Transcript O - s3.amazonaws.com

Chapter 31
Post-transcriptional
Processing
(pages 1057-1066)
Learning objectives: Understand the following
• What are capping, polyadenylation, splicing?
• What is the functional importance of each process?
• Why does each process work on transcripts produced by
RNAP II?
Post-transcriptional Processing
of mRNA in Eukaryotes
• Translation closely follows transcription
in prokaryotes
• In eukaryotes, these processes are
separated - transcription in nucleus,
translation in cytoplasm
• On the way from nucleus to cytoplasm,
the mRNA is converted from a "primary
transcript" to "mature mRNA"
Post-transcriptional Modifications of
mRNA in Eukaryotes
1) 5’ end = capping
2) 3’ end = polyadenylation
3) internal = splicing
pre-mRNA
(heteronuclear RNA )
Order
of
events
Mature mRNA
(gets translated)
Overview of post-transcriptional modifications
Capping and Methylation
• Primary transcripts (pre-mRNAs or
heterogeneous nuclear RNA) are usually first
"capped" by a guanylyl group
• This is a modification of the 5’ triphosphate of
the mRNA
• The reaction is catalyzed by the enzymes
phosphohydrolase, guanylyl transferase and
methyl transferases
Capping and Methylation
• Starting with the 5’ triphosphate of the mRNA
the gamma phosphate group is hydrolyzed off.
g
b a
O
O
O
5’
O-P-O-P-OCH2
O
O O
-
O
-
5’
O-P-O-P-O-P-OCH2
O
O O O
base
base
phosphohydrolase
Pi
O
O
OH
RNA chain
O
OH
RNA chain
• The remaining 5’ diphosphate attacks GTP,
forming a covalent link with the GMP portion
and generating PPi from the GTP as the
leaving group.
O
RNA chain
O
-
5’
O-P-O-P-O-P-OCH2
O
O O O
G
O
5’
O-P-O-P-OCH2
O
O O
-
G
O
O
base
-
O
OH
Unusual 5’-5’
triphosphate linkage
O
5’
-P-OCH2
O
O
O
base
OH
-
5’
O-P-O-P-OCH2
O
O O
OH
O
Guanylyl transferase
O
PPi
OH
OH
RNA chain
OH
Hydrolyzed
Attack
• The guanine is subsequently methylated at the
N-7 position by a methyltransferase, using
S-adenosylmethionine as the methyl donor
OH
OH
5' m(7)GpppN cap
O
-P-OCH2
O
O
-
O
base
-
O-P-O-P-OCH2
O
O O
OH
O
O
Methyltransferase
O
O-P-O-P-OCH2
O
O O
base
-
G
O
RNA chain
-P-OCH
2
O
O
OH
OH
O
CH3
O
RNA chain
OH
G
Capping and Methylation
• All eukaryotic mRNAs have 5’-N7
methylguanosine CAP structure.
• This is referred to as a “CAP 0” structure
• Additional methyl groups are added in the
majority of mRNAs:
• CAP1 structure has a second methylation (not
found in yeast mRNAs)
• CAP2 structure has a third methylation (found
mainly in vertebrate mRNAs)
Additional
methylations
are possible
CAP 0
CAP 1
CAP 2
Capping and Methylation
summary
• Primary transcripts (pre-mRNAs or
heterogeneous nuclear RNA) are "capped" by
a guanylyl group
• The reaction is catalyzed by three different
enzymes
• The capping G residue is methylated at 7position
• Additional methylations occur at 2'-O positions
of next two residues and at 6-amino of the first
adenine
Why have a Cap?
• The CAP structure promotes stability of the
mRNA (prevents degradation by 5’
exonucleases)
• The CAP structure promotes translation of the
mRNA
• A ribosomal protein subunit interacts with the
CAP and recruits mR NA to the ribosome
Why are only mRNAs Capped?
• The CAP structure is only added to transcripts
synthesized by RNAP II (i.e. mRNA transcripts)
• This is because the capping enzymes bind to a
part of RNAP II that is unique to that enzyme
(not found in RNAP I or RNAP III)
• Capping enzymes bind to the CTD of RNAP II
Review of CTD
•Diagram shows an alignment of amino acids from the largest
subunits of RNAP II and III from yeast, and from E. coli.
