RNA splicing

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RNA splicing

• By xiao yi
• ??????
• 200431060010

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In most cases of eukaryotic gene, the coding
sequences is interrupted by noncoding sequences

• The coding sequences are called exons
• The noncoding sequences are called introns

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Before translation, the introns of pre-RNA must
be removed, and this process is called RNA
splicing
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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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The chemistry of RNA splicing

• 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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• the exon-intron boundary at the 5 end of the
  intron is called 5 splicing site
• the exon-intron boundary at the 3 end of the
  intron is called 3 splicing site

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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

• RNA splicing is achieved by two successive
  transesterification reactions

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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.

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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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Exons from different RNA molecules can be fused
by Trans-splicing

• The only difference is that the other
  product---the lariat in the standard
  reaction---is, in trans splicing, is a Y shaped
  branch structure instead

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THE SPLICESOME MACHINERY

• RNA splicing is carried out by a large complex
  called 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 are called small nuclear
  RNAs (snRNAs)
• The complexes of snRNA and proteins are called
  small nuclear ribonuclear proteins (snRNP)

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Three roles of snRNPs in splicing

• They recognize the 5 splicing site and the
  branch site
• They bring those site together as required
• They catalyze (or help catalyze) the RNA cleavage
  and joining reactions

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SPLICING PATHWAYS

• Assembly
• Rearrangement
• catalysis

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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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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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Assembly step 4

• 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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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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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.

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Self-splicing introns reveal that RNA can
catalyze RNA splicing
TABLE 13-1 Three class of RNA Splicing TABLE 13-1 Three class of RNA Splicing TABLE 13-1 Three class of RNA Splicing TABLE 13-1 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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• When we examine the group 1 and group 2
  self-splicing, we find the intron itself folds
  into a specific conformation within the precursor
  RNA and catalyze the chemistry of its own release

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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

• Instead of a branch point A residue, they use a
  free G nucleotide or nucleoside. This G species
  is bound by the RNA and its 3 OH group is
  presented to the 5 splicing site. Here fuses the
  G to the 5 end of the intron. The freed 3 end
  attacks the 3 splicing site. In this case the
  intron is linear rather than a lariat structure

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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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Two ways to enhance the accuracy

• RNA polymerase carries with it various proteins
  with roles in RNA processing
• SR (serine argenine anthentic) proteins bind to
  sequences to called exonic splicing enhancers
  (ESEs) within the exons

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SR proteins are essential for splicing

• They ensure the accuracy and efficiency of
  constitutive splicing.
• They also regulate alternative splicing
• They come in many varieties, some controlled by
  physical signals, others constitutively active.
  Some are expressed preferentially in certain cell
  types and control splicing in cell-type specific
  patterns.

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Alternative splicing

• RNAs can be spliced in alternative ways to
  generate different mRNAs and, thus, different
  protein products

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There are five ways to splice a RNA
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Alternative splicing can be either constitutive
or regulated

• Constitutive alternative splicing more than one
  product is always made from a pre-mRNA
• Regulative alternative splicing different forms
  of mRNA are produced at different time, under
  different conditions, or in different cell or
  tissue types

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Alternative splicing is regulated by activators
and repressors

• Proteins that regulate splicing bins to specific
  sites called exonic (or intronic) splicing
  enhancers (ESE or ISE) or silencers (ESS and ISS)
• The former enhance and the latter repress,
  splicing at nearby splice sites

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• The SR proteins bind RNA using one
  domain----RNA-recognition motif (RRM)
• Each SR protein use RS domain which is rich in
  arginine and serine to mediate interactions
  between the SR protein and proteins within the
  splicing machinery

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• Heterogeneous nuclear ribonucleoprotein (hnRNP)
  bind to RNA but lack the RS domain so cannot
  recruit splicing machinary, instead, they repress
  the use of those sites

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hnRNPI protein repress splicing by two ways

• hnRNPI bind to each end of exon, then interact
  with each other, looping out the exon
• hnRNPI coat the RNA across the whole exon, making
  the exon invisible to the splicing machinary

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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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Exon shuffling

• Exons are shuffled by recombination to produce
  gene encoding new proteins

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• 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?

• 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.

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

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• 3. Related exons are sometimes found in unrelated
  genes.

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RNA EDITING

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

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1. 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.

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• 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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2 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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Guide RNAs

• gRNAs range from 40 to 80 nucleotides in length
  and are encoded by genes distinct from those that
  encode the mRNAs they act on

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• gRNAs have three regions
• 1.Anchor
• 2.Editing region
• 3.poly-U stretch

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mRNA transport

• Once processed, mRNA is packaged and exported
  from the nucleus into the cytoplasm for
  translation

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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.

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• 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.
• 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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Thank you

• By xiao yi
• 200431060010