Transcript Document

Post-Transcriptional
Regulation
• Biochemistry – Third Edition by Garrett &
Grisham
• Chapter 29 – Pages 974-981
Post-transcriptional processing of
mRNA in eukaryotes
Translation closely follows transcription in prokaryote
– no nucleus
In eukaryotes, these processes are separated transcription occurs in the nucleus, translation
occurs in the cytoplasm
On the way from nucleus to cytoplasm, the mRNA is
converted from "primary transcript" (or pre-mRNA)
to "mature mRNA"
What is post-transcriptional regulation ?
• Transcription: The processes of RNA
synthesis.
• Post-transcriptional regulation: The
regulation AFTER transcription, but not
translation.
post-transcriptional
regulation
mRNA degradation
mRNA localization
Comparison between procaryotic
and eucaryotic mRNA
polycistronic
monocistronic
Eukaryotic genes are split
Because contain introns and exons
•
Introns intervene between exons doesn’t code for a protein
•
Exon: protein coding region; intron: noncoding region
•
Exon size is much smaller than intron size
•
Examples: actin gene has 309-bp intron separates first three amino
acids and the other 350 or so
•
Chicken pro-alpha-2 collagen gene is 40-kbp (40,000 bp) long, with 51
exons of only 5 kbp total; the exons range in size from 45 to 249 bases
•
Most introns (lengths vary from 60-10,000 bps) have stop codons in all 3
reading frames, thus they are untranslatable; they need to be removed splicing
•
Mechanism by which introns are excised and exons are spliced
together is complex and must be precise
The organization of split eucaryotic genes
The organization of the mammalian
dihydrofolate reductase (DHFR) gene
The gene is split into 6 exons spread over 31-kbp
The six exons are spliced together to give a 6-kb mRNA
Note that the size of the exons are much shorter than the introns,
and the exon pattern is more highly conserved than the intron pattern
Capping and Methylation
• Primary transcripts (pre-mRNAs) are “capped” as soon
as they are transcribed by RNA polymerase II
• The reaction is catalyzed by guanylyl transferase using
GTP as a substrate
• Capped G residue is methylated at N7-position
• Additional methylation occurs at C2'-O positions of
next two residues and at 6-amino group of the first
adenine (if A is the initial nucleotide)
Figure 29.37
The capping of eukaryotic pre-mRNAs. Guanylyl transferase catalyzes the addition of a guanylyl
residue (Gp) derived from GTP to the 5-end of the growing transcript, which has a 5-triphosphate
group already there. In the process, pyrophosphate (pp) is liberated from GTP and the terminal
phosphate (p) is removed from the transcript. Gppp + pppApNpNpNp…  GpppApNpNpNp… + pp +
p (A is often the initial nucleotide in the primary transcript).
Figure 29.38
Methylation of several specific sites located at the 5-end of eukaryotic pre-mRNAs is an essential
step in mRNA maturation. A cap bearing only a single CH3 on the guanyl is termed cap 0. This
methylation occurs in all eukaryotic mRNAs. If a methyl is also added to the 2-O position of the first
nucleoside after the cap, a cap 1 structure is generated. This is the predominant cap form in all
multicellular eukaryotes. Some species add a third CH3 to the 2-O position of the second
nucleoside after the cap, giving a cap 2 structure. Also, if the first base after the cap is an adenine, it
may be methylated on its 6-NH2. In addition, approximately 0.1% of the adenine bases throughout
the mRNA of higher eukaryotes carry methylation on their 6-NH2 groups.
Enzymes involved in the 5’ Capping
Phosphatase
Guanylyl transferase
Guanine 7-Methyl transferase
2’ O-Methyl transferase
Why do cells need to cap their mRNA?
