Chap. 8 Post-transcriptional Gene Control

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Chap. 8 Post-transcriptional Gene Control

• Topics
• Processing of Eukaryotic Pre-mRNA
• Regulation of Pre-mRNA Processing
• Cytoplasmic Mechanisms of Post-transcriptional
  Control

• Goals
• Learn the mechanisms of 5' capping and
  polyadenylation.
• Learn the mechanism of pre-mRNA splicing by the
  spliceosome complex.
• Learn the general functions of splicing
  repressors and activators in regulation of
  pre-mRNA splicing.
• Learn about mechanisms for translation control
  via targeted RNA degradation.

hnRNP-stained lampbrush chromosome
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Post-transcriptional Gene Control
Post-transcriptional gene control refers to all
of the processes that regulate gene expression
subsequent to transcription initiation (Fig.
8.1). These processes include regulation of
alternative splicing, RNA editing, and RNA
degradation. With the exception of alternative
splicing, these mechanisms typically are involved
in the regulation of only a relatively small
fraction of RNAs in a cell. However, they can be
highly important for regulation of a given gene.
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Overview of Pre-mRNA Processing
Pre-mRNA processing includes 5' capping, 3
polyadenylation, and intron splicing (Fig. 8.2).
These reactions occur in the nucleus, and begin
while the primary transcript is being elongated
(co-transcriptional). Mature mRNAs then are
transported to the cytoplasm for translation.
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Eukaryotic pre-mRNA Processing Capping
Bacterial mRNAs are functionally active as
transcribed. Eukaryotic pre-mRNAs must be
extensively processed to attain their final
functional forms. The modification that occurs at
the 5' end of the primary transcript is called
the 5' cap (m7Gppp) (Fig. 4.14). In this
modification, a 7-methylguanylate residue is
attached to the first nucleotide of the pre-mRNA
by a 5'-5' linkage. The 2'-hydroxyl groups of the
ribose residues of the first 2 nucleotides may
also be methylated. The 5' cap is important for
transport of the mRNA to the cytoplasm,
protection against nuclease degradation, and
initiation of translation.
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Mechanism of 5' Capping
The synthesis and structure of the 5' cap that is
added to most vertebrate mRNAs is illustrated in
Fig. 8.3. Caps are added to mRNAs and snRNAs
transcribed by RNA Pol II. Capping enzyme removes
the ? phosphate from the 5 end of the pre-mRNA
and adds the 5'-5'-linked guanylate residue to
the end of the RNA. Capping enzyme associates
with RNA Pol II via its phosphorylated CTD. Other
enzymes add the methyl groups to N7 of the 5'
guanylate and to 2'-hydroxyl groups of the first
one or two nucleotides in the primary transcript.
(S-Ado-Met S-adenosylmethionine).
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Heterogeneous Ribonucleoprotein Particles
Pre-mRNAs and other nuclear RNAs are collectively
known as heterogeneous nuclear RNA (hnRNA). hnRNA
is extensively bound to binding proteins, and
complexes between hnRNA and protein are called
heterogeneous ribonucleoprotein particles
(hnRNP). Binding proteins function by preventing
hnRNA from forming tangled 2 structures that
would otherwise interfere with processing
reactions. As illustrated in Fig. 8.5, many
RNA-binding proteins contain an RNA recognition
motif (RRM) that binds RNA via positively charged
amino acids. The cover figure for Chap. 8 shows
the extensive hnRNP content of highly transcribed
lampbrush chromosomes in newt oocytes.
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Intro to pre-mRNA Splicing
In higher eukaryotes, nearly all genes contain
intron sequences that must be spliced out of
pre-mRNA to form mature mRNA species. One of the
earliest (1977) experiments showing that introns
are present in genes is shown in Fig. 8.6. In
this experiment, a double-stranded DNA fragment
containing most of the adenovirus hexon gene was
denatured, hybridized with the hexon mRNA, and
then viewed under the electron microscope. As
shown in the micrograph and the schematic diagram
on the right, DNA loop sequences corresponding to
introns removed from the mRNA can be seen looping
out from the DNA/RNA hybrid.
