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 most of the eukaryotic genes are mosaic , the
  coding sequences are separated by noncoding
  sequences

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Exons the coding sequences Introns the
intervening sequences( uncoding streches )
(1)The number of introns found within a gene
varies enormously.(2)The sizes of the exons and
introns vary..
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Critical questions

• How are the introns and exons distinguished from
  each other?
• How are introns removed?
• How are exons joined with high precision?

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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
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Sequence 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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• Specific nucleotide sequences within the
  pre-mRNA
• 5splice site the exon-intron boundary at the
  5 end of the intron(GU)
• 3 splice site the exon-intron boundary at the
  3 end of the intron(AG)
• Branch point site an A close to the 3 end of
  the intron, which is followed by a polypyrimidine
  tract (A)

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Figure 13-2 The consensus sequences at the
intron -exon boundary
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The intron is removed in a Form called a Lariat
(??) as the Flanking Exons are joined

• Two successive transesterification reactions

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?
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Three-way junction

• The OH of the conserved A attacks the phosphoryl
  group of the conserved G.
• Exon is released and the 5-end of the intron
  forms a three-way junction structure.

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The structure of three-way function
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?The newly liberated 3OH of the 5exon attacks
the phosphoryl group at the 3splice site. ? The
newly liberated intron form a lariat.
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Overview
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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.
• The Difference
• A Y-shaped branch structure is formed instead
  of a lariat.

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Trans-splicing
Y-shaped branch
?
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THE SPLICESOME MACHINERY
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• RNA Splicing is carried out by a large complex
  called the spliceosome
• The spliceosome comprises about 150 proteins and
  5 snRNAs
• It is believed that the RNA components rather
  than the proteins carry out the functions of the
  spliceosome.

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Components involved snRNAs The five RNAs (U1,
U2, U4, U5, and U6) are called small nuclear RNAs
. snRNPsThe complexes of snRNA and proteins are
called small nuclear ribonuclear proteins. Other
proteins.
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Three roles of snRNPs in splicing

• Recognizing the 5 splice site and the branch
  site.
• Bringing those sites together.
• Catalyzing (or helping to catalyze) the RNA
  cleavage and rejoining reaction.
• RNA-RNA, RNA-protein and protein-protein
  interactions are all important during splicing.

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Some RNA-RNA hybrids formed during the
splicing reaction
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Splicing pathways
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Assembly, rearrangement, and catalysis within the
spliceosome the splicing pathway

• Assembly pathway
• Formation of early (E) complex
• Formation of A complex
• Formation of B complex
• Rearrangement
• Formation of C complex
• Catalysis
• Formation of the active site

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Formation of E complex 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
?E complex
?A complex
U2 binds to the branch site, and then A complex
is formed
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U4, U5 and U6 form the tri- snRNP Particle With
the entry of the tri- snRNP, the A complex is
converted into the B complex.
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B complex
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?U1 leaves the complex, and U6
replaces it at the 5 splice site. ? U4 is
released from the complex, allowing
U6 to interact with U2 This arrangement called
the C complex
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• Formation of the C complex produces the active
  site.
• 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.
• U5 snRNP helps to bring the two exons together,
  and aids the second transesterification reaction
• Release of the mRNA product and the snRNPs

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Overview of splicesome -mediated splicing
reactions
E complex
A complex
Tri- snRNP particle
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.
• Practical definition for self-splicing introns
  the introns that can remove themselves from RNAs
  in the test tube in the absence of any proteins
  or other RNAs.

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There are two classes of self-splicing introns,
group I and group II self-splicing introns in
eukaryotes. They are not enzymes, because they
mediate only one round of RNA prodessing.
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Group ? Introns
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 ? Introns
Group ? Introns release a linear rather a
lariat. They use a free G nucleotide or
nucleoside instead of a branch site A
residue. 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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Three forms of splicing
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How does spliceosome find the splice sites
reliably?

