Elongation and pre-mRNA processing
MBV4230
Odd S. Gabrielsen
Introduction
Regulation of elongation most transcription units are probably regulated during elongation
because the elongation machinery must coordinate with so many other nuclear processes while navigating a nucleoprotein template.
Regulation can be general, applying to many genes, or selective.
Themes RNA polymerase II Elongation factors - Specific proteins affecting elongation Chromatin and elongation Pre-mRNA processing
Capping, Splicing and Termination/3´-end formation
The elongating RNAPII
MBV4230
Odd S. Gabrielsen
Control of elongation by RNAPII Two basic features
RNAPII has a remarkable processitivity
RNAPII is susceptible to transient pausing and arrest
MBV4230
Odd S. Gabrielsen
The Elongation phase - RNAPII
Processivity explained by 3D of RNAPII RNAP closes upon”promoter
clearance” and transition to TEC (trx elongation complex)
Contacts to PIC disrupted and new contacts with elongation factors formed
CTD phosphorylated Conformational change to a ternary complex of high
stability Closed chanel around DNA-RNA hybrid in the active site
Encouter obstacles that lead to to pausing or arrest
MBV4230
Odd S. Gabrielsen
Early evidence for elongation factors
Early evidence for general elongation factors Elongation rate of RNAPII in vitro << in vivo
In vitro: 100-300 nt per min, frequent pauses, some times full arrest
In vivo: 1200-2000 nt per min, probably because elongation-factors suppress pausing
The DRB-inhibitor: nucleotide-analogue causing strong inhibition of hnRNA synthesis, acts by enhanced arrest of RNAPII, but has no effect on purified RNAPII, targets probably an elongation factor
DRB = 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole
MBV4230
Odd S. Gabrielsen
Several protein-factors isolated that stimulate elongation
P-TEFb
TFIIS
FACT
TFIIF
Elongin
ELL
MBV4230
Odd S. Gabrielsen
Elongation
RNAPII target
Chromatin target
SuppressPausingIncrease rate
Suppressarrest
Stimulation of elongation - multiple mechanisms
Several possible mechanisms stimulating elongation Factors that facilitate elongation through
chromatin FACT - ”facilitates chromatin transcription” SWI/SNF-type chromatin remodelling
Factors that facilitate elongation by supression of RNAPII pausing
TFIIF, Elongin (SIII), ELL, ELL2, CSB Factors that facilitate elongation by liberating
RNAPII from arrest TFIIS
Elongation factors that target RNAPII
Phosphorylation of CTD
- early stages of elongation
MBV4230
Odd S. Gabrielsen
Elongation factors that target RNAPII: P-TEFb phosphorylates CTD The P-TEFb (positive transcription elongation
factor) complex, which contains the cyclin-dependent kinase CDK9 and cyclin T, couples RNA processing to transcription by phosphorylating Ser2 of the RNAP II CTD
Identified biochemically Based on its ability to protect RNAPII aginst arrest in a Drosophila tr.system
structure: Heterodimer = 124 kDa + 43 kDa activity: a CTD-kinase
Cdk9 (også kalt PITALRE) + cyclin T1, T2 or K Kinase-inactive form without effect on elongation
Distinctive feature: inhibited by the nucleotide analogue DRB
MBV4230
Odd S. Gabrielsen
Elongation - phosphorylation cycle
MBV4230
Odd S. Gabrielsen
P-TEFb action - release from DSIF/NELF induced pause
Mechanism of action Ser 2 phosphorylation of CTD with P-TEFb blocks binding of the elongation -inhibitors NELF
and DSIF (DRB-sensitivity inducing factor) NELF = Negative ELongation Factor, a multiprotein complex (5 polypeptides 46-66 kDa) DSIF = DRB Sensitivity Inducing Factor, a heterodimer (14 + 160 kDa) = Spt4 + Spt5 in yeast Stop and wait for capping: DSIF interacts with hypophosphorylated CTD. NELF recognizes the
RNAP II–DSIF complex and halts elongation. This pause allows the recruitment of the capping enzyme by the CTD and DSIF (Spt5 subunit), which adds a 5 -cap to the nascent transcript.