- cap is recognized by cap-binding proteins
- cap distinguishes mRNAs from other types of RNA molecules
(RNA pol I and III produce uncapped RNAs)
- mRNAs need a cap (and poly A tail) for export from the nucleus
- cap is necessary for translation
- cap stabilizes mRNA in the cytoplasm
RNA factory - mRNA processing
is coupled to transcription
CTD
1. Requires C-terminal domain (CTD)
of the largest subunit of RNA
polymerase II
2. CTD has repeats (52 copies) of heptapeptide
‘YSPTSPS’. 5 of these 7 have –OH; which
are differentially phosphorylated
during elongation
3.
Phosphorylation of CTD provides binding
site for factors involved in capping, splicing,
and 3’ end formation
4.
These mRNA processing factors are
transferred to the nascent RNA at the
appropriate time
5.
Dephosphorylation of CTD occurs at
the end of transcription; necessary
for re-initiation
3'-Polyadenylation and
transcription termination
3'-Polyadenylation
• Termination of transcription occurs only after RNA
polymerase has transcribed a consensus AAUAAA
sequence - the poly(A) signal
• 10-30 nucleotides after this site [the poly(A) signal], the
mRNA is cleaved and a string of ~200 adenine residues is
added to the mRNA transcript - the poly(A) tail
• poly(A) polymerase adds these A residues
• poly(A) tail bound by PABP:
• stimulates translation and
• governs the stability of mRNA
Signals required for the formation
of the 3’ end of mRNA
Mammalian pre-mRNA 3’
end processing complex
CPSF- cleavage, polyadenylation
specificity factor
recognizes the poly(A) signal
CstF- cleavage
stimulation factor
CF- cleavage factor
recognizes the downstream GU-rich sequences
Figure 29.39
Poly(A) addition to the 3-ends of
transcripts occurs 10 to 35
nucleotides downstream from a
consensus AAUAAA sequence,
defined as the polyadenylylation
signal. CPSF (cleavage and
polyadenylylation specificity factor)
binds to this signal sequence and
mediates looping of the 3-end of
the transcript through interactions
with a G/U-rich sequence even
further downstream. Cleavage
factors (CFs) then bind and bring
about the endonucleolytic cleavage
of the transcript to create a new 3end 10 to 35 nucleotides
downstream from the
polyadenylylation signal. Poly(A)
polymerase (PAP) then
successively adds 200 to 250
adenylyl residues to the new 3end. (RNA polymerase II is also a
significant part of the
polyadenylylation complex at the
3-end of the transcript, but for
simplicity in illustration, its
presence is not shown in the lower
part of the figure.)
Mechanism of poly A tail formation
- coupled to transcription
Polyadenylation of mRNA
A. Where is the template?
- does not require a template
- the poly(A) tail is not encoded in the genome
B. What’s the function of the polyA tail?
- by interaction with poly(A) binding protein (PABP),
it is necessary for efficient translation and protection
from mRNA degradation (2 purposes)
Pre-mRNA splicing
splicing
translation
Splicing of pre-mRNA
•
Within the nucleus, pre-mRNAs form ribonucleoprotein particles (RNPs) through
association with a characteristic set of nuclear proteins
•
In "splicing", the introns are excised and the exons are sewn together to form
mature mRNA
•
The substrate for splicing is the capped primary transcript emerging from the
RNA polymerase II in the form of RNP complex
•
Splicing occurs only in the nucleus; mature mRNA is then exported to the
cytoplasm
•
Consensus sequences defining the exon/intron junctions are derived from
analysis of the splice sites in vertebrate genes
•
The 5'-end of an intron (5’ splice site) in higher eukaryotes is always GU and the
3'-end (3’ splice site) is always AG
•
All introns have a "branch site" 18 to 40 nucleotides upstream from 3'-splice site
•
5’ and 3’ splice sites and branch site are essential for splicing
What makes an intron?
