Bio 402/502Section II, Lecture 3

Transcription & mRNA splicing

Dr. Michael C. Yu

• Lectures powerpoint files & readings (in PDF files) are located in http://biology.buffalo.edu/Faculty/Yu/yu.html

• Office hours: Weds & Thurs, 9am-12pm, or by appt (email for appointment)

Transcriptional elongation

(Orphanides & Reinberg, 2000)

CTD phosphorylation status of RNA pol II

Steps leading to transcriptional activation

Promoter escape/clearance

Transition to elongation phase

What happens during transcriptional elongation?

• Original contacts within pre-initiation complex abolished

(Orphanides & Reinberg, 2000)

• Formation of new contacts with elongation factors

• Phosphorylation of CTD

• Change of RNA pol II to a ternary complex = high stability

Model of nucleosome dynamics during transcription

• Phosphorylation of the CTD defines the stage of transcription

(Workman, 2006)

• CTD consists of heptad repeats of the consensus sequence: YSPTSPS

• # of repeats differ in organisms

• Promoter clearance: Ser #5 gets phosphorylated

CTD: Not phosphorylated

• Transition to elongation: Ser #2 gets phosphorylated

CTD: phosphorylated

Experimental evidence for elongation factors

• Comparison of RNAPII elongation rate

• in vitro: 100-300 nt/min, frequent pauses, and sometimes full arrest

• in vivo: 1200-2000 nt/min

Why the discrepancy?

• Use of pharmacological agents

• DRB(5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole

• DRB, nucleotide-analogue, cause inhibition of hnRNA transcription by arresting RNA pol II in vivo, but not purified RNA pol II. Possible target?

These evidence suggest existence of factors that facilitate transcriptional elongation

Biochemical purification identified elongation factors

In vitro transcription assays

pTEFb

TF-IIS

Elongin

• Elongating through chromatin (such as chromatin remodelers). Ex = SWI/SNF, FACT

• Suppression of RNA pol II pausing, ex=elongin, TF-IIF

• Liberating RNA pol II from transcriptional arrest, ex = TF-IIS

Mechanisms by which elongation factors work:

RNA polymerase II often encounters pauses & arrests

• Arrest (irreversible backsliding 7-14 nts)

(Sims et al, 2004)

• Pause (back-tracking 2-4 nts)

• Function of elongation factors: minimize these pauses & arrests

HIV virus can transactivate by hijacking elongation machinery

(Karn, HIV database)

HIV can bypass pre-initiation complex and head straight for elongation by hijacking RNA pol II from host

P-TEFb phosphorylates RNA polII CTD

Tat: HIV’s own elongation factor

Nascent RNAs are processed co-transcriptionally

(Lewin, Genes IX)

• During the message production, processing takes place simultaneously

• What are the common mRNA processing events?

• Capping• Splicing• 3’-end cleavage/processing• Polyadenylation

Capping of pre-mRNAs

• Capping= first mRNA processing event - occurs during transcription

• Cap=modified guanine nucleotide

• Cap structure is recognized by CBC• stablize the transcript• prevent degradation by exonucleases• stimulate splicing and processing

• CTD recruits capping enzyme as soon as it is phosphorylated

• Pre-mRNA modified with 7-methyl-guanosine triphosphate (cap) when RNA is only 25-30 bp long

Enzymes involved in mRNA capping

(Proudfoot et al; 2002)

1. RNA 5’-triphosphatase (RTP): removal of a single phosphate

2. Guanylyl transferase (GT) - attaches GMP (guanosine 5’-monophosphate)

3. 7-methyltransferase (MT): modifies terminal guanosine

12

3

Purpose of pre-mRNA capping

• Protects mRNA from ribonucleases • Distinguish mRNAs from other RNAs

• Directs mRNA for transport

• Promotes efficient translation

• Aid in interaction with transcription machineries

(Proudfoot et al; 2002)

Processing of pre-mRNAs Cont’d - splicing

(McKee & Silver, 2007)

• a typical eukaryotic gene has ~4 introns/kb

• Why splicing?• Multiple proteins from a single gene (alternative splicing)• Facilitate evolution of new genes (“exon shuffling”)

(www.wisc.edu/pharm)

Splicing factors are co-transcriptionally recruited

(Lei & Silver, 2004)

Why co-transcriptional?

