RNA Splicing

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Chapter 13?????? ?? 200431060017

• RNA Splicing

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OUTLINE

• The Chemistry of RNA Splicing
• The Spliceosome Machinery
• Splicing Pathways (important)
• Alternative Splicing (important)
• Exon Shuffling
• RNA Editing
• mRNA Transport

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Key Words

• Exon coding sequences
• Intron intervening sequences
• Pre-mRNA the primary transcript of DNA
• RNA splicing process that intron are
  moved from the pre-RNA
• Spliceosome a huge molecular machine catalyse
  RNA splicing
• Alternative splicing some pre-mRNAs can be
  spliced in more than one way,and produce
  alternative mRNAs.

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Topic 1 The Chemistry of RNA Splicing

• Sequences within the RNA determine where
  splicing occurs
• The intron is moved in a form called Lariat as
  the flanking exons are joined
• Exons from different RNA molecules can be fused
  by trans-splicing

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one Sequences within the RNA determine
where splicing occurs

• The borders between
• introns and exons are
• marked by specific
• nucleotide sequences
• ( GUAG Law )
• within the pre-mRNAs.
• Py-tract rich in pyrimidine

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Notice

• GU in 5 splicing site, AG in 3 splicing site
  and A in branch point site are the most conserved
  sequences, and they are all in the intron.
• These sequences are important for the distinguish
  between intron and exon ,remove of intron
  ,linkage of exons and delineate where splicing
  will occur.

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Two The intron is moved in a form called Lariat
as the flanking exons are joined

• RNA splicing consists of two successive
  transesterification reaction.

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• Reaction 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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• Reaction 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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The structure of three-way junction
In addition to the 5 and 3 backbone linkages,a
third phosphodiester extends from the 2OH of
that A to create a three-way junction.
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Reaction 2

• Ensure the splicing only goes forward
• One an increase in entropy.
• Two the excised intron lariat is rapidly
  degraded after its removal.

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Notice

• In the two reactions, there is no net gain in the
  number of chemical bonds. So no energy is
  demanded by the process.
• But, we see a large amount of ATP is consumed
  during the splicing reaction. Why? This energy is
  required to properly assemble and operate the
  splicing machinery, not for the chemistry.

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Three 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.
• This process is rare .But all mRNAs in
  the nematode worm undergo trans-splicing.

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Topic 2 The Spliceosome Machinery

• RNA splicing is carried out by a large complex
  called spliceosome
• Spliceosome is a complex that mediates splicing
  of introns from pre-mRNA.
• And it 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 (snRNPs).
• The spliceosome is the largest snRNP, and the
  exact makeup differs at different stages of the
  splicing reaction.
• Different snRNPs come and go at different
  times,each carrying out particular functions in
  the 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
Branch-point binding protein
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Topic 3 Splicing Pathways

• Assembly, rearrangement, and catalysis within the
  spliceosome the splicing pathway
• Self-splicing introns reveal that RNA can
  catalyze RNA splicing
• Group I introns release a linear intron rather
  than a lariat
• How does spliceosome find the splice sites
  reliably

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One Assembly, rearrangement, and catalysis
within the spliceosome the splicing pathway

• Steps of splicing pathway
• Assembly step one
• 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 two
• 1.With the help of U2AF, U2 binds to the branch
  site replacing of BBP, 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 three
• 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 four
• 1 , U1 leaves the complex, and U6 replaces it
  at the 5 splice site.
• 2 , U4 is released from the complex, allowing
  U6 to interact with U2 .This arrangement called
  the C complex.

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Catalysis

• Step one
• 1 , Formation of the C complex produces the
  active site, with U2 and U6 RNAs being brought
  together

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• 2 , 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 two
• 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.
• Step three
• Release of the mRNA product and the
  snRNPs

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Two Self-splicing introns reveal that RNA can
catalyze RNA splicing

• Self-splicing introns the intron itself folds
  into a specific conformation within the pre-mRNA
  and catalyzes the chemistry of its own release
  (recall RNA enzyme ) and the exon ligation.
• 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.
• Two classes of self-splicing introns, group I and
  group II self-splicing introns.

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Three classes of RNA splicing
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Notice 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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Three Group I introns release a linear intron
rather than a lariat

• 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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Three classes of RNA splicing
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Group 1 intron structure
Share a conserved secondary structure, which
includes an internal guide sequence
base-pairing with the 5 splice site sequence in
the upstream exon.