5’ splice site
Branch site (usually
closer to 3’ss)
3’ splice site
R: Purine A or G
Y: Pyrimidine U or C
The branch site and lariat
•
Branch site is usually YNYRAY, where Y = pyrimidine (C or U), R = purine (A or G)
and N is anything
•
The intron is excised as a lariat structure after the completion of splicing
•
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 lariat is excised when the 3’-OH of the consensus G at the 3’ end of the 5’
exon joins with the 5’-P at the 5’ end of the 3’ exon
•
The lariat product is unstable; the 2'-5' phosphodiester is quickly cleaved and the
linear intron is degraded in the nucleus
•
The reactions that occur are transesterification reactions where an OH group
reacts with a phosphoester bond, displacing an OH to form a new phosphoester
link
•
Because the reactions lead to no net change in the number of phosphodiester
linkages, no energy input is needed
Figure 29.41
Splicing of mRNA precursors. A representative precursor mRNA is depicted. Exon 1 and Exon 2 indicate two exons
separated by an intervening sequence (or intron) with consensus 5, 3, and branch sites. The fate of the
phosphates at the 5- and 3’-splice sties can be followed by tracing the fate of the respective ps. The products of
the splicing reaction, the lariat form of the excised intron and the united exons, are shown at the bottom of the
figure. The lariat intermediate is generated when the invariant G at the 5-end of the intron attaches
via its 5-phosphate to
the 2-OH of the
invariant A within the
branch site. The
consensus guanosine
residue at the 3-end of
Exon 1 (the 5-splice
site) then reacts with the
5-phosphate at the 3splice site (the 5-end of
Exon 2), ligating the two
exons and releasing the
lariat structure.
Although the reaction is
shown here in a
stepwise fashion, 5cleavage, lariat
formation, and exon
ligation/lariat excision
are believed to occur in
a concerted fashion.
(Adapted from Figure 1
in Sharp,P.A., 1987.
Splicing of messenger
RNA precursors.
Science 235:766.)
The spliceosome - RNA/protein complex
Composition:
Small nuclear RNAs (snRNAs):
5 snRNAs (U1, U2, U4, U5, U6), called U because it is uridine rich, range
in size from 106-189 nucleotide long (small).
Proteins:
10 identified associated with each of the snRNAs to form the small
nuclear ribonucleoproteins (snRNPs).
non-snRNP splicing factors: SR-family proteins and other splicing
factors
(current estimate of total proteins is >50 different)
The importance of snRNP
• Splicing depends on small nuclear ribonucleoprotein
particles - snRNPs, pronounced "snurps"
• A snRNP consists of a small nuclear RNA (100-200
nucleotides long) and about 10 different proteins
• Some of the 10 proteins form a core set common to all
snRNPs, whereas others are unique to a specific
snRNP
Assembly of the spliceosome
•
snRNPs and pre-mRNA form a multicomponent complex called
spliceosome
•
Spliceosome is about the size of ribosome and its assembly requires
ATP
•
U1 snRNP binds at the 5'- splice site and U2 snRNP binds at the
branch site
•
Interactions between the snRNPs brings 5'- and 3'- splice sites
together so lariat can form and exon ligation can occur
•
In addition to the snRNPs, a number of proteins with RNA-annealing
functions as well as ATP-dependent RNA-unwinding activity
participate in spliceosome function
•
The transesterification reactions that join the exons may in fact be
catalyzed by snRNAs themselves, but not by snRNP proteins
Figure 29.42
Mammalian U1 snRNA can be arranged in a secondary structure where its 5-end is singlestranded and can base-pair with the consensus 5-splice site of the intron. (Adapted from Figure
1 in Rosbash, M., and Seraphin, B., 1991. Who’s on first? The U1 snRNP-5- splice site interaction
and aplicing. Trends in Biochemical Sciences 16:187.)
Figure 29.43
Events in spliceosome assembly.
