Elongation and pre-mRNA processing

RNAPII

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Introduction

Themes Stage 1 - first steps - RNA polymerase II is released Stage 2 - promoter proximal events - pausing, checkpoint control Stage 3 - productive elongation

Chromatin and elongation Pre-mRNA processing

Capping, Splicing and Termination/3´-end formation

main rate-limiting stages of

transcription elongation

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Regulation of trx elongation

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.

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

Elongation stage 1

Promoter escape

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Promoter escape - early steps to break up contacts to the promoter ITC

At the onset ITC: the initially transcribing complex (ITC). ITC undergoes abortive initiation

ITC cycles through several rounds of abortive initiation, releasing large amounts of 2–3-nucleotide-long RNA transcripts

Escape commitment After synthesis of the first 4 nucleotides, the B-finger of TFIIB and a

switch domain of Pol II stabilize the short RNA. Clash with B-finger of TFIIB

After 5 nucleotides are added, the nascent RNA collides with the B-finger of TFIIB, inducing stress within the ITC.

probably contributes to the rate-limiting step of promoter escape escape: Transition ITC - EEC (early elongation complex)

Stress from the growing transcription bubble and the production of a 7-nucleotide-long RNA trigger collapse of the transcription bubble, providing the energy to remodel the transcription complex and eject the B-finger from the RNA-exit tunnel and TFIIB is released. 5

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Stage 1 - the early steps: from ITC to EEC

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Elongation stage 2

Promoter-proximal pausing

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Pausing

Promoter proximal pausing a phenomenon whereby RNAPII pauses in the 5′ region of the

transcription unit. Requires a signal to proceed. Regulatory function: pausing constitutes an important regulation step in

vivo. A stalled RNAPII can escape rapidly from the pause into the productive elongation phase providing highly dynamic and rapid response.

Examples: heat-shock-inducible genes and the proto-oncogenes MYC+FOS Checkpoint control: pausing functions as a checkpoint before

committing to productive elongation. The Dm hsp70 example

Paused Pol II fully occupies the promoter-proximal region of Hsp70 under conditions in which the gene is not induced. RNAPII paused after synthesis of about 25 nt of RNA. Pausing mapped to several sites from +20 to +40. 8

RNAPII

pause

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RNAPII

Several elongation-factors isolated that control pausing or stimulate elongation

P-TEFb

TFIIS

FACT

TFIIFElongin

ELL

NELF

DSIF

-

+

+

+

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Two negative elongation factors DSIF and NELF induce the pause

Mechanism of action NELF = Negative ELongation Factor, multiprot complex (4 polypeptides 46-66 kDa) DSIF = DRB Sensitivity Inducing Factor, heterodimer (14+160 kDa) = Spt4+Spt5 yeast In vivo, DSIF and NELF are present at uninduced paused D.m. heat-shock genes. Stop and wait for capping: DSIF interacts with hypophosphorylated CTD. NELF

recognizes the RNAP II–DSIF complex (may also bind RNA) 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.

Elongation stage 3

Productive elongation

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Release of RNAPII from the pause: P-TEFb phosphorylates CTD The P-TEFb (positive transcription elongation factor) complex,

which contains the cyclin-dependent kinase CDK9 and cyclin T, relieves the negative effects of DSIF and NELF

P-TEFb 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

P-TEFb is inhibited by the nucleotide analogue DRB

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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) 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.

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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).

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Elongation - phosphorylation cycle

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HIV and P-TEFb

TAR

Tat

Cdk9Cyclin T

P-TEFb

5´-

RNAPII

CTDCTD-kinase

Stimulated elongation

Human specific

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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

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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

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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

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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.

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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

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Pausing and arrest

Pausing = rate-limiting step during elongation RNAPII susceptible to pausing at each

step RNAPII cycles between active and

inactive (paused) conformations

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Elongation factors affecting pausing or arrest Pausing = rate-

limiting step during elongation RNAPII susceptible to pausing

at each step RNAPII cycles between active

and inactive (paused) conformations

Pausing = reversible Arrest = not reversible

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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 stimulates elongation 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)

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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

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Summary so far

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Elongation factors helping RNAPII through chromatinChromatin is an obstacle for the elongating RNAPII

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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

In vivo cellular factors helps to disrupt the chromatin block to elongation

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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

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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

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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

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FACT

FACT functions to destabilize the nucleosome by selectively removing one H2A/H2B dimer, thereby allowing RNAP II to traverse a nucleosome.

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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.

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Pattern of histone modifications on active genes Methylation and elongation

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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

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HKMT(SET1)

HKMT(SET2)

Histone methylation:

RNAPII dynamicprocess

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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.

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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

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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

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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

caAAAAAAAAAAAAA

Pre-mRNA(hnRNA)

mRNA

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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

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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

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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

.

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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

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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

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Splicing - excision of lariat

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Cotranscriptionalsplicing

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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

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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

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Trimming of 3´-end

capAAAAAAAAAAAAA

Inprecisetermination

cap

Precise end aftercleavage and polyadenylation

0 0

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Poly-adenylation - two-step process

1.cleavage 15-25 downstream of AAUAAA within 50 nt before a less conserved (G)U-rich element (DSE) cleavage preferentially in a CA nucleotide

2. 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

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Cleavage and polyadenylation

6 multimeric protein factors involved PAP (poly (A) polymerase) PABP II (poly(A)-binding protein) CPSF CstF CF-I CF-II

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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

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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)

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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

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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.

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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.

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RNAPII = mRNA factory that is orchestrating a coupled series of events including transcription, capping, splicing and processing of 3´-end

Cotranscriptionalprocessing