MBV4230
Odd S. Gabrielsen
The first steps in elongation
The kinase action of TFIIH phosphorylates Ser5 of CTD DSIF and NELF followed by the capping machinery (CE) are
recruited into the stalled transcription complex CE caps the nascent mRNA (see later) SCPs (small CTD phosphatases) dephosphorylate Ser5. P-TEFb phosphorylates Ser2 of CTD + the SPT5 subunit of DSIF,
which may facilitate the release NELF. Trx elongation is resumed through the association of elongation
factors (EFs).
MBV4230
Odd S. Gabrielsen
HIV and P-TEFb
TAR
Tat
Cdk9Cyclin T
P-TEFb
5´-
RNAPII
CTDCTD-kinase
Stimulated elongation
Human specific
MBV4230
Odd S. Gabrielsen
HIV and P-TEFb
HIV-1 produces its own”elongation factor” Tat Tat is a sequence-specific RNA-binding protein encoded by HIV Tat binds to a sequence-element TAR (transactivation response element) in the 5´-end
of HIV-transcripts Tat+TAR promote effective elongation of HIV transcripts
P-TEFb + CTD is required for Tat-function Ternary complex formed with human cyclin T1+ Tat + TAR (human, not murine T1) Murine cells become HIV-infectable after transfection with human cyclin T1 Mechanism: Tat facilitates HIV expression by recruiting P-TEFb to TAR, which
improves specifically elongation of HIV-transcripts
Elongation factors
Helping arrested RNAPII
MBV4230
Odd S. Gabrielsen
Pausing and arrest
RNAPII encounters obstacles during elongation leading to pausing or arrest
This stage of trx is subject to control and several genes may be regulated also on the level of elongation
Pausing and arrest result from aberrant backward movement of
RNAPII, leading to displacement of the 3´-OH end of the growing RNA from the catalytic site
Pausing = reversible Arrest = not reversible
MBV4230
Odd S. Gabrielsen
Elongation factors that suppress RNAPII arrest : TFIIS
structure: monomer = 38 kDa TFIIS binds arrested RNAPII and TFIIS strongly enhances a weak intrinsic nuclease
activity of RNAPII
TFIIS induces the polymerase to cleave its nascent transcript, repositioning the new RNA 3´-end within the polymerase catalytic center
MBV4230
Odd S. Gabrielsen
TFIIS helps RNAPII to recover from an arrested state & resume elongation
Arrest and resuce When RNAPII
approaches an arrest site, it may stop, reverse direction (backtracking), and extrude RNA, leading to transcriptional arrest.
TFIIS can rescue arrested Pol II by inducing cleavage of the extruded RNA fragment. Transcription is then resumed and continued past the arrest site.
MBV4230
Odd S. Gabrielsen
RNAPII active site switches from polymerizing to cleavage RNAPII contains a single active site for
both RNA polymerization and cleavage
polymerizing
TFIIS inducedcleavage
Elongation factors
Helping paused RNAPII
MBV4230
Odd S. Gabrielsen
Pausing and arrest
Pausing = rate-limiting step during elongation RNAPII susceptible to pausing at each
step RNAPII cycles between active and
inactive (paused) conformations
MBV4230
Odd S. Gabrielsen
Elongation factors affecting pausing or arrest
MBV4230
Odd S. Gabrielsen
Elongation factors that suppress pausing:
TFIIF, Elongin (SIII), and ELL TFIIF
Protects the elongation complex against pausing Acts probably by a direct but transient interaction with the elongating
RNAPII
phosphorylation of RAP74 stabilizes binding to RNAPII and stimulates elongation
MBV4230
Odd S. Gabrielsen
Elongation factors that suppress pausing:
TFIIF, Elongin (SIII), and ELL Elongin
Heterotrimer of subunits A, B and C where A is active, B and C regulatory Elongins activity can probably be regulated by the von-Hippel-Lindau (VHL)
tumor supressor protein which binds Elongin BC and blocks their binding to Elongin A.