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Splice Site Consensus Sequences
Pre-mRNA splice site consensus sequences located
at the extreme ends of introns help direct
splicing reactions (Fig. 8.7). The identities of
these sequences were learned by comparing the
sequences of genes to their spliced mRNA
products. The GU dinucleotide at the 5' splice
site of the intron and the AG dinucleotide at the
3' splice site are highly conserved. Also highly
conserved within the intron is a branch point
sequence containing the branch-point A residue
located 20-50 nucleotides upstream of the 3'
splice site. The remaining central region of the
intron (not shown) generally is unimportant for
splicing.
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Mechanism of the Splicing Reaction
The splicing reaction occurs via 2
transesterification reactions, for which ?Gsum
0 (Fig. 8.8). Thus no energy input is required
for splicing. In the first reaction, the free
2'-hydroxy group of the branch point A residues
attacks and cleaves the phosphodiester linkage at
the 5' splice site. In the second reaction, the
3'-hydroxy group of the 5' exon attacks and
cleaves the phosphodiester linkage at the 3'
splice site. The products of the second reaction
are the spliced mRNA product and the excised
intron, which is called the lariat product. The
lariat intron RNA is degraded.
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Small Nuclear RNAs (snRNAs) and Splicing
The splicing reaction requires 5 snRNAs (U1, U2,
U4, U5, U6) that range from about 100-200
nucleotides in length. Each snRNA forms a complex
with 6-10 proteins which are called small nuclear
ribonucleoprotein particles (snRNPs, pronounced
"snurps"). snRNAs bind to pre-mRNA and each other
within a larger splicing complex known as the
spliceosome (next slide). Interactions between
the U1 snRNA and the 5' splice site, and the U2
snRNA and the branch point sequence are crucial
in selecting where splicing occurs (Fig. 8.9a).
Note that the branch point A residue bulges out
of the U2-pre-mRNA duplex. Sm sites indicate
where snRNP proteins bind to the snRNAs.
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Spliceosome Reactions (I)
Spliceosomes are large supramolecular complexes
consisting of 5 snRNPs and the pre-mRNA. The
assembly of the spliceosome and splicing
reactions begin with a complex between the
pre-mRNA intron, the U1 snRNP bound to the 5'
splice site, and the splicing factors SF1 and
U2AF bound to the branch point A and pyrimidine
tract/3 AG of the intron, respectively (Fig.
8.11, top ). In Step 1, SF1 departs and the U2
snRNP adds to the complex. In Step 2, the
U4/U6/U5 complex adds on forming the fully
assembled spliceosome. In Step 3, the U1 and U4
snRNPs depart, and the pre-mRNA is repositioned
in the complex for splicing. (Continued on the
next slide.)
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Spliceosome Reactions (II)
The transesterification reactions occur in Steps
4 5 via the mechanism shown in Fig. 8.11.
Following splicing, the remaining components of
the complex disassemble. In Step 6, a nuclease
known as debranching enzyme cleaves the 2'-5'
branch point linkage in the lariat. Degradation
of the lariat to individual nucleotides by
3'-to-5' exonucleases then ensues (not shown). It
is estimated that 95 of the polymerized
nucleotides within pre-mRNAs ultimately are
degraded back to single nucleotides following
splicing.
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RNA Pol II CTD Binds Pre-mRNA Processing Factors
Enzymes involved in 5' capping, polyadenylation,
and splicing bind to the long phosphorylated CTD
of RNA Pol II (Fig. 8.12) while it is
transcribing a gene. This ensures that these
factors are delivered to the pre-mRNA sites where
they are needed. Current research indicates that
the binding of these factors to phosphorylated
CTD is required to ensure that the enzyme remains
processive. Thus, transcription will occur only
if these factors are present in sufficient supply.
