Part ? Expression of the Gene

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Part ? Expression of the Gene Chapter 13 RNA
Splicing
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OUTLINE Abstract Contents The Chemistry of RNA
Splicing The Spliceosome Machinery Splicing
Pathways Alternative Splicing Exon Shuffling RNA
Editing mRNA Transport Summary
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Abstract
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The constructive ways of the coding sequence 1.
In the vast majority of cases in bacteria and
their phage the coding sequence is contiguous,
the codon for one amino acid is immediataly
adjacent to the codon for the next amino acid in
the polypeptide. 2. In eukaryotic genes the
coding sequence is periodically interrupted by
stretches of non-coding sequence.
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In eukaryotic genes ?Coding sequences
exons ?Intervening sequences introns mosaics
As a consequence of this alternating pattern of
exons and introns, genes bearing non-coding
interruptions are often said to be in pieces or
split.
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Primary transcript
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Introns 1. Number varies enormously. E.g.
One in the case of most intron-containing yeast
genes and a few human genes. 50 in the case
of the chicken proa2 collagen gene. 363 in
the case of the Titin gene of humans. 2. The
sizes of the exons and introns vary introns are
very often much longer than the exons they
separate. E.g. ?150 nucleotides in exons 800kb
in introns. ?The mammalian gene for the enzyme
dihydrofolate reductase 6 exons correspond to
2kb of mRNA (?) 31kb long of the whole
genes i.e. the coding portion of the gene is
less than 10 of its total length.
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?RNA splicing the process by which introns are
removed from the pre-mRNA. ? Alternative
splicing some pre-mRNAs can be spliced in more
than one way , generating alternative mRNAs. 60
of the human genes are spliced in this manner.
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Topic ? The Chemistry of RNA Splicing
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? Sequences within the RNA Determine Where
Splicing Occurs
The borders between introns and exons are marked
by specific nucleotide sequences within the
pre-mRNAs.
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?5splice site the exon-intron boundary at the
5 end of the intron ?3 splice site the
exon-intron boundary at the 3 end of the
intron ?Branch point site an A close to the 3
end of the intron, which is followed by a
polypyrimidine tract (Py tract).
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? The intron is removed in a Form Called a Lariat
as the Flanking Exons are joined

• Two successive transesterification
• Step 1 The OH of the conserved A at the branch
  site attacks the phosphoryl group of the
  conserved G in the 5 splice site. As a result,
  the 5 exon is released and the 5-end of the
  intron forms a three-way junction structure.
• Step 2 The OH of the 5 exon attacks the
  phosphoryl group at the 3 splice site. As a
  consequence, the 5 and 3 exons are joined and
  the intron is liberated in the shape of a lariat.

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Three-way junction
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The structure of three-way function
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? Exons from different RNA molecules can be fused
by Trans-splicing
Trans-splicing the process in which two exons
carried on different RNA molecules can be spliced
together.
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Trans-splicing
Not a lariat
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Topic ? The Spliceosome Machinery
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RNA splicing is carried out by a large complex
called spliceosome
?The above described splicing of introns from
pre-mRNA are mediated by the spliceosome. ?The
spliceosome comprises about 150 proteins and 5
snRNAs. ?Many functions of the spliceosome are
carried out by its RNA components.
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?The five RNAs (U1, U2, U4, U5, and U6, 100-300
nt) are called small nuclear RNAs (snRNAs). ?The
complexes of snRNA and proteins are called small
nuclear ribonuclear proteins (snRNP, pronounces
snurps). ?The spliceosome is the largest
snRNP, and the exact makeup differs at different
stages of the splicing reaction.
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Three roles of snRNPs in splicing 1. Recognizing
the 5 splice site and the branch site. 2.
Bringing those sites together. 3. Catalyzing (or
helping to catalyze) the RNA cleavage. RNA-RNA,
RNA-protein and protein-protein interactions are
all important during splicing.
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RNA-RNA interactions between different snRNPs,
and between snRNPs and pre-mRNA
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Topic ? The Spliceosome Machinery
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? Assembly, rearrangement, and catalysis within
the spliceosome the splicing pathway
Assembly step 1 1. U1 recognize 5 splice site.
2. One subunit of U2AF binds to Py tract and
the other to the 3 splice site. The former
subunits interacts with BBP and helps it bind to
the branch point. 3. Early (E) complex is formed
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Assembly step 2 1. U2 binds to the branch site,
and then A complex is formed. 2. The
base-pairing between the U2 and the branch site
is such that the branch site A is extruded. This
A residue is available to react with the 5
splice site.
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E complex
A complex
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Assembly step 3 1. U4, U5 and U6 form the
tri-snRNP Particle. 2. With the entry of the
tri-snRNP, the A complex is converted into the B
complex.