Genetic disease VHL dispose for several cancers, where mutated VHL binds Elongin BC less avidly
ELL 80 kDa elongation factor found fused with MLL (mixed lineage leukemia) in
certain leukemias with translocation between chromosome 11 and 19 (ELeven-nineteen Leukemia)
MBV4230
Odd S. Gabrielsen
Mechanims of action
Pausing = rate-limiting step during elongation RNAPII susceptible to pausing at each step RNAPII cycles between active and inactive (paused) conformations
Elongation factors that suppress pausing, probably act by decreasing the fraction of time RNAPII spends in an inactive paused conformation For many factors supressing pausing and increasing rate of trx, our
understanding of mechanism is incomplete
Elongation factors helping RNAPII through chromatin
MBV4230
Odd S. Gabrielsen
Elongation through chromatin
Chromatin is not only an obstacle to TFIID binding and PIC assembly, but also for the elongating RNAPII
MBV4230
Odd S. Gabrielsen
Through arrays of nucleosomes - propagation of chromatin disruption Nucleosome arrays
more difficult to pass Inter-nucleosome contacts
repress elongation Induce pausing Some elongation factors
stimulate elongation on free DNA in vitro, but cannot overcome the chromatin block
MBV4230
Odd S. Gabrielsen
Through arrays of nucleosomes - propagation of chromatin disruption In vivo cellular
factors helps to disrupt the chromatin block to elongation
MBV4230
Odd S. Gabrielsen
Elongation factors acting on chromatin
Factors that facilitate elongation through chromatin SWI/SNF-type chromatin remodellering through ATPdependent mechanisms
Swi-Snf and Chd1 remodel nucleosomes Proteins that acetylate (e.g. Gcn5 and Elp3) or methylate histones FACT - ”facilitates chromatin transcription”- can bind to and destabilize nucleosomes
a heterodimer where SPT16 encodes the large subunit HMG1-like factor SSRP1 Proposed that FACT transiently binds and removes H2A+H2B
Spt4+Spt5 (DSIF) and SPT6 proteins
Reassembly of chromatin after passage of RNAPII important To suppress trx initiation from cryptic initiation site (noise) FACT and SPT6 probably acts by enabling chromatin structure to be disrupted and
then reestablished during trx
MBV4230
Odd S. Gabrielsen
The targeting problem again
How are these factors targeted to the transcribed regions of the genome?
Hitching a ride on the RNAPII
Likely through recognizing hyperphosphorylated CTD P/CAF (HAT) binds specifically to the
hyperphosphorylated RNAPII An ”elongator” isolated in yeast that
associates only with the hyperphosphorylated elongating form of RNAPII
One of the subunits, Elp3 = HAT
MBV4230
Odd S. Gabrielsen
FACT facilitates chromatin transcription
FACT is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro.
FACT specifically interacts with nucleosomes and histone H2A/H2B dimers
FACT appears to act as a histone chaperone to promote H2A/H2B dimer dissociation from the nucleosome and allow RNAPII transcription on chromatin
Trx correlates with the generation of a nucleosome depleted for one H2A/H2B dimer
MBV4230
Odd S. Gabrielsen
FACT
FACT functions to destabilize the nucleosome by selectively removing one H2A/H2B dimer, thereby allowing RNAP II to traverse a nucleosome.
MBV4230
Odd S. Gabrielsen
The ebb and flow of histones The histone chaperone activity of Spt6
helps to redeposit histones on the DNA, thus resetting chromatin structure after passage of the large
RNAPII complex. FACT enables the displacement of the
H2A/H2B dimer from the nucleosome, leaving a “hexasome” on the DNA. The histone chaperone activity of FACT might help to
redeposit the dimer after passage of RNAPII, thus resetting chromatin structure.
A possible relationship between histone acetylation and transcription through the nucleosome. In this scenario, HATs associated with RNAPII acetylate
the histone that is being traversed, facilitating its disruption and displacement.
Upon redeposition of the displaced histone dimer or octamer, HDACs immediately deacetylate the histones, resetting chromatin structure.
MBV4230
Odd S. Gabrielsen
1. A decompaction of chromatin surrounding the activator site Implies a specialized ”pioneer”
polymerase to do the first trip Implies that elongation itself could
play a role in chromatin modification
2. Activators promote decompaction of chromatin over the whole gene
3. Histone methylation
Three possible disruption mechanisms
MBV4230
Odd S. Gabrielsen
Histone Lys methylation
PIC assembly Upstream and downstream of
the PIC, nucleosomes are dimethylated on H3-K4 and not methylated at H3-K36.
Promoter clearance CTD-kinase of TFIIH
phosphorylates ser-5 of the CTD resulting in disengagement from the promoter and recruitment of the Set1 complex (HKMT) and the capping machinery.
Elongation CTK1 kinase complex (or P-
TEFb) is recruited to the trx apparatus resulting in phosphorylation of ser-2 of the CTD.