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Exon Recognition in Long Pre-mRNAs
The average human intron is 3,500 nucleotides in
length, while the average exon is only 150
nucleotides long. The longest introns are 500 kb
in length. As shown in Fig. 8.7, splice site
consensus sequences are fairly degenerate, and in
long introns, multiple potential 3' acceptor
sites occur. Remarkably, exon sequences play an
important role in splice site selection in many
long introns (Fig. 8.13). Exons contain exonic
splicing enhancers (ESEs) that bind SR proteins
which recruit the U2 snRNP U2AF factor to 3'
splice sites, and the U1 snRNP to 5' splice sites
flanking exons. These assemblies are known as
cross-exon recognition complexes. Through this
mechanism, the correct splice junctions within a
long pre-mRNA are accurately selected.
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Self-splicing Introns
Introns in some protozoan rRNA primary
transcripts (group I introns) are self-splicing.
Likewise, introns in some protein, rRNA, and tRNA
transcripts produced from mitochondrial and
chloroplast genes in plants and fungi (group II
introns) also carry out self-splicing reactions.
The study of intron self-splicing lead to the
discovery of catalytic RNA (ribozymes).
Self-splicing introns have strongly conserved
secondary and tertiary structures. Because the
structure of snRNAs in the spliceosome complex
resembles that of group II introns (Fig. 8.14),
it is speculated that the spliceosome machinery
evolved from group II introns. Early in
evolutionary history, when catalytic RNAs may
have been much more prevalent, all introns may
have been excised by self-splicing. The transfer
of splicing reactions to snRNA would have removed
constraints on the structure of introns, and
thereby facilitated exon shuffling and gene
evolution.
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3' Cleavage and Polyadenylation of Pre-mRNAs (I)
3' cleavage and polyadenylation of mRNAs are
tightly coupled processes that are signaled by 2
sequences near the 3' end of pre-mRNA. These
sequences serve as binding sites for 4 nuclear
factors (Fig. 8.15). In Step 1, CPSF (cleavage
and polyadenylation specificity factor), CStF
(cleavage stimulatory factor), and CFI/II
(cleavage factors I II) bind to these sites.
Then in Step 2, PAP (poly(A) polymerase) binds to
the complex. In Step 3, the pre-mRNA is cleaved
just downstream of the AAUAAA poly(A) signal.
(Continues on the next slide)
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3' Cleavage and Polyadenylation of Pre-mRNAs (II)
In Step 4, the CStF, CFI, and CFII factors and
the 3' fragment from the pre-mRNA are released.
The RNA fragment is rapidly degraded. PAP then
begins slow polymerization of the poly(A) tail.
In Step 5, PABII (PABPII, poly(A)-binding protein
II) adds to the complex and stimulates rapid
polymerization of the remainder of the poly(A)
tail (Step 6). PABPII also controls the length of
the poly(A) tail which typically ranges from
200-250 residues. PABPII binds the RNA via a RRM
binding sequence. As discussed in Chap. 4, the
poly(A) tail functions in translation and mRNA
turnover.
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Intro to Control of Alternative Splicing
The most common mechanism by which
post-transcriptional gene control is achieved is
the regulation of alternative splicing. In
humans, 95 of genes are specified by complex
transcription units that produce different
protein isoforms due to alternative splicing.
Alternative splicing is very common in the
nervous system. Alternative splicing is regulated
by splicing repressors and activators that
control splice site selection.
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Regulated Splicing in Drosophila Sexual
Differentiation (I)
One of the best understood systems where
alternative splicing is used to regulate gene
expression is that used in the control of sexual
differentiation in Drosophila embryos. Sexual
differentiation is controlled by the sex-lethal
(sxl), transformer (tra), and double-sex (dsx)
genes (Fig. 8.16). Sxl is a female specific
splicing repressor that is not synthesized in
males. Sxl not only regulates the splicing of its
own primary transcript
but also regulates splicing of the pre-mRNA
encoding the Tra protein in females. The Sxl and
Tra isoforms produced in males are non-functional
due to the presence of stop codons in exons 3 and
2 of these respective genes. These exons are
skipped in alternative splicing of the sxl and
tra transcripts in females.