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A complex
B complex
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B complex
C complex in which the catalysis has not occurred
yet
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Catalysis Step 1 ?Formation of the C complex
produces the active site, with U2 and U6 RNAs
being brought together. ?Formation of the active
site juxtaposes the 5 splice site of the
pre-mRNA and the branch site, allowing the
branched A residue to attack the 5 splice site
to accomplish the first transesterfication
reaction.
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Catalysis Step 2 U5 snRNP helps to bring the two
exons together, and aids the second
transesterification reaction, in which the 3-OH
of the 5 exon attacks the 3 splice site. Final
Step Release of the mRNA product and the
snRNPs.
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C complex
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splicesome-mediated splicing reactions
E complex
A complex
B complex
C complex
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? Self-splicing introns reveal that RNA can
catalyze RNA splicing
Self-splicing introns the intron itself folds
into a specific conformation within the precursor
RNA and catalyzes the chemistry of its own
release and the exon ligation.
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Adams et al., Nature 2004, Crystal structure of a
self-splicing group I intron with both exons
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?Practical definition for self-splicing introns
the introns that can remove themselves from
pre-RNAs in the test tube in the absence of any
proteins or other RNAs. ?There are two classes
of self-splicing introns, group I and group II
self-splicing introns.
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Three class of RNA Splicing Three class of RNA Splicing Three class of RNA Splicing Three class of RNA Splicing
Class Abundance Mechanism Catalytic Machinery
Nuclear pre-mRNA Very common used for most eukaryotic genes Two transesterification reactions branch site A Major spliceosome
Group II introns Rare some eu-Karyotic genes from organelles and prokaryotes Same as pre-mRNA RNA enzyme encoded by intron (ribozyme)
Group I introns Rare nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genes Two transesterific-ation reactions exogenous G Same as group II introns
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The chemistry of group II intron splicing and RNA
intermediates produced are the same as that of
the nuclear pre-mRNA.
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? Group I introns release a linear intron rather
than a lariat
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?Instead of using a branch point A, group I
introns use a free G to attack the 5 splice
site. ?This G is attached to the 5 end of the
intron. The 3-OH group of the 5 exon attacks
the 5 splice site. ?The two-step
transesterification reactions are the same as
that of splicing of the group II intron and
pre-mRNA introns.
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G instead of A
a linear intron
a Lariat intron
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Group I introns 1. Smaller than group II
introns. 2. Share a conserved secondary
structure, which includes an internal guide
sequence base-pairing with the 5 splice site
sequence in the upstream exon. 3. The tertiary
structure contains a binding pocket that will
accommodate the guanine nucleotide or nucleoside
cofactor.
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The similarity of the structures of group II
introns and U2-U6 snRNA complex formed to
process first transesterification
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? How does spliceosome find the splice sites
reliably
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?Two kinds of splice-site recognition errors
Splice sites can be skipped. ?Pseudo splice
sites could be mistakenly recognized,
particularly the 3 splice site.
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Reasons for the recognition errors (1) The
average exon is 150 nt, and the average intron is
about 3,000 nt long (some introns are near
800,000 nt). It is quite challenging for the
spliceosome to identify the exons within a vast
ocean of the intronic sequences. (2) The splice
site consensus sequence are rather loose. For
example, only AG/G tri-nucleotides is required
for the 3 splice site, and this consensus
sequence occurs every 64 nt theoretically.
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Two ways to enhance the accuracy of the
splice-site selection 1. Because the C-terminal
tail of the RNA polymerase II carries various
splicing proteins, co-transcriptional loading of
these proteins to the newly synthesized RNA
ensures all the splice sites emerging from RNAP
II are readily recognized, thus preventing exon
skipping. 2. There is a mechanism to ensure
that the splice sites close to exons are
recognized preferentially. SR proteins bind to
the ESEs (exonic splicing enhancers) present in
the exons and promote the use of the nearby
splice sites by recruiting the splicing machinery
to those sites
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SR proteins, bound to exonic splicing enhancers
(ESEs), interact with components of splicing
machinery, recruiting them to the nearby splice
sites.
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SR proteins are essential for splicing 1. Ensure
the accuracy and efficacy of constitutive
splicing. 2. Regulate alternative splicing. 3.
There are many varieties of SR proteins. Some are
expressed preferentially in certain cell types
and control splicing in cell-type specific
patterns.
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Topic ? Alternative Splicing
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? Single genes can produce multiple products by
alternative splicing
Many genes in higher eukaryotes encode RNAs that
can be spliced in alternative ways to generate
two or more different mRNAs and, thus, different
protein products.
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Drosophila DSCAM gene can be spliced in 38,000
alternative ways
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There are five different ways to alternatively
splice a pre-mRNA
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? Alternative splicing can be either constitutive
or regulated
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An example of constitutive alternative splicing
Splicing of the SV40 T antigen RNA
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Alternative splicing is regulated by activators
and repressors ?The regulating sequences
exonic (or intronic) splicing enhancers (ESE or
ISE) or silencers (ESS and ISS). The former
enhance and the latter repress splicing. ?Protein
s that regulate splicing bind to these specific
sites for their action.