• EM evidence

• Efficiency (zero vs. first order reaction)

• Specificity

Mechanism of pre-mRNA splicing

Exon 1 Intron Exon 2

5’ splice site 3’ splice site

5’- -3’

2’

branch-point adenosine

3’

3’

ligated exons lariat intron

pre-mRNA

trans-esterification

trans-esterification

(www.wisc.edu/pharm)

Cut at 5’ site, lariat formation

Cut at 3’ site, exon joining, lariat release

5’ splice site Branch point sequence (Bp)

3’ splice site

exon intron exonintron

• The bigger the nucleotide = more frequent it appears at that position

• Black-colored nucleotides are thought to be involved in intron recognition

• Splice sites are not always conformed to this consensus

polypyrimidine tract (Py)

Splice sites are short consensus sequences

(www.wisc.edu/pharm)

Spliceosome assembly is a step-wise event

complex

complex

complex

complex

U2 binds branch pt

C1. 5’-site cleaved & lariat formedC2. 3’-site cleaved

U1 initates splicing by binding to 5’-splice site

U1

U2

U4

U5

U6

5 small nuclear RNAs (snRNAs) participate in pre-mRNA splicing

orange-interaction with the 5’ splice sitegreen-interaction with the branch siteblue-interaction between U2 and U6tan-Sm-binding site (PuAU4-6GPu) flanked by two stem-loop structures(www.wisc.edu/pharm)

1. A radiolabeled pre-mRNA is incubated in a nuclearextract in the presence of ATP

2. Reactions are deproteinized and isolated RNA isfractionated on a denaturing polyacrylamide gel

Result: Nuclear extracts are competent for splicinga pre-mRNA and the reaction intermediates andproducts can be visualized after electrophoresis

1. Similar reactions are carried out in the presence of RNaseH (which cuts the RNA strand of a RNA:DNA hybrid) and a DNA oligonucleotide thatis complementary to a specific snRNA

2. Examine whether the loss of the snRNA affects production of reaction products or intermediates

GTTCACATCATCGACA-5’ CAAGUGUAGUAGCUGU

DNA oligo

RNaseH

Experimental support for the requirement of snRNA in splicing

(www.wisc.edu/pharm)

Spliced product

Pre-mRNA

lariat

intermediate

(Kambach, C. et al. 1999)

• These core snRNP proteins are called Sm because of their reactivity with antibodies of the Sm serotype from patients with systemic lupus erythematosus

• Sm proteins play a key role in hypermethylation of the m7G snRNA cap to m3G, 3’ end maturation, and nuclear import of the assembled snRNP

(www.wisc.edu/pharm)

Sm proteins assembles with U-snRNAs

SR proteins RRM X RSRNA recognition

motif arginine/serine-rich domain

exon-dependent functions exon-independent functions

regulated 3’ splice site selection

regulated 5’ splice site selection

(SR proteins bind to exon sequences and enhance splicing of the adjacent intron)

(Graveley, 2000) (U2AF65 binds thepolypyrimidine tract)

splicing enhancer

SR proteins play important role in pre-mRNA splicing

(www.wisc.edu/pharm)

Facilitate U-snRNP interactions

•13-15% of all genes in C. elegans are expressed as part of an operon

Some lower eukaryotes employ a different type of splicing

(www.wisc.edu/pharm)

(Dorn, 2001)

TGCCCACTAaACCCCATGCTTTCGGTTTTCCTCGACTCTCGAG ATACGGAGATCAGTT

5’ splice sitebranchpoint polypyrimidine tract

Trans-splicing have also been found in higher eukaryotes

(www.wisc.edu/pharm)

Cis- vs. trans-splicing of pre-mRNAs

(Blumenthal, WormBook)

Single RNA substrate

Two RNA substrates

pre-mRNA splicing trans-mRNA splicing

spliced leader

Same splicing mechanism is employed in trans-splicing

(www.wisc.edu/pharm)

Spliced leader contains the cap structure!

Alternative splicing: a way to increase total number of genes

Alternative splicing: a single gene can encode many messages depending on how the message is spliced

• Alternative possibilities for 4 exons leave a total number of possible mRNA variations at 38.016

Drosophila Dscam gene contains thousands of possible splice variants

Common forms of alternative splicing

CTD of RNA pol II plays important role in pre-mRNA splicing

(Kornblihtt et al, 2004)

Effect of transcriptional elongation on alternative splicing

(Kornblihtt et al, 2004)

How do you experimentally test this?

Lecture 3 Summary