U1 snRNP binds at the 5-splice
site, followed by the association
of U2 snRNP with the UACUAA*C
branch-point sequence. The
triple U4/U6-U5 snRNP complex
replaces U1 at the 5-splice site
and directs the juxtaposition of
the branch-point sequence with
the 5-splice site, whereupon U4
snRNP is released. Lariat
formation occurs, freeing the 3end of the 5-exon to join with the
5-end of the 3-exon, followed by
exon ligation. U2, U5, and U6
snRNPs dissociate from the lariat
following exon ligation.
Spliceosome assembly,
rearrangement, and disassembly
require ATP as well as various
RNA-binding proteins (not
shown). (Adapted form Figure 2
in Staley,J.P., and Guthrie,C.,
1998. Mechanical devices of the
spliceosome: Motors, clocks,
springs, and things. Cell 92:315326.)
Pre-mRNA splicing is coupled to transcription
Constitutive pre-mRNA Splicing
Every intron is removed and every exon is incorporated into mature RNA
without exception – results in a single form of mature mRNA
A cell
I
B cell
II
I
II
III
III
I
II
I
II
III
III
Alternative pre-mRNA splicing
Pre-mRNA can be spliced in different ways – gives rise to multiple forms of mature mRNAs
and increases the coding capacity of the genome
A single gene makes possible a set of related polypeptides, protein isoforms
Occurs because there is intron sequence ambiguity;
different choices are made by chance on different transcripts
Several versions of the protein encoded by the gene are made in all cells constitutive alternative splicing
Regulated alternative splicing - many cases
Switch from production of a nonfunctional protein to the production of a functional one
Cell-type specific splicing - generate different versions of a protein in different cell types
RNA splicing can be regulated negatively, which prevents access to a particular splice site,
or positively, which activates an otherwise overlooked splice site.
Constitutive alternative splicing
A cell
I
B cell
II
I
I
III
II
III
III
I
II
I
I
III
II
III
III
Regulated alternative splicing
A cell
I
B cell
II
I
II
III
III
I
C cell
II
I
I
III
II
III
III
I
II
I
III
III
Alternative splicing expands the coding potential of
the genome
It contains 18 exons
Eleven constitutive exons (exon 1-3, 9-15, and 18) are found in all mature mRNAs (always there)
Five exons (4-8) are combinatorial; (none, 1,2, etc or different combinations) individually included or
excluded; 32 possible combinations
Two exons (16 and 17) are mutually exclusive; one or the other is always present never together. Only
2 possibilities
Each mRNA includes all constitutive exons, one of the 32 possible combinations from exon 4-8, and
exon 16 or 17.
32 X 2 = 64 possible mature mRNAs can be generated from the primary transcript
Alternative splicing expands the coding potential of
the genome – Tissue-specific splicing
Negative and positive control of splicing
mRNA QUALITY CONTROL
mRNA processing
normal
7mG
AAAAAA
ppp
abnormal
7mG
ppp
7mG
ppp
7mG
ppp
AAAAAAlong
mRNA export
mRNA Quality Control:
Nuclear Retention and
Degradation
mRNA transport through the nuclear pore complex
phenylalanineglycine repeats
5’ end of mRNA leaves the nucleus first. Note exchange of some of
the proteins which do not leave the nucleus.
Two mechanisms by which mRNA export
factors are recruited onto mRNAs : two mechanisms
1.
Splicing-dependent recruitment
2.
Transcription-dependent recruitment
Splicing-dependent recruitment
of mRNA export factors
UAP56: a spliceosomal component;
deposited onto mRNAs during splicing
ALY forms a complex with UAP56 and
is recruited onto the mRNA
TAP/p15 binds ALY and releases UAP56
The mRNP is exported from the nucleus
Transcription-dependent recruitment
of mRNA export factors
THO complex: interacts with
RNAPII and functions in
transcription elongation.
THO complex associates with
Sub2p and Yra1p to form TREX
(Transcription/EXport) complex.