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Regulated Splicing in Drosophila Sexual
Differentiation (II)
Tra protein is a splicing activator. Its
expression in females results in the synthesis of
the female isoform of Dsx. Its absence in males,
results in the synthesis of the male isoform of
Dsx. The female form of Dsx is a transcriptional
repressor of male differentiation genes. The male
form of Dsx is a transcriptional repressor of
female differentiation genes. Thus alternative
splicing of the sxl gene ultimately determines
sex.
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Mechanism of Action of Tra Protein
The Tra splicing activator regulates splice site
selection in female embryos by binding to a
complex between the Rbp1/Tra2 SR proteins bound
to exonic splicing enhancer sequences in the 4th
exon of the dsx primary transcript (Fig. 8.17).
Binding directs the assembly of the U2 snRNP and
the U2AF protein at the 3' end of the intron
preceding exon 4. Thus, the 4th exon is spliced
into the dsx mRNA in females. This exon is
skipped over in splicing of the male dsx
transcript. The protein domain encoded by the 4th
exon is important in determining the repressor
activity of the Dsx TF.
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Gene Repression by miRNA siRNA
Two post-transcriptional mechanisms for
inhibition of gene expression by small
single-stranded RNAs were discovered relatively
recently in C. elegans. Micro RNAs (miRNAs)
inhibit gene expression by blocking the
translation of complementary mRNAs. Humans
express about 500 miRNAs, and some plants express
over 106 miRNAs. Because a single miRNA can bind
to more than one target mRNA, it is estimated
that about 1/3 of all human genes may be
regulated by miRNAs. Short interfering RNAs
(siRNAs) inhibit gene expression by specifically
targeting a complementary mRNA for degradation.
The mechanism of gene silencing by siRNA is known
as RNA interference (RNAi) and is an important
research tool. RNAi is thought to play a natural
role in protection of cells from RNA viruses and
retrotransposons.
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Structures of miRNA siRNA
Both miRNAs and siRNAs are 21-23-nucleotide
single-stranded RNAs. miRNAs bind to the 3' UTR
regions of complementary mRNAs via imperfect
base-pairing (Fig. 8.25a). Thus they often can
inhibit translation of more than one mRNA. siRNAs
hybridize perfectly without any mismatches to the
coding region of their target mRNAs (Fig. 8.25b).
Thus they typically regulate only a single mRNA
species.
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Mechanism of Action of mi- and siRNAs
miRNAs are produced by the mechanism shown in
Fig. 8.26. RNA Pol II transcribes pri-miRNA
transcripts that are partially double-helical.
The pri-miRNA is processed to a shorter 70 nt
pre-miRNA that is then transported to the
cytoplasm. The pre-miRNA, which folds into a
hairpin structure, is bound by a protein complex
containing the enzyme known as Dicer. Dicer
cleaves the molecule producing a 21-23-nt double
stranded miRNA. Finally, one of the strands is
bound by a protein complex known as the RISC
complex (RNA-induced silencing complex). The
RISC/miRNA complex subsequently binds to the 3
UTR of a target mRNA leading to its sequestration
away from ribosomes. siRNAs are generated in the
Dicer reaction from double-helical RNAs
introduced into cells or produced from cleavage
of viral RNAs. They also are bound by the RISC
complex. However,
RISC/siRNA complexes bind to the coding region of
a target mRNA, and ultimately the target mRNA is
cleaved at a site within the perfect siRNA-mRNA
duplex (Fig. 8.25b, arrow).
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RNA Interference (RNAi)
In RNA interference, short interfering RNAs
(siRNAs, 21 nts) produced from longer dsRNAs
specifically block gene expression by binding to
a target mRNA and triggering its degradation.
dsRNAs can be transcribed in vitro and injected
into an embryo, for example, where processing by
the enzyme known as dicer produces the siRNA
(Fig. 5.45 a b). Alternatively, dsRNA can be
expressed in vivo in response to some signal.
Subsequent processing to siRNA by dicer then
triggers mRNA degradation (Fig. 5.45c).
RNAi-mediated gene inactivation is commonly
applied to silence gene expression in C. elegans,
Drosophila, plants, and even mice. The mechanism
by which siRNAs cause mRNA degradation is covered
in Chap. 8.