• The active site of the spliceosome is only formed
  on RNA 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
• It seems inevitable that many errors would occur

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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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Errors produced by mistakes in splice-site
selection
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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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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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Alternative Splicing
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Single genes can produce multiple products by
alternative splicing. Exons can be extended ,or
skipped, also, introns can be retained in some
messages.
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Single genes can produce multiple products by
alternative splicing.
Drosophila DSCAM gene can produces 38016
different mRNA and proteins.
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Exons can be extended ,or skipped, also, introns
can be retained in some messages.
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The previous mechanisms described that exons are
not skipped and splice sits not ignored. So, how
does alternative splicing occur so often? The
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.
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There are two forms of alternative splicing
Regulated alternative splicing different
forms are generated at different times, under
different conditions, or in different cell or
tissue types. Constitutive alternative splicing
more than one product is always made from the
transcribed gene. (See picture 13-17 for details)
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Constitutive alternative splicing in the troponin
T antigen (generating two different T- ag )
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Alternative splicing is regulated by activators
and repressors Proteins regulating
splicing bind to specific sites called Exonic (or
intronic) splicing enhancers (ESE or ISE) or
silencers( ESS or ISS) The former enhance and
the latter repress splicing. Proteins that
regulate splicing bind to these specific sites
for their actions.
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Repressors and activators are respond for
alternative splicing, As a result, a particular
exon is chosen in a certain cell.
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• Alternative splicing as a way in which multiple
  protein product can be produced from a single
  gene, these different proteins are called
• isoforms.
• Some gene that encodes only a single functional
  protein also show alternative splicing.
• Why???
• As a way of switching expression of the gene on
  and off (by two pathway)

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Inhibition of splicing by hnRNPI
Coats the entire Exon.
Binds at each end of the exon and conceals it.
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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.
• Using a low- abundance form of splicing.
• The chemical pathway is the same as the major
  spliceosome.
• (Read page 400 for more details)

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Exon Shuffling
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Exons are shuffled by recombination to produce
gene encoding new proteins

• Why introns are rare
• almost nonexistent in bacteria?
• Are they lost or never existent?
• Why does it happen?

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Two Models

• Introns early model introns existed in all
  organisms but have been lost from bacteria.
• (Bacteria become gene- rich responding to
  selective pressure)
• Intron late model introns never existed in
  bacteria but rather arose later in evolution.

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Advantages of the presence of introns First,
the presence of introns, and the need to remove
them, allow for alternative splicing which can
generate multiple proteins from a single
gene. Second, having the coding sequences of
genes divided into several exons allow new genes
to be created by reshuffling exons.
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The evidence of

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

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RNA Editing
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RNA editing is another way of changing the
sequence of an mRNA

• 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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A tissue- specific manner by deamination of a
specific cytodine to generate a uridine.
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Guide RNA-directed uridine insertion or
deletion. ?Multiple Us are inserted into
specific region of mRNAs after transcription (or
US may be deleted). ? This form of RNA editing is
found in the mitochondria of trypanosomes. ? Us
are inserted into the message by so-called guide
RNAs(gRNAs).
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Three regions of gRNA
?
?
?
?
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poly-U stretch
anchor directing the gRNAs to the region of
mRNAs it will edit.
editing region determining exactly where the Us
will be inserted
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RNA-DNA duplex with looped out single strand
regions
?
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RNA ligase
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RNA Transcription
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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. So, how are RNA selected and transport
achieved???
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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. A typical
mature mRNA carries a collection of proteins
that identifies it as being ready for
transport. Export takes place through the
nuclear pore complex.
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Summary

• ?Critical concepts exon, intron, RNA splicing,
  trans-splicing, self-splicing, RNA editing
• ? The chemistry and pathway of RNA splicing
• ? The ways to enhance accuracy
• ? The mechanism of selecting RNA to be
  transported
• ? Understanding ways to increase diversity of
  proteins generated by the same gene
• (1)Alternative splicing (most important of
  all)
• (2)Exon shuffling
• (3)RNA editing (more economic?)

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Wish you enjoy this chapter ? Thank you !
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