Ser-5
Ser-2
MBV4230
Odd S. Gabrielsen
HKMT(SET1)
HKMT(SET2)
Histone methylation
= stable epi-markRNAPII dynamic
process
MBV4230
Odd S. Gabrielsen
In yeast two separate HKMT-containing complexes associate with RNAP II and are implicated in histone methylation at mRNA coding regions
Set1 is implicated in establishing H3-K4 histone methylation.
Set2 is implicated in establishing H3-K36 histone methylation.
tri-methylation of H3-K4 catalyzed by Set1 accumulate near the 5´-mRNA coding region of genes
and is associated with the early stages of transcription.
Set2 specifically associates with the elongating form of RNAP II.
Set2-mediated H3-K36 methylation, along with di-methyl H3-K4, corresponds to later stages of elongation.
MBV4230
Odd S. Gabrielsen
PAF complex
The yeast Set1 and Set2 HKMTs are recruited by the PAF trx elongation complex in a manner dependent upon the phosphorylation state of the CTD of RNAPII The PAF complex directly recruits Set1 to the trx machinery by bridging the interaction
between RNAP II and Set1
PAF has five subunits Paf1, Rtf1, Cdc73, Leo1, and Ctr9 Evidence suggests that PAF integrates transcriptional regulatory signals and coordinates
modifications affecting chromatin
MBV4230
Odd S. Gabrielsen
A possible logic?
The CTD of RNAPII has been found to anchor several proteins with a role in elongation and pre-mRNA processing
A histone code of methylated histone-tails may provide additional anchorage sites for elongation factors or processing enzymes
Ass factors
Ass factors
Pre-mRNA processing
Processes tightly linked to elongation
MBV4230
Odd S. Gabrielsen
A role for CTD in mRNA processing?
Several novel CTD-binding proteins identified the last few years with functions in splicing and termination
Tight coupling : transcription - pre-mRNA processing
capAAAAAAAAAAAAA
Pre-mRNA(hnRNA)
mRNA
MBV4230
Odd S. Gabrielsen
CTD-mediated coupling : transcription - pre-mRNA processing
Pre-mRNA processing Capping Splicing Cleavage/polyadenylation
Physical contact between the machines for for transcription and pre-mRNA processing through CTD
Capping
MBV4230
Odd S. Gabrielsen
Cap-2
Cap-1
Cotranscriptional ”Capping”
Pre-mRNA modified with 7-methyl-guanosine triphosphate (cap) when RNA is only 25-30 bases long
Cap: 3 modifications 7-met-guanosine coupled to 5´-end
Coupling by 5´-5´triphosphate bridge Takes place co-transcriptionally
O2´-methylation of ribose Cap2, Cap1 (multicellulær), Cap0 (unicellulær)
N6-methylation of adenine
Capping occurs co-transcriptionally
MBV4230
Odd S. Gabrielsen
Capping
3 enzymes involved 1. RNA 5´-
Triphosphatase (RTP) removes a phosphate
2. Guanylyl transferase (GT) attach GMP
Enzyme 1+2 coupled: in multicellular organisms: in same polypeptid, in yeast heterodimers
3. 7-methyltransferase (MT) modifies the terminal guanosine
1. 2.
3.
MBV4230
Odd S. Gabrielsen
Cotranscriptional ”Capping”
CTD recruits capping enzyme as soon as it is phosphorylated CTD required for effective capping Guanylyl transferase (mammalian + yeast) binds directly to phosphorylated
CTD, not to non-phosphorylated 7-methyltransferase (yeast) binds also phosphorylated CTD
phosphorylated CTD may also regulate the activity of the enzymes
Cap structure is recognized by CBC (Cap binding complex) Composed of two proteins CBP20 and CBP80 Major role in stabilization, block exonucleases CBC stimulates subsequent splicing and 3´-end processing
Splicing
MBV4230
Odd S. Gabrielsen
Splicing
Splicing of introns occurs cotranscriptionally EM evidence Half-life BR1 intron only 2.5 min ≈ 5 kbs elongation of RNAPII
Splicing depends on CTD Inhibited by CTD truncation In vitro splicing stimulated by added phosphorylated CTD
CTD binds probably splicing-factors Not fully characterized CTD associated with SR- and Sm-splicing factors
MBV4230
Odd S. Gabrielsen
Splicing - excision of lariat
MBV4230
Odd S. Gabrielsen
Cotranscriptionalsplicing
MBV4230
Odd S. Gabrielsen
Association CTD-splicing factors
CTD binds probably splicing-factors CTD associated with SR- and Sm-splicing factors CASP (CTD-associated SR-like proteins) and SCAF (SR-like CTD-associated
