-protein
complexes alter interactions between PABP
poly(A), or between eIF4E and 5 cap ?
providing access to PARN (deadenylase) 2)
ARE-binding proteins interact directly w/ and
regulates deadenylase. Cis-acting elements
regulating mRNA stability also found in 5 UTR
and coding region Evidence that mRNA
stability can be regulated by signalling pathways
that trigger stability or decay
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Cap-dependent deadenylation of mRNA in mammals
Experiment 1) Synthesized m7G-capped poly
adenylated RNA substrate as before. 2)
Incubated RNA substrates in presence of HeLa cell
cytoplasmic extract and m7GpppG or GpppG
competitor. 3) Ran RNA products of incubation on
polyacrylamide gels.
Experiment 1) An appropriate plasmid was
transcribed from a SP6 RNA pol-based expression
vector RNA transcripts were synthesized w/
32P-UTP ( to uniformly label RNA), w/ 3 poly(A)
tails, and w/ 5 cap (m7GpppG or GpppG in rxn).
2) Incubated RNA substrates in presence of HeLa
cell cytoplasmic extract for different time
periods. 3) Ran RNA products of incubation on
polyacrylamide gels.
Free competitor
Completely deadenylated RNA
Conclusion Free 7mG competitor inhibits
deadenylation rxn. better
than unmethylated competitor
only bone fide 7mG cap stimulates
deadenylation.
Conclusion RNA decay retarded in absence of
7mG cap 7mG cap stimulates
deadenylation in vitro.
Dehlin et al. EMBO J 191079 2000
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Experiment 1) Synthesized 32P-labelled
polyadenylated ß-globin 3 UTR RNA with m7GpppG,
GpppG, ApppG, or pppG at 3 end. 2) Added
purified PARN, a mammalian poly(A)-specific exo-
ribonuclease (i.e., putative deadenylase). 3)
Incubated for different time periods removed
aliquot of rxn. 4) Purified RNA in rxn. mixture
and ran on acryl. gels.
(Fully deadenylated)
Quantitated amount of fully deadenylated product
vs incubation time.
Conclusion Deadenylation by PARN is 7mG
cap-dependent.
Dehlin et al. EMBO J 191079 2000
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In yeast, major cytoplasmic mRNA decay mechanism
is 1) deadenylation 2) decappinng
3) 5 ? 3 exo digestion (can also detect
minor 3 ? 5 degradation pathway) In mammals,
major cytoplasmic mRNA decay mechanism is
1) deadenylation 2) 3 ? 5 digestion by
exosome 3) decapping by scavenger decapping
enzyme (can also detect minor 5 ? 3
degradation pathway)
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Functional Link Between Mammalian Exosome and
mRNA Decapping
Exptal RNA
Exosome - complex of 10-11 subunits w/ 3 ?5 exo
act. accessory factors inc.
RNA helicases, GTPase,
proteins that target exosome to approp. RNA
Control unadenylated RNA
Control oligo
Experiment 1) A random plasmid polylinker
region was transcribed from a T7 RNA pol-based
expression vector RNA transcripts were
synthesized w/ 32P-UTP ( to uniformly label
RNA), A60 or G16 at 3 end, w/ and w/out 5 cap
( m7GpppG I rxn). 2) Incubated RNA substrates
in presence of cytosolic S130 extracts from
human erythroleukemia K562 cells or HeLa
cells for different time periods. 3) Ran RNA
products of incubation on polyacrylamide gels.
capped
(Same as A60)
(Same as A60)
Conclusion 3 ? 5 RNA degradation is more
prominent than 5 ? 3 degradation in vitro.
A. Capped or uncapped polyadenylated RNA
capped RNA more stable suggests that 5
? 3 exo activity contributes to decay in this
rxn. B. Capped or uncapped RNAs w/ G16 track on
3 end both capped and un- capped
RNAs more stable w/ G16 3 end, but still
difference between capped uncapped. C.
Capped or uncapped RNA unadenylated RNA both
capped uncapped RNAs are unstable in
absence of polyadenylation. D. Data from B C
plotted. E. Capped or uncapped RNA w/ internal
G16 track at postion 96 in RNA shows
directionality of decay degradation from 5?3
would yield 56 nt band degradation from 3?5
would yield 112 nt band. Major band is 112
nt ? most degradation from 3 ? 5 direction.
Wang Kiledjian Cell 1077512001
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Functional Link Between Mammalian Exosome and
mRNA Decapping (cont.)
