RNA Splicing

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Chapter 13

• RNA Splicing

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OUTLINE

• Introduction
• The chemistry of RNA Splicing
• The Splicing Machinery
• Splicing Pathway
• Alternative Splicing
• Exon Shuffling
• RNA Editing
• mRNA Transport

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Introduction

• In almost all bacterial and phage genes,the
  opening-reading frame is a single stretch of
  condons with no break.

• But the coding sequence of many eukaryotic genes
  is split into streches of condons interrupted by
  streches of noncoding sequence.

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The coding streches in these split genes are
called exons (for
expressed sequences) The uncoding streches are
called introns (for
intervening sequences)

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Note that
(1)The number of introns found within a gene
varies enormously. (2)The site of the exons and
introns vary.
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The primary transcripts(pre-mRNA) must have their
introns removed before they can be translated
into protein.
Introns are removed from the pre-mRNA by a
process called RNA Splicing. RNA Splicing
converts the pre-mRNA into mature messanger RNA.
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The chemistry of RNA Splicing
Sequence within the RNA determine where splicing
occurs.

• Specific nucleotide sequences within the
  pre-mRNA
• 5 splicing sitethe exon-intron boundary at
  5end of the intron.(GU)
• 3 splicing sitethe exon-intron boundary at
  3end of the intron.(AG)
• Branch point sitewithin the intron and is
  followed by Py tract.(A)

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The intron is removed in a form called a lariat.
Two transesterification reactions
1)The 2OH of the conserved A at the branch site
attack the phosphoryl of the conserved G in the
5splice site
2)The newly liberated 3OH of the 5exon attack
the phosphoryl group at the 3splice site.
The newly liberated intron has the shape of a
lariat.
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A three-way junction is created.
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Note that
1)the chemistry of the process demand no energy
input as it is just a question of shuffling
bonds.
2)two features contribute to the direction of the
splicing reaction 1.An increase in
entropy 2.The excise intron is rapidly
degraded.
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In some cases, exons from different RNA molecules
can be fused by Trans-Splicing.
The Difference A Y-shaped branch structure is
formed instead of a lariat.
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The splicing machinery
RNA Splicing is carried out by a large complex
called the spliceosome.
Components involved
snRNPs proteins not part of the
snRNPs proteins loosely bound to the spliceosome.
It is believed that the RNA components rather
than the proteins carry out the functions of the
spliceosome.
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snRNPs (small nuclear ribonuclear proteins)
snRNP is composed of snRNAs and several
proteins. The five snRNAs U1,U2,U4,U5,U6
The snRNPs have 3 roles in splicing

• They recognise the 5splice site and the branch
  site

• They bring these sites together as required

• They catalyze the RNA cleavage and joinging
  reactions.

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Other proteins
U2AF Recognizes the Py tract/3splice site and
help BBP bind to the branch site. BBP RNA-anneali
ng facters DEAD-box helicase proteins and ------
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Splicing Pathway
The splicing Pathway involves assembly,rearrangeme
nts and catalysis within the spliceosome.
The action of the spliceosome is particularly
interesting in two regards
First,the RNA components have a central role in
recognizing introns and catalyzing their
removal. Second,the complex is very dynamic.
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Early complex
Py tract
A complex
A is extruded and unpaired
Tri-snRNP particle
B complex
U6 replace U1
U6 interacts with U2
C complex
producing the active sit
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The active site of the spliceosome is only formed
on TNA sequences that pass the test of being
recognized by multiple elements during
spliceosome assembly.
Yet,the problem of appropriate splice-site
recognition in the pre-mRNA remains formidable
The splice sites are defined by rather short
sequences with low levels of conservation.It thus
represents a significant challenge for the
splicing machinery to recognize and splice only
at correct sites.
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Splice-site recognition is prone to two kinds of
errors
First, splice sites can be skipped Second,other
sites,close n sequence but not legitimate splice
site,could be mistakenly recognized.
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Two ways in which the accuracy of splice-site
selection can be enhanced
First,it assembles on the sites soon after they
have been synthesized. Second,there are other
proteins---SR proteins---that bind near
legitimate splice sites and help recruit the
splicing machinery to those sites.
So-called SR proteins bind to sequences called
exonic splicing enhancers(ESEs)
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Self-splicing Introns
Apart from the pre-mRNA splicingmediated by
Spliceosome found in eukaryotes,there are
group?and group ? introns.
They can remove themselves from RNAs in the tube
in the absence of any proteins or other RNA
molecules.
But they are not enzymes,because they mediate
only one round of RNA prodessing.
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Group ? Introns
The chemistry of splicing ,and the RNA
intermediates produced,are the same as for
nuclear pre-mRNAs
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Group ? Introns
Group ? Introns release a linear rather a
lariat. They use a free G nucleotide or
nucleoside instead of a branch site A residue.
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Group ? Introns share a conserved secondary
structure

• A binding pocket that will accommodate any
  guanine nucleotide or nucleoside.