Fates of mRNAs in the cytoplasm
• Translation
• Degradation
• Localization → translation
mRNA stability
Procaryotes
- Bacterial mRNAs are very unstable
- They are synthesized and degraded rapidly
- Bacteria can adapt quickly to environmental changes
Eucaryotes
- Most eucaryotic mRNAs are more stable; half-life > 10 hr
- Some mRNAs, however, have half-life = 30 min or less;
encode regulatory proteins whose production rates need to
be changed rapidly in order to respond to environmental changes
- Why some mRNAs are unstable?
- How mRNAs are degraded in eucaryotic cells?
Determinants of mRNA stability
m7G
AAAAA
Stabilizer
m7G
AAAAA
Destabilizer
m7G
AAAAA
m7G
AAAAA
Very stable
Stable
Unstable
a-Globin mRNA
Most mRNAs
Cytokine and
protooncogene mRNAs
Two mechanisms of eucaryotic mRNA decay
General decay pathway: most mRNAs
Certain mRNAs: requires specific sequences
and specific proteins and specific endonucleases
mRNA localization
Generally mRNAs are translated in the cytoplasm by free ribosome; their
products may be directed to other sites in the cell
mRNA encoding secreted or membrane-bound proteins are directed to
endoplasmic reticulum (ER) by a signal at the amino terminus of the protein
Some mRNAs are directed to specific intracellular locations before
translation begins, i.e. translated at the site where the protein functions
RNA localization may be an ancient mechanism for producing cytoplasmic
asymmetry
Basic features of mRNA localization include cis-acting elements within the
mRNA that targets that message to a subcellular region, a protein-RNA
complex that effects localization, and the cytoskeleton that acts as a “road”
for RNA movement
Most localization signal sequences appear to be in the 3'UTR (untranslated
region) of mRNAs
The importance of 3’ UTR in mRNA localization
mRNA encoding hairy protein is
normally localized to the apical
site of nuclei
Injected hairy RNA containing the 3’ UTR
Injected hairy RNA lacking the 3’ UTR
RNA Editing
RNA Editing is the change in RNA sequence after transcription
by processes other than splicing
mRNAs in the mitochondria of Trypanosome: one or more U are inserted
(or, less frequently, removed); so extensively that over half of the
nucleotides in the mature mRNA are U that were inserted
mRNAs in the mitochondria of chloroplast: changes from C to U,
without nucleotide insertions or deletions
Much more limited kind occurs in mammals
RNA editing – A to I editing in mammals
A ----- U
I ----- C
1.
Altering amino acid codon
2.
Changing splice site
3.
Introducing premature stop
codons
Carried out by ADAR- adenosine deaminase acting on RNA, which recognizes
a double-stranded RNA region
Such regions form when an exon region containing the A to be edited base pairs with a
complementary base sequences in an intron known as the editing site complementary
sequence
Occurred in transcripts encoding glutamate receptors; changes a glutamine codon (CAG)
to an arginine codon (CGG); altering the conductance properties of the receptor
Editing of apolipoprotein B (apoB) mRNA – C to U
Non-edited mRNA encodes 550-kDa protein;
functions to make liver-derived VLDL
Carried out on a single-stranded region of
transcript by an editosome whose core structure
consists of a cytosine deaminase and an adapter
protein that brings the deaminase and the transcript
together
Editing of CAA codon to UAA stop site at codon
2152; edited mRNA encodes 250-kDa protein;
functions to make intestine-derived lipid complexes
Unified theory of gene expression
Stages: transcription → transcript processing → mRNA export → translation
Traditionally they have been presented as a linear series of events
(a pathway of discrete and independent steps) – each going to completion
before the next begins
Now it is clear that each stage is part of a continuous process with
physical and functional connections between the transcriptional and
processing machineries.
Capping, RNA splicing, 3’ end formation and polyadenylation, and nuclear
export are coupled to transcriptional machinery
Regulation occurs at multiple levels in this continuous process
in a coordinated fashion
Eucaryotic cells have elaborate mRNA surveillance systems to destroy any
messages containing errors
Traditional View of Gene Expression
Contemporary view of gene expression