Transcript Bio 402/502 Section II, Lecture 1

Bio 402/502
Section 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
Steps leading to
transcriptional activation
CTD phosphorylation
status of RNA pol II
Promoter
escape/clearance
Transition to
elongation phase
(Orphanides & Reinberg, 2000)
What happens during transcriptional elongation?
• Original contacts within pre-initiation
complex abolished
• Formation of new contacts with
elongation factors
• Change of RNA pol II to a ternary
complex = high stability
• Phosphorylation of CTD
(Orphanides & Reinberg, 2000)
Model of nucleosome dynamics during transcription
• Phosphorylation of the CTD
defines the stage of transcription
• CTD consists of heptad repeats of
the consensus sequence: YSPTSPS
CTD: Not phosphorylated
CTD: phosphorylated
• # of repeats differ in organisms
• Promoter clearance: Ser #5 gets
phosphorylated
• Transition to elongation: Ser #2
gets phosphorylated
(Workman, 2006)
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
Mechanisms by which elongation factors work:
• 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
RNA polymerase II often encounters pauses & arrests
• Arrest (irreversible backsliding 714 nts)
• Pause (back-tracking 2-4 nts)
• Function of elongation factors:
minimize these pauses & arrests
(Sims et al, 2004)
HIV virus can transactivate by hijacking elongation machinery
P-TEFb phosphorylates RNA polII CTD
Tat: HIV’s own
elongation factor
(Karn, HIV database)
HIV can bypass pre-initiation complex and head straight
for elongation by hijacking RNA pol II from host
Nascent RNAs are processed co-transcriptionally
• During the message production, processing
takes place simultaneously
• What are the common mRNA processing
events?
•
•
•
•
Capping
Splicing
3’-end cleavage/processing
Polyadenylation
(Lewin, Genes IX)
Capping of pre-mRNAs
• Cap=modified guanine nucleotide
• Capping= first mRNA processing
event - occurs during transcription
• 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
• Cap structure is recognized by CBC
• stablize the transcript
• prevent degradation by exonucleases
• stimulate splicing and processing
Enzymes involved in mRNA capping
2
1
(Proudfoot et al; 2002)
3
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
Purpose of pre-mRNA capping
(Proudfoot et al; 2002)
• Protects mRNA from ribonucleases
• Distinguish mRNAs from other RNAs
• Directs mRNA for transport
• Promotes efficient translation
• Aid in interaction with transcription machineries
Processing of pre-mRNAs Cont’d - splicing
• a typical eukaryotic gene has ~4 introns/kb
(www.wisc.edu/pharm)
(McKee & Silver, 2007)
• Why splicing?
• Multiple proteins from a single gene
(alternative splicing)
• Facilitate evolution of new genes
(“exon shuffling”)
Splicing factors are co-transcriptionally recruited
• EM evidence
Why co-transcriptional?
• Efficiency (zero vs. first order
reaction)
• Specificity
(Lei & Silver, 2004)
Mechanism of pre-mRNA splicing
branch-point adenosine
5’ splice site
5’-
Exon 1
3’ splice site
Intron
Exon 2
-3’
pre-mRNA
trans-esterification
Cut at 5’ site, lariat
formation
2’
3’
trans-esterification
ligated exons
(www.wisc.edu/pharm)
Cut at 3’ site, exon
joining, lariat release
lariat intron
3’
Splice sites are short consensus sequences
5’ splice site
3’ splice site
Branch point polypyrimidine
sequence (Bp)
tract (Py)
exon intron
intron
exon
(www.wisc.edu/pharm)
• 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
Spliceosome assembly is a step-wise event
complex
U1 initates splicing by binding to 5’-splice site
complex
U2 binds branch pt
C1. 5’-site cleaved &
lariat formed
C2. 3’-site cleaved
complex
complex
5 small nuclear RNAs (snRNAs) participate in pre-mRNA splicing
U5
U1
U2
U6
U4
(www.wisc.edu/pharm)
orange-interaction with the 5’ splice site
green-interaction with the branch site
blue-interaction between U2 and U6
tan-Sm-binding site (PuAU4-6GPu) flanked
by two stem-loop structures
Experimental support for the requirement of snRNA in splicing
1.
intermediate
2.
A radiolabeled pre-mRNA is incubated in a nuclear
extract in the presence of ATP
Reactions are deproteinized and isolated RNA is
fractionated on a denaturing polyacrylamide gel
lariat
Result: Nuclear extracts are competent for splicing
a pre-mRNA and the reaction intermediates and
products can be visualized after electrophoresis
Pre-mRNA
1.
Spliced product
2.
DNA oligo
GTTCACATCATCGACA-5’
CAAGUGUAGUAGCUGU
RNaseH
(www.wisc.edu/pharm)
Similar reactions are carried out in the presence
of RNaseH (which cuts the RNA strand of a
RNA:DNA hybrid) and a DNA oligonucleotide that
is complementary to a specific snRNA
Examine whether the loss of the snRNA affects
production of reaction products or intermediates
Sm proteins assembles with U-snRNAs
(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)
SR proteins play important role in pre-mRNA splicing
SR proteins
RRM
RNA recognition
motif
exon-dependent functions
(SR proteins bind to exon sequences and
enhance splicing of the adjacent intron)
regulated 3’ splice
site selection
X
RS
arginine/serine-rich
domain
exon-independent functions
regulated 5’ splice
site selection
(U2AF65 binds the
polypyrimidine tract)
(Graveley, 2000)
Facilitate U-snRNP interactions
splicing enhancer
(www.wisc.edu/pharm)
Some lower eukaryotes employ a different type of splicing
(www.wisc.edu/pharm)
•13-15% of all genes in C. elegans are expressed as part of an operon
Trans-splicing have also been found in higher eukaryotes
branchpoint
polypyrimidine tract 5’ splice site
TGCCCACTAaACCCCATGCTTTCGGTTTTCCTCGACTCTCGAG ATACGGAGATCAGTT
(Dorn, 2001)
(www.wisc.edu/pharm)
Cis- vs. trans-splicing of pre-mRNAs
Single RNA substrate
Two RNA substrates
(Blumenthal, WormBook)
Same splicing mechanism is employed in trans-splicing
pre-mRNA splicing
trans-mRNA splicing
spliced leader
Spliced leader contains the cap structure!
(www.wisc.edu/pharm)
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
Drosophila Dscam gene contains thousands of possible splice variants
• Alternative possibilities for 4 exons leave a total
number of possible mRNA variations at 38.016
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