RNA Splicing

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CHAPTER 13

• RNA Splicing

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• 2 conceptions
• What is intron ?
• And what is exon ?

Recall the structuer of DNA
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• In the vast majority of cases in bacteria and
  their phage the codon for one amino acid is
  immediately adjacent to the codon for the next
  amino acidin the polypeptide chain.
  
• But in many eukaryotic gene consisting of
  blocks of coding sequences separated by blocks
  of non-coding sequences.
• The coding sequences are called extron
• The non-coding sequences are called intron

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Question

• Why do eukaryotic gene contain so much non-coding
  sequences ? Is that a waste?

Try to find out the answer after finish the study
of this chapter!
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What is RNA splicing?

• The primary transcript for a typical eukaryotic
  gene contained introns as well as exon calls pre-
• mRNA
• The process which removes the introns from the
  pre-mRNA is called RNA Splicing

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Typical eukaryotic gene
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• Objectives
• Understand the mechanism of splicing by
  splicesome.
• Understand how the splicesome direct the splicing.

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Topic 1
The chemistry of RNA splicing
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Sequences within the RNA determing where splicing
occurs

• 5 splice site (contain GU)
• 3 splice site (contain AG)
• Branch point site (contain A)

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How the intron is removed?

• Two transesterification reactions
• 1.The 2OH of the conserved A at the branch site
  attack the phosphoryl group of the conserved G in
  the 5 splice site so the freed 5 end is joined
  to the A.
• 2. The 5exon attacks the phosphoryl group at the
  3splice site so the 5 is joined to the 3.

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Three way junction
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• Notice that the newly liberated intron has the
  shape of a lariat

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The structure of the three-way junction formed
during the splicing reaction
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What ensures that splicing only goes forward

• First ,the forward reaction involves an increase
  in entropy .
• Second ,the excised intron is rapidly degraded
  after its removal .

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Exon from different RNA molecules can be fused by
thans-splicing
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Topic 2The spliceosome machinery
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Splicing is carried out by spliceosome

• The spliceosome complex contain
• 1.150 proteins
• 2.5 small nuclear RNAs(U1 U2 U4 U5 U6)
  complexed with small nuclear ribonuclear
  protein(snRNPs)

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snRNPs
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Three roles of snRNPs in splicing

• Recognize the 5splice site and the branch site
• Bring those sites together as required
• Catalyzed the RNA cleavages and joining reaction.
• To perform these functions, RNA-RNA,RNA-protein,
  and protein-protein interactions are all
  important

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RNA-RNA hybrids formed during the splicing
reaction
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Topic 3splicing pathways
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Assembly, rearrangements, and catalysis within
the spliceosome

• Assembly
• 1.U1 snRNP recognize the 5 splice site
• 2.One subunit of U2AF binds to the Py and the
  other to the 3splice site
• 3.The BBP bind to the branch site
• This arrangement of RNA and protein are called
  early (E) complex.

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E complex
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• 4.U2 snRNPs then binds to the branch site ,aided
  by U2AF and displacing BBP.
• This arrangement is called A comlex

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A complex
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• 5.The U4 and U6 snRNPs, alone with the U5 snRNPs,
  joined the complex. The three snRNPs are called
  the tri-snRNP particle.
• The A complex is converted into B complex

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B complex
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• 6.U1 leaves the complex, and U6 replaces it at
  the 5 splice site .This the base-pairing between
  the U1 and the pre-mRNA be broken, allowing the
  U6 to anneal with the same region.
• This steps complete the assembly pathway .

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Assembly is completed
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• Rearrangement
• 1.U4 is released from the complex ,allowing U6
  to interact with U2
• This arrangement is called C complex ,producing
  the active site, it also ensure the substrate RNA
  is the active site primarily formed of RNA

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C complex
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• Catalysis
• 1.The formation of the active site juxtaposes
  the 5 splice site of the pre-mRNA and the branch
  site.
• 2.The second reaction ,the U5 snRNPs helps to
  bring the two exons together.
• 3.Final step involves release of the mRNA
  product and the snRNPs.

