Transcript Slide 1

COUPLING BETWEEN TRANSCRIPTION
AND mRNA PROCESSING
Mouse (n = 52)
Budding / Fission yeast (n = 26 / 29)
Structure of RNA Pol II
CTD appears to be of sufficient size and flexibility to interact with
multiple components of pre-mRNA processing machinery and to
localize this machinery close to the pre-mRNA as it emerges from the
exit groove of the pol II.
Patrick Cramer, David A. Bushnell, Roger D. Kornberg
Science (2001) 249, 1863
Initiation factor TFIIH (Cdk 7/cyclin H) phosphorylates Ser-5
of CTD shortly after initiation.
Before transcription initiation, the CTD is unphosphorylated and
is associated with the mediator and components of the initiation
machinery.
( Tyr Ser Pro Thr Ser Pro Ser ) n
RNA Pol II is arrested by Negative Transcritption Elongation
Factor (NELF/DSIF).
P-TEFb (Cdk 9/cyclin T) phosphorylates Ser-2 of CTD during
elongation and neutalizes the repressive action by NELF.
Binding of Mammalian Capping
Enzyme to CTD Phosphopeptides
Mce1 (Mouse Capping Enzyme) binds avidly to CTD containing PO4
on Ser2 or Ser5 of each heptad.
Stimulation of Guanylyltransferase
Activity by CTD-PO4
Allosteric effector function of Ser5 phosphorylation is “dominant” over
non-activating Ser2 phosphorylation.
Capping enzyme is recruited
to the transcription complex
via Pol II CTD.
Pol II CTD and transcription
elongation factor Spt5
(subunit of DSIF) stimulates
MCE1 GTase activity (Wen &
Shatkin).
Cap structure is added (as
short as 22 nts). P-TEFb is
recruited to the elongation
complex, phosphorylate CTD
and neutalizes the repressive
action by NELF.
40
HOW IS SPLICING POSSIBLE?
SUR2 gene:
exon
9.6 kb intron
intron
40 kb
Human Dystrophin gene
260 kb intron
2.4 Mb
Like transcription factors, hnRNP proteins are modular
proteins containing two types of domains:
RNA-binding domain
Protein-protein
interaction domain
The RNA-binding domains have much less specificity for
binding to a specific RNA sequence than do the DNAbinding domains of activators and repressors.
Searching for proteins binding to RNA Pol II by two-hybrid screen identified several
SR-like proteins known as SCAFs (SR-related CTD associated factors).
SR (Ser-Arg) protein family:
•Characterized by one or two RBDs at their N-terminal and an Arg-Ser (RS)
repeat domain at the C-terminal.
•Can bind the pre-mRNAs via their RBDs and interact with proteins via their RS
domain simultaneously.
•It has been proposed that SR proteins help to bridge the two splice-sites.
Targeting of pre-mRNA Splicing Factor to Transcription Sites in Vivo
Misteli T. and Spector D. (1999) Mol Cell 3: 697
In higher eukaryotes, exons encode
more than just amino acid sequence.
Exons in long pre-mRNAs also
contain binding sites for SR proteins
called exonic splicing enhancers.
SR proteins bound to exonic splicing enhancers that span
an exon interact with U1 snRNP bound to the 5’ splice site
of the downstream intron and to U2AF and U2 snRNP
bound to the 3’ end of the upstream intron.
These protein-protein and RNA-protein interactions form a
network of intermolecular interactions that span the length
of an exon called a cross exon recognition complex.
The cumulative effect of these multiple interactions
specifies an exon, distinguishing it from intron sequence, a
process called exon definition.
pre-mRNA
Coupling between Splicing Machinery and Transcription.
 In vitro splicing reactions are 1000-fold slower than in vivo.
 Transcription by a Pol II with only 5 copies of CTD repeats
inhibits splicing.
 Pol IIO (phosphorylated form) has been shown to enhance in
vitro splicing in the absence of transcription.
 Overexpression of CTD fusion proteins interferes with mRNA
splicing in vivo.
 CTD peptides or antibodies against the CTD inhibit in vitro
splicing.
Coupled
transcription and
splicing
(Adapted from
T. D. Pollard and
W. C. Earnshaw
Cell Biology
(2002))
snRNPs, SR proteins
and splicing factors
associate with phosphorylated CTD
Phosphorylated CTD
U1snRNP
Cross-exon recognition
complex
U2snRNP
Pol II
FUNCTIONAL LINK BETWEEN TRANSCRIPTION AND SPLICING??
Coupling between 3’ Cleavage and Transcription.
Multi subunit protein complex:
 CPSF (cleavage/polyadenylation
specificity factor): Required for
cleavage and polyadenylation. Binds
to AAUAAA sequence just upstream of
poly A site.
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Coupling between 3’ Cleavage and Transcription.
Multi subunit protein complex:
 CPSF (cleavage/polyadenylation
specificity factor): Required for
cleavage and polyadenylation. Binds
to AAUAAA sequence just upstream of
poly A site.
 CstF (cleavage stimulation factor):
Required for cleavage only. Binds to
downstream G/U rich sequence.
Coupling between 3’ Cleavage and Transcription.
Multi subunit protein complex:
 CPSF (cleavage/polyadenylation
specificity factor): Required for
cleavage and polyadenylation. Binds
to AAUAAA sequence just upstream of
poly A site.
 CstF (cleavage stimulation factor):
Required for cleavage only. Binds to
downstream G/U rich sequence.
 CF1 and CF2 (cleavage factor) :
Required for pre-cleavage assembly
and stability.
Coupling between 3’ Cleavage and Transcription.
Multi subunit protein complex:
 CPSF (cleavage/polyadenylation
specificity factor): Required for
cleavage and polyadenylation. Binds
to AAUAAA sequence just upstream of
poly A site.
 CstF (cleavage stimulation factor):
Required for cleavage only. Binds to
downstream G/U rich sequence.
 CF1 and CF2 (cleavage factor) :
Required for pre-cleavage assembly
and stability.
 PAP (Poly A Polymerase): Catalyze
Pol(A) synthesis. Also required for
cleavage.
Coupling between 3’ Cleavage and Transcription.
Ryan K etal. (2002) MCB 22: 1684
Coupling between 3’ Cleavage and Transcription.
 Pol II CTD stimulates in vitro
cleavage reaction.
 RNA transcribed by truncated CTD
was not efficiently polyadenylated in
vivo.
Coupling between 3’ Cleavage and Transcription.
 Purified Pol II stimulates in vitro
cleavage reaction.
 RNA transcribed by truncated CTD
was not efficiently polyadenylated in
vivo.
 CPSF is brought to the pre-initiation
complex by the general transcription
factor TFIID and dissociate from
TFIID in elongating polymerase.
 CPSF and CstF both bind the
unphosphorylated and
phosphorylated form of Pol II.
Less Processing
Less Transcription
Before transcription initiation, the CTD is
unphosphorylated and is associated with the mediator
and components of the initiation machinery.
Partial phosphorylation of CTD during transcription
initiation recruits the capping enzymes to cap the 5’ end
of nascent transcript. CTD also activate the capping
activity.
Further phosphorylation of the CTD recruits components
of the pre-mRNA splicing machinery.
RNA Pol II reaches termination signals and factors required
for clavage and 3’ polyadenylation of the transcript
associated with the CTD and recognized sequences in the
RNA.