•The boxes are domains of high amino acid similarity
•RNAPII largest subunit has a C-terminal extension - the
CTD!!
RPB1
RPC1
b’
RPB1 = largest subunit of RNAP II
RPC1 = largest subunit of RNAP III
The sequence of the CTD is an
unusual 7 amino-acid repeat
with an “extended” structure
26
• deletion studies showed that
yeast require at least 13 repeats
to survive
• therefore the CTD is essential
for RNAP II function
• the CTD has a lot of S, T amino
acids which can be
phosophorylated
• it is known that RNAP II in an
initiation complex has a nonphosphorylated CTD
52
• elongating RNAP II has a
phosphorylated CTD
Why are only mRNAs Capped?
• Capping enzymes bind to the CTD of RNAP II
• They bind only to the elongating form of the
CTD( I.e. the phosphorylated CTD)
• As soon as the transcript emerges from
RNAP II it is in contact with the capping
enyzmes
3'-Polyadenylation
25-30 bp
mRNA
Stop
ATG
TATA box
Coding sequence of the gene
+1
5’UTR
3’UTR
Translated region
A poly(A) tail is added to the 3’ end of the transcript
(defines the 3’ end) in 2 steps…
1) cleavage: the RNA is cut 10-30 nucleotides
downstream of a specific sequence in the 3’UTR
2) addition of A’s (100-200 are added) to generate a
poly(A) tail
CPSF
CFI CFII
PAP
5’ CAP
AAUAAA
mRNA
A ribonucleoprotein complex
1) CPSF = “cleavage/polyadenylation specificity
factor” (3 subunits) recognizes the AAUAAA
sequence in the RNA 3’UTR and binds to it
2) Recruits CFI and CFII = “cleavage factors”
3) Recruits PAP = “poly(A) polymerase”
CPSF
CFI CFII
PAP
5’ CAP
AAUAAA
mRNA
• The RNA is cleaved 10-30 nucleotides
downstream of the AAUAAA by CFI/II
• This generates a 3’OH to which PAP adds A
residues. It doesn’t copy from a template - just
needs a 3’OH of an RNA
• The poly(A) tail is bound by poly(A)-binding
protein
AAUAAA
AAAAAAAAAAAAA100-200
Why have a Poly(A) tail?
• The poly(A) tail promotes stability of the mRNA
(prevents degradation by 3’ exonucleases)
• The poly(A) tail promotes translation of the
mRNA
• A ribosomal protein subunit that interacts with
the CAP structure is stimulated in CAP binding
by poly(A) binding protein
• Therefore the CAP and the poly(A) tail work
synergistically to recruit mRNA to the ribosome
Why are only mRNAs
polyadenylated?
• The poly(A) tail is only added to transcripts
synthesized by RNAP II (I.e. mRNA
transcripts)
• This is because the essential polyadenylation
factor CPSF binds to the CTD of RNAP II
• Therefore, only transcripts made by RNAP II
are in close proximity to CPSF which starts off
the polyadenylation process
3'-Polyadenylation
Summary
• Termination of transcription occurs only after
RNA polymerase has transcribed past a
consensus AAUAAA sequence - the poly(A)+
addition site
• 10-30 nucleotides past this site, a string of
100 to 200 adenine residues are added to
the mRNA transcript - the poly(A)+ tail
• poly(A) polymerase adds these A residues
• Function still not completely worked out, but
poly(A) tail increases stability of the mRNA
Intron = “Intervening sequence”
Exon = “Expressed sequence”
Eukaryotic Genes are Split
• Introns intervene between exons
• Examples: actin gene has 309-bp intron
separates first three amino acids and the other
350 or so
• But chicken pro-alpha-2 collagen gene is 40kbp long, with 51 exons of only 5 kbp total.