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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.
  Strictly speaking, they are not enzymes for they
  mediate only one round of RNA processing.

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Self-splicing intron
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The self-splicing introns are grouped into two
classes on the basis of their structure and
splicing mechanism

• Group I
• Group II
• In the case of group II introns, the chemistry of
  splicing, and the RNA intermediates produced ,are
  the same as for nuclear pre-mRNA.

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Group I introns forms a complex structure
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• The secondary structure folds into a tertiary
  stureture
• The Guanine-binding pocket
• Internal guide sequence.

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Guanine-binding pocket can bind any G-containing
ribonucleotide
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The path way of group I introns splicing

• 1.It use a free G nucleotide instead of a branch
  point A residue. The same type of transesterific-
• ation reaction that leads to the lariat
  formation in the earlier example fuses the G to
  the 5end of the intron
• 2.The freed 3end of the exon attack the
  5splice site and fuse the two exon and release
  the intron

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Group I introns release a linear intron rather
than a lariat
Linear
?
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Group II introns

• The chemistry is essentially the same as in the
  spliceosome case, with a highly reactive Adenine
  within the intron initiating splicing, and
  leading to the formation of a lariat product.

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Proposed folding of the RNA catalytic region for
splicing of group II introns and pre-mRNA
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How does the spliceosome find the splice site
reliably

• The two kinds of errors in splice-site
  recognition
• 1.The splice sites can be skipped
• 2.Other site, close in sequence but not
  legitimate splice sites, could be mistakenly
  recognized.

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Exon skipping
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Pseudo splice-site selection
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Why does the problem of appropriate splice site
recognition remain formidable?

• The average extron is only some 150 nucleotides
  long, whereas the average intron is approximately
  3,000 nucleotides long. Thus ,the extron must be
  identified within a vast ocean of intronic
  sequences ,so it seems inevitable that many
  errors would occur.

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The accuracy of splice-site selection can be
enhanced in two ways

• First ,while transcribing , RNA polymerase II
  carries with it various proteins. Once in place,
  the 5 splice site components are poised to
  interact with those that binds to the next 3
  splice to be synthesized. This co-transcriptional
  loading process greatly diminishes the likelihood
  of exon skipping.

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• Secondary , the so-called SR (Serine Argenine
  rich) proteins bind to sequences called exonic
  splicing enhancers (ESEs) within the exons. Then
  they interact with components of the splicing
  machinery, recruiting them to the nearby splice
  sites than to the incorrect sites not close to
  exons.

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SR proteins are essential for splicing

• They are not only ensure the accuracy and
  efficiency of constitutive splicing but also
  regulate alternative splicing . They come in many
  varieties, some controlled by physiological
  signals, others constitutively active.

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SR proteins recruit spliceosome components to the
5 and 3 splice sites
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Topic 4Alternative splicing
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• Single genes can produced multiple products by
  alternative splicing
• Many genes in higher eukaryote encode RNAs
  that can be spliced in alternative ways to
  generate two or more different mRNAs and in some
  cases the number of potential alternatives is
  breathtakinghundreds or even thousands.

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How does alternative splicing occur so often?

• Some splice site are used only some of the time,
  leading to the production of different versions
  of RNA from different transcripts of the same
  gene.

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Alternative splicing
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Ways to splicing an RNA
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Two kinds of alternative splicing

• Constitutive
• In this case, more than one product is
  always made from the transcribed gene.
• Regulated
• Different forms are generated at different
  times, under different conditions, or in
  different cell or tissue types.

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Constitutive alternative splicing of the SV40 T
antigen RNA
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Alternative splicing is regulated by activators
and repressors

• Specific sites related in regulating splicing are
  called exonic (or intronic) splicing enhancer
  (ESEs or ISE) or silencers (ESS and ISS). The
  former enhance, and the latter repress, splicing
  at nearby splice site.