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SR proteins binding to enhancers act as
activators. (1) One domain is the
RNA-recognition motif (RRM). (2) The other
domain is RS domain rich in arginine and serine.
This domain mediates interactions between the SR
proteins and proteins within the splicing
machinery.
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hnRNPs binds RNA and act as repressors 1. Most
silencers are recognized by hnRNP ( heterogeneous
nuclear ribonucleoprotein) family. 2. These
proteins bind RNA, but lack the RS domains.
Therefore, (1) They cannot recruit the splicing
machinery. (2) they block the use of the
specific splice sites that they bind.
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Regulated alternative splicing
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An example of repressors inhibition of splicing
by hnRNPI
Coats the RNA and makes the exons invisible to
the splicing machinery
Binds at each end of the exon and conceals it
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The outcome of alternative splicing 1.
Producing multiple protein products, called
isoforms. 2. Switching on and off the expression
of a given gene. In this case, one functional
protein is produced by a splicing pattern, and
the non-functional proteins are resulted from
other splicing patterns.
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? A small group of intron are spliced by minor
spliceosome
?This spliceosome works on a minority of exons,
and those have distinct splice-site sequence.
?The chemical pathway is the same as the major
spliceosome.
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U11 and U12 are in places of U1 and U2,
respectively
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Topic ? Exon Shuffling
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Exons are shuffled by recombin-ation to produce
gene encoding new proteins
All eukaryotes have introns, and yet these
elements are rare in bacteria. Two likely
explanations for these situation 1. Introns
early model introns existed in all organisms
but have been lost from bacteria. 2. Intron late
model introns never existed in bacteria but
rather arose later in evolution.
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Why have the introns been retained in eukaryotes?
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1. The need to remove introns, allows for
alternative splicing which can generate multiple
proteins from a single gene. 2. Having the
coding sequence of genes divided into several
exons allows new genes to be created by
reshuffling exon.
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Three observations suggest exon shuffling
actually occur 1. The borders between exons and
introns within a gene often coincide with the
boundaries between domains within the protein
encoded by that gene. 2. Many genes, and
proteins they encode, have apparently arisen
during evolution in part via exon duplication and
divergence. 3. Related exons are sometimes found
in unrelated genes.
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For example DNA-binding protein
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Exons have been reused in genes encoding
different proteins
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Topic ? RNA Editing
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RNA editing is another way of changing the
sequence of an mRNA
I. Site specific deamination 1. A specifically
targeted C residue within mRNA is converted into
U by the deaminase. 2. The process occurs only in
certain tissues or cell types and in a regulated
manner. 3. Adenosine deamination also occurs in
cells. The enzyme ADAR (adenosine deaminase
acting on RNA) convert A into Inosine. Insone can
base-pair with C, and this change can alter the
sequence of the protein. 4. An ion channel
expressed in mammalian brains is the target of
Adenosine deamination.
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The human apolipoprotein gene
Stop code
In liver
In intestines
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II Guide RNA-directed uridine insertion or
deletion 1. This form of RNA editing is found in
the mitochondria of trypanosomes. 2. Multiple Us
are inserted into specific region of mRNAs after
transcription (or US may be deleted).
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3. The addition of Us to the message changes
codons and reading frames, completely altering
the meaning of the message. 4. Us are inserted
into the message by guide RNAs (gRNAs) .
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gRNAs Having three regions anchor directing
the gRNAs to the region of mRNAs it will
edit. editing region determining where the Us
will be inserted. poly-U stretch
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Topic ? mRNA Transport
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Once processed, mRNA is packaged and exported
from the nucleus into the cytoplasm for
translation
All the fully processed mRNAs are transported to
the cytoplasm for translation into proteins.
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?Movement from the nucleus to the cytoplasm is an
active and carefully regulated process. ?The
damaged, misprocessed and liberated introns are
retained in the nucleus and degraded. 1. A
typical mature mRNA carries a collection of
proteins that identifies it as being ready for
transport. 2. Export takes place through the
nuclear pore complex.
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3. Once in the cytoplasm, some proteins are
discarded and are then imported back to the
nucleus for another cycle of mRNA transport. Some
proteins stay on the mRNA to facilitate
translation.
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Summary
1. Why RNA splicing is important? 2. Chemical
reaction determination of the splice sites, the
products, trans-splicing. 3. Spliceosome
splicing pathway and finding the splice
sites. 4. Self-splicing introns and
mechanisms. 5. Alternative splicing and
regulation, alternative spliceosome. 6. Two
different mechanisms of RNA editing. 7. mRNA
transport-a link to translation.