• The exons range in size from 45 to 249 bases
• Mechanism by which introns are excised and
exons are spliced together is complex and
must be precise
Organization of the mammalian DHFR gene
Splicing of Pre-mRNA
Capped, polyadenylated RNA, in the form of a RNP
complex, is the substrate for splicing
• In "splicing", the introns are excised and the
exons are sewn together to form mature mRNA
• Splicing occurs only in the nucleus
• The 5'-end of an intron in higher eukaryotes is
always GU and the 3'-end is always AG
• All introns have a "branch site" 18 to 40
nucleotides upstream from 3'-splice site
• Branch site is essential to splicing
Sequence requirements for
splicing
2. 3’ splice site
1. 5’ splice site
5’ Exon AG GU
Intron
AG G 3’ Exon
18 - 40
nucleotides
Y= pyrimidine
R= purine
YNYRAY
N = anything
3. Branch point
Splicing occurs through two
Transesterification reactions
• In a transesterification reaction a phosphodiester bond
is transferred to a different hydroxyl group
OO
R - OH
-
+
X - OH +
O
O
-
R
OO
P
P
O
-
-
RNA Chain
X
Y
Y
• There is no hydrolysis and no energy loss
O
The Branch site and Lariat
Summary
• Branch site is usually YNYRAY, where Y =
pyrimidine, R = purine and N is anything
• The "lariat" a covalently closed loop of RNA is
formed by attachment of the 5'-P of the intron's
invariant 5'-G to the 2'-OH at the branch A site
• The exons then join, excising the lariat.
• The lariat is unstable; the 2'-5' phosphodiester is
quickly cleaved and intron is degraded in the
nucleus.
The Importance of snRNP
• Small nuclear ribonucleoprotein particles snRNPs, pronounced "snurps" - are involved in
splicing
• A snRNP consists of a small RNA (100-200
bases long) and about 10 different proteins
• snRNPs and pre-mRNA form the spliceosome
• Spliceosome is the size of ribosomes, and its
assembly requires ATP
Spliceosome
5 snRNPs (“snurps”)
snRNA
•Small (100-200 ntds)
•Rich in U’s
•U1, U2, U4, U5, U6 snRNAs
~ 10 different
proteins
U1, U2, U4,
U5, U6
snRNPs
snRNAs
• snRNAs are very important in splicing
• mediate the sequence specificity and the
accuracy of the splicing reaction
• fold into secondary structures (stemloops) that are important for catalysis
•In snRNPs it is the RNA part that is
catalytic, not the protein part
Example of snRNAs mediating sequence specificity of
splicing
Assembly of the Spliceosome
1) U1 snRNP binds to the 5’ splice junction
• This helps to recruit
2) U2 snRNP binds to the intron branch point
• This helps to recruit:
3) A trimeric snRNP composed of U4/U6-U5
• U4 and U6 RNAs are base-paired together to
keep U6 RNA in the correct format for binding
• U6 RNA binds to the 5’ splice site and
replaces U1
Assembly of the Spliceosome
(continued)
4) U6 RNA now peels off the U4 RNA (it leaves
the spliceosome)
5) This allows U6 RNA to pair with U2 RNA
• This brings the 5’ splice junction right next to
the branch point
6) Catalysis!! The first transesterification occurs
• This generates the intron lariat + free 3’OH of
the 5’ exon
• The free 5’exon doesn’t diffuse away since it
is held in place by interactions with U5 snRNP
Assembly of the Spliceosome
(continued)
7) The second transesterification occurs
• This generates a free lariat structure which is
rapidly degraded, plus the spliced exons.
8) Now the spliceosome components all
disassemble. And the U6 snRNP reassociates
with a U4sNRNP for delivery to the next
spliceosome.
RNA interactions at
time of recruiting
the U4/U6-U5 snRNP
RNA interactions
after U1 and U4 have
left the complex
Differential splicing can generate multiple proteins
from a single gene
Why are only mRNAs spliced by
snRNPs?
• The CTD of RNAP II is required for splicing
• Therefore it is proposed that splicing factors
from the spliceosome interact with the CTD
• This ensures that transcripts emerging from
RNAPII are in the correct location for
interaction with the splicing machinery
We have now finished Chapter
31!!
For next class please read:
Chapter 32 sections 1 and 2
Pages 1069-1074
Elucidation of the genetic code