• An internal fuide sequence

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Self-and spliceosome-mediated splicing
The similar chemistry seen in self-and
spliceosome-mediated splicing is believed to
reflect an evolutionary relationshipperhaps
ancestral group ?-like self-splicing introns were
the starting point for the evolution of modern
pre-mRNA splicing.
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RNA might carry out catalytic functions before!
The structure of the catalytic region that
performs the first transesterification reaction
is very similar in the group ? introns and the
pre-mRNA/snRNA complex. So, RNA might carry out
catalytic functions before!
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Alternative Splicing
Single genes can produce multiple prodcts by
alternative spling. Exons can be extended ,or
skipped,also,introns can bbe retained in some
messages.
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Since there are mechanisms that ensure variations
of this sort do not take place,how does
alternative splicing occur so often?
The basic answer is that some splice sites are
used only some of the time ,leading to the
production of different versions of the RNA from
different transcriptions of the same gene.
Constitutive alternative splicingmore than one
product is always made from the transcribed
gene. Regulated alternative splicingdifferent
forms are generated at different conditions.
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Examples
1)Alternative splicing in the troponin T gene.
2)Constitutive alternative splicing.
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Alternative spling is regulated by activators and
repressors
Proteins regulating splicing bind to specific
sites called Exonic(or intronic) splicing
enhancers(ESE or ISE) Exonic(or intronic)
splicing silencers(ESS or ISS)
The presence or activity of a given SR protein
can determine whether a particular splice site is
used in a particular cell type,or, at a
particular stage of development,using its RS
domain.
An Examplethe Drosophila Half-pint protein
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Most silencers are recognized by members of the
heterogeneous nuclear ribonucleoprotein(hnRNP)
family which bind RNA but lack the RS domain ,by
blocking specific splice sites.(acting as
repressors)
Examples 1)hnRNPA1 2)the hnRNPI proein
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A small group of introns are spliced by an
alternative spliceosome compose of a different
set of snRNPs
In some organisms,certain pre-mRNAs are spliced
by a low-abundance form of spliceosome----the
AT-AC spliceosome.
This rare form contains some components common to
the major spliceosome but other unique components
too. These unique components recognise different
branch site and splice site squences ,but they
both use the same chemical pathway.
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A small group of introns are spliced by an
alternative spliceosome compose of a different
set of snRNPs
In some organisms,certain pre-mRNAs are spliced
by a low-abundance form of spliceosome----the
AT-AC spliceosome.
This rare form contains some components common to
the major spliceosome but other unique components
too. These unique components recognise different
branch site and splice site squences ,but they
both use the same chemical pathway.
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It is suggested that the AT-AC introns evolved
from the group ? introns and gives rise to the
major pre-mRNA introns.
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It is suggested that the AT-AC introns evolved
from the group ? introns and gives rise to the
major pre-mRNA introns.
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Exon Shuffling
Having the coding sequence of genes divided into
several exons allows new genes to be be created
by reshuffling exons.

• The borders between exons and introns within a
  given gene often coincide with the boundaries
  between domains within the protein encoded by
  that gene.

• Many genes,and the proteins they encode,have
  apparently arisen during evolution in part via
  exon dupllication and divergence.

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• Related genes are sometimes found in otherwise
  unrelated genes.

An example LDL receptor gene
We can see from all above that it has been
relatively easy ,through evolution,to generate
new proteins by shuffling existing exons between
genes.
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RNA Editing
RNA Editing is another mechanism that allows an
RNA to be changed after transcription so as to
encode a different protein from that encoded by
the gene.

• There are two mechanisms that mediate editing
• Site-specific deamination
• Guide RNA-directed uridine insertion or deletion

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Site-specific deamination
In one form of site-specific deamination,a
specifically targeted cyosine residue within mRNA
is converted into uridine by deamination.
An Example Mammalian apolipoprotein-B gene
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Other forms of deaminations include adenosine
deamination which is carried out by the enzyme
ADAR(adenosine deaminase acting on RNA).
ADAR
adenosine
Inosine
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Guide RNA-directed uridine insertion or deletion
direct gRNA to the region it will edit
determine where Us are inserted
Poly-U streches
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The editing reaction
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mRNA transport
RNA export from the nucleus is an active process.
Only certain RNAs are selected for transport.
The RNA has the correct collection of proteins
bound to it Protein that recognise exonexon
boundaries,indicating an mature mRNA
appropriately splicedsuch as SR protein. Protein
that bind introns indicate an RNA to be retained .
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Export takes placethrough a special structure in
the nuclear membrame calle the nuclear pore
complex.
Small molecular unaided Larger molecular
require active transport which is supplied by
hydrolysis of GTP
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A three-way junction
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Some RNA-RNA hybrids formed during the splicing
reaction
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Pre-mRNA
Group 2
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Errors produced by mistakes in splice-site
selection
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SR protein recruit spliceosome components to the
5and 3splice sites
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LDL receptor gene is made up of exons shuffled
between other genes
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RNA editing deamination
A stop codon is introduced
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Transport of mRNA out of the nucleus
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Structure of an antibody molecule