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SR proteins bind RNA performing as an activator

• The SR proteins use one domainfor example, the
  well-characterized RNA-recognition motif (RRM),
  to bind to RNA/
• They use another domain which is rich in arginine
  and serine to find the C-terminal end of the
  protein, mediates interactions between the SR
  protein and proteins within the splicing
  machinery.

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hn RNP protein binds to RNA acting as repressors

• They lack RS domain so cannot recruit the
  splicing machinery .
• They block specific splice sites so to repress
  the use of those sites.

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The structure of hnRNP
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Regulated alternative splicing
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The two way alternative splicing can be used

• This process can produce multiple protein from a
  single gene which are called isoform.
• They can be used as an on/off switch by
  regulating the use of an intron

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A small group of introns are spliced by an
alternative splieosome composed of a different
set of snRNPs

• Some pre-mRNAs of higher eukaryotes are spliced
  by a low-abundance form of spliceosome. The rare
  form contains some components common to the major
  spliceosome but other unique as well.
• The minor spliceosome recognize rarely occurring
  introns having consequences distinct from the
  sequences of most pre-mRNA introns.

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The minor spliceosome works on a minority of exon
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Topic 5exon shuffling
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Exon are shuffled by recombination to produce
genes encoding new proteins
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Two models for intron existence

• Introns early model
• Introns existed in all organisms but have
  lost from bacteria.
• Introns late model
• Introns were inserted into genes that
  previously had no introns.

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Why have the introns been retained in eukaryotes,
and in particular, in the extensive form seen in
multicellular eukaryotes?
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• First ,the borders between exons and introns
  within a given gene often coincide with the
  boundaries between domains within the protein
  encoded by that gene.

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Exons encode protein domain
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• Second, many genes ,and the proteins they encode,
  have apparently arisen during evolution in part
  via exon duplication and divergence.
• Third, related exons are sometimes found in
  otherwise unrelated genes. That is, there is
  evidence that exons really have been reused in
  genes encoding different proteins.

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Gene made up of parts of other genes
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Topic 6RNA editing
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RNA editing is another way of alternating the
sequence of an mRNA

• It can change the sequence of an RNA after it has
  been transcribed. There are two mechanisms that
  mediate editing
• 1.site-specific deamination
• 2.guide RNA-directed uridine insertion or
  deletion

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deamination

• In one form, a specifically targeted cytosine
  residue within mRNA is converted into uridine.
• Other examples include adenosine deamination.
  This reaction carried out by the enzyme ADAR
  (adenosine deaminase acting on RNA)

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Deamination
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RNA editing from human apolipoprotein
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RNA-directed uridine insertion or deletion

• This very different form is found in the RNA
  transcripts in the mitochondria of trypanosomes.
  In this case, multiple Us are inserted into
  specific regions of mRNAs after transcription

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How are these additional bases inserted?

• Us are inserted into the message by so-called
  guide RNAs ( gRNA)

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Each gRNAs range is divided into three regions

• The first, at the 5 end, is called anchor and
  directs the gRNA to the region of the mRNA it
  will edit.
• The second determines exactly where the Us will
  be inserted within the edited sequences.
• The third, at the 3 end, is a poly-U stretch.

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Topic 7mRNA transport
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• Once processed, mRNA is packaged and exported
  from the nucleus into cytoplasm for translation.
• The fully processed mRNA represent only a small
  proportion of the RNA found in the nucleus for
  many other would be detrimental to the cell if
  exported.

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How are RNA selection and transport achieved?

• Once the RNA molecule starts to be transcribed,
  it becomes associated with proteins of various
  sorts.
• It is the set of proteins, not any individual
  kind of protein, that makes RNAs for either
  export or retention in the nucleus.

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• Exports takes place through a special structure
  in the nuclear membrane called nuclear pore
  complex

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End enjoying your study!