Is 3 ? 5 exo activity also the predominant
mechanism of decay in vivo (in intact cells)?
YES.
Experiment 1) Synthesized same labelled RNAs
as before. 2) Electroporated labelled RNA into
K562 cells. 3) Cells harvested a time points
after electroporation. 4) Purified RNA and ran on
polyacrylamide gel. 5) For panel E) Used
endogenous c-myc RNA that has short ½ life
isolated RNA at time points after
Actinomycin D treatment of cells used RNase
protect. assay to determine if 3 end or 5
end of RNA more abundant.
A. RNAs lacking poly(A or G ) structure at 3
end RNA w/ cap more stable and uncapped
RNA in absence of 3 tail structure. B.
RNAs w/ a G18 track at 3 end RNAs w/ G16
tail more stable than without (in A)
capped RNA w/tail more stable than
uncapped w/ tail. C. RNAs containing a
poly(A) tail at 3 end RNAs w/A60 tail
also more stable than w/out, and capped RNA
/w tail more stable than uncapped w/ tail. D.
Data from A B plotted. E. RNase protection
assay to detect 5 end and 3 end of
endogenous c-myc RNA in cytoplasm from K562
or HeLa cells ratio of 3 end to 5 end
decreased over time ? c-myc RNA in cells
is predominantly degraded from the 3 end.
Wang Kiledjian Cell 1077512001
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Paper also showed 1) A methylated cap-specific
decapping activity present in mammalian S130
extract and releases m7GMP. 2) This decapping
activity was dependent on 3 ? 5 degradation
unadenylated RNA is decapped at a rate 5X
greater for polyadenylated RNA. 3) Using
extracts depleted for poly(A) binding protein
(which facilitates deadenylation), saw greater
decapping activity. 4) This decapping activity
assoc. w/ subset of exosome proteins. Conclusions
The decapping activity acts on degraded RNA and
is a scavenger activity.
One potential pathway for mRNA decay in
mammalian cells
Wang Kiledjian Cell 1077512001
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Eukaryotic Transcription Termination
Polyadenylation site required for termination
Pol pause sites down- stream of polyA site
1) Anti-termination conformation change in pol
upon recognition of poly(A) signal involvement of

anti-terminator factor 2) RNA
cleavage/torpedo cleavage of transcript at
poly(A) site promotes 5 ? 3 degradation of
uncapped
downstream portion of transcript still
attached to pol II and triggers termination
Xrn2 required for transcription termination
(from RNAi studies)
Fr. Luo Bentley Cell 2004
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1) Cleavage polyadenylation factors bind to CTD
of pol II 2) Cleavage polyadenylation factors
required for termination 3) Mammalian CTD
required for termination
Ser2 phosphorylation important for 3
co-transcriptional end processing
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Alternative Model for Eukaryotic Transcription
Termination Modified Torpedo
Fr. West et al. Nat. 2004 Tollervey Nat 2004
Co-transcritional \ cleavage site
ß-globin gene (only gene identified w/ CoTC)
Self-cleaving
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Nonsense-Mediated Decay (NMD) Also targets large
number of normal mRNAs
(Exon junction complex)
In yeast, NMD appears to occur exclusively in
cytoplasm. Not as clear In mammals evidence for
both nuclear and cytoplasmic NMD. NMD appears
to be linked to splicing (in mammals) by the
binding of marker proteins to regions just
upstream of exon-exon junctions (where splicing
just took place). However, evidence for one
round of pioneer translation of mRNAs in
nucleus because NMD requires translation
termination If translation terminates at a
nonsense codon gt50-55 nucleotides upstream of
exon-exon junction, Upf1 thought to initiate
NMD by interacting w/ EJC intronless genes dont
undergo NMD
Wilusz et al. Nat Rev Molec Cell Biol. 2237
2001
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(No Transcript)
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Nonstop Decay (NSD)
Eliminates mRNAs that lack a stop codon These
nonstop RNAs are as labile as mRNAs with
premature termination codons Nonstop RNAs
degraded by exosome in 3 to 5 direction
beginning at end of poly(A) tail NSD pathway
distinct from normal mRNA decay pathways and NMD
pathway Unlike NMD, NSD occurs in cytoplasm
NSD requires translation of the mRNA appears
to be triggered by ribosome reaching the 3 end
of a mRNA Prevents translation of aberrant
mRNAs including those with premature pol(A)
tails (due to presence of poly(A) addition
signals within normal coding region) prevents
synthesis of truncated proteins