factors) RNA-binding proteins due to
RRM-domains target the factor to exon enhancer sequences RS-domains acting as ”glue” by forming RS-RS interactions
Promoter-context can determine associated SR proteins and hence splicing Fibronectin: one intron included or excluded depending on the promoter Model: SR-CTD interaction set up during intiation, thus priming the elongation
complex
Elongation rate can determine choice of alternative splice sites
3´-end formation
MBV4230
Odd S. Gabrielsen
Modification of 3´- end:poly-adenylation Defined 3´-end is formed not by
precise termination, but as a result of processing Pre-mRNA heterogenous 3´-ends, mRNA well defined 3´-ends
Poly(A) tails added in 3´-end Ca 200x adenosines in a stretch of As added in a
particular process I.e. poly(A) not gene encoded
capAAAAAAAAAAAAA
MBV4230
Odd S. Gabrielsen
Trimming of 3´-end
capAAAAAAAAAAAAA
Inprecisetermination
cap
Precise end aftercleavage and polyadenylation
0 0
MBV4230
Odd S. Gabrielsen
Poly-adenylation - two-step process
cleavage 15-25 downstream of AAUAAA within 50 nt before a less conserved (G)U-rich element (DSE) cleavage preferentially in a CA nucleotide
Poly(A) tail made by a poly(A) polymerase Recognition:
AAUAAA binds CPSF through its largest subunit (of four in total) Cleavage and polyadenylation specificity factor
DSE binds Cleavage stimulatory factor CstF In addition two other ”cleavage factors” CF-I and -II
Coupled processes: CPSF and CstF stimulates each other bound CPSF stimulates the poly(A) polymerase
MBV4230
Odd S. Gabrielsen
Cleavage and polyadenylation
6 multimeric protein factors involved PAP (poly (A) polymerase) PABP II (poly(A)-binding protein) CPSF CstF CF-I CF-II
MBV4230
Odd S. Gabrielsen
Processing of 3´-end: ”Cleavage/polyadenylation” When RNAPII is approaching the 3´-end of the
transcript, several coupled processes are taking place Splicing of terminal intron cleavage at poly(A)-site, addition of poly(A) tail, termination downstream of poly(A)-site and liberation of RNAPII These av difficult to separate in time
These processes depend on CTD Splicing, processing of 3´-end and termination downstream of poly(A) site are all
inhibited by CTD truncations ”Cleavage-polyadenylation specificity factor” CPSF and ”cleavage stimulation
factor” CstF bind specifically to CTD and are found associated with holoRNAPII.
Poly(A) polymerase is NOT associated with RNAPII CPSF is TBP-associated - becomes at some stage transferred from TFIID to CTD
MBV4230
Odd S. Gabrielsen
Molecular interactions between mRNA processing reactions Several steps stimulates other steps in the process
Eks 1: Cap stimulates splicing of first intron Eks 2: Cap stimulates 3´-end cleavage (but not polyadenylation)
MBV4230
Odd S. Gabrielsen
Models for trx termination - A
The allosteric model (A) During elongation, RNAPII is in a
highly processive conformation (green oval).
RNAPII is transformed into a nonprocessive form (red octagon) after transcribing through the poly(A) site (AATAAA).
The RNA transcript red upstream of and blue downstream of the poly(A) cleavage site (lightening
bolt). Dotted blue line = degraded RNA. 5´cap, added cotrx, = pale blue hat
MBV4230
Odd S. Gabrielsen
Models for trx termination - B
The torpedo model (B) RNA downstream of the
poly(A) cleavage site (blue line) is digested by a 5´-3´exonuclease (Rat1 in yeast and hXrn2 in humans (blue pacman), which tracks with RNAPII throughout the length of the gene.
After poly(A) site cleavage, the exonuclease torpedo is guided along the RNA to its polymerase target and dissociates it from the DNA template.
MBV4230
Odd S. Gabrielsen
A combined model
where the exonuclease cooperates with an unknown helicase and/or allosteric modulator of the polymerase, converting it from processive to nonprocessive form, ultimately disrupting the RNA-DNA hybrid and releasing the polymerase.
MBV4230
Odd S. Gabrielsen
RNAPII = mRNA factory that is orchestrating a coupled series of events including transcription, capping, splicing and processing of 3´-end
Cotranscriptionalprocessing