• A complex secondary structure

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Group 1 intron structure
The tertiary structure contains a binding
pocket that will accommodate the guanine
nucleotide or nucleoside cofactor
Bind any G-containing ribonucleotide.
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Steps of group 1 intron splicing

• free guanosine binds in the guanine-binding
  pocket
• 3OH of guanosine attacks phosphate at 5end of
  intron
• 3OH of exon 1 attack 5phosphate of exon 2
• Intron is released

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• Internal guide sequence base pairs with 5end of
  intron
• 3guanosine binds in guanine-binding pocket
• 3OH of bound guanosine attacks phosphate to the
  right of IGS
• Bond between 3G and 5 phosphate hydrolyzes,
  leaving inactive intron

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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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Four How does spliceosome find the splice sites
reliably

• splice-site recognition is prone to 2 kinds of
  errors
• ?Splice sites can be skipped.
• ?some site close in sequence but not legitimate
  splice site ,could be mistakenly recognized.
• For example ,Pseudo splice sites could be
  mistakenly recognized and pair with component at
  5site, particularly the 3 splice site.

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Error produced by mistakes in splice-site
selection
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Two ways to enhance the accuracy of the
splice-site selection

• 1 , the C-terminal tail of the RNA polymerase II
  carries various splicing proteins.
• when a 5splice site is encountered in the
  newly synthesized RNA, those components are
  transferred from the Pol II C-terminal tail onto
  the RNA .

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• Once in place ,the 5splice site components are
  poised to interact with those that bind to the
  next 3splice siteto be synthesized. Thus ,the
  correct 3splice site can be recognized before
  any competing sites further downstream have been
  transcribed.
• This co-transcriptional loading process greatly
  diminishes the likelihood of exon skipping.

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• 2 , a second mechanism guides against the use of
  incorrect sites by ensuring that splice sites
  close to exons are recognized preferentially.
• SR proteins bind to sequences called exonic
  splicing enhancers (ESEs) within the exons.
• SR bound to ESE interacts with components of the
  splicing machinery ,recruiting them to the nearby
  splicing sites. In this way , the machinery binds
  more efficiently to the nearby sites than to
  incorrect sites not close to exons.

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SR protein recruits spliceosome components to the
5and 3 splice sites
SR proteins bind to ESEs ,recruit U2AF and
U1snRNP to the downstream 5and upstream 3splice
sites respectively. This initiates the assembly
of the splice machinery on the correct sites and
splice can proceed as outlined earlier.
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SR proteins function

• Ensure the accuracy and efficiency of
  constitutive splicing.
• Regulate alternative splicing.
• They come in many varieties ,controlled by
  physiological signals and constitutively active.
  And some express preferential in certain cell
  types and control splicing in cell-type specific
  patterns.

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Topic 4 Alternative splicing

• Single gene can produce multiple products by
  alternative splicing
• Alternative splicing is regulated by activators
  and repressors
• A small group of introns are spliced by an
  spliceosome composed of a different set of snRNPs

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One Single gene can produce multiple products
by alternative splicing

• Alternative splicing
• many genes in higher eukaryotes encode
  RNAs that can be spliced in alternative ways to
  generate two or more different mRNAs and
  ,different protein products.

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• As we know ,exons are not skipped and splice
  sites not ignored, why does alternative splicing
  occur so often ?
• Answer some splice sites are used only some of
  the time ,leading to the production of different
  versions of the RNA from different transcripts of
  the same gene.

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Five ways to splice an RNA
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• Alternative splicing can be either constitutive
  or regulated.
• Constitutive more than one product from the
  same gene
• Regulated different products are generated at
  different times, under different conditions , or
  in different cell or tissue type.

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Constitutive alternative splicing
high level SF2/ASF
Splicing of the monkey SV40 T antigen RNA.
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Two Alternative splicing is regulated by
activators and repressors

• Proteins that regulate splicing bind to specific
  sites called exonic (or intronic ) splicing
  enhancers or silencers.
• Protein specific sequence can guide elements of
  spliceosome to exons.

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• SR protein family is large and diverse, has
  specific roles in regulated alternative splicing
  by directing the splicing machinery to different
  splice sites under different conditions.
• The presence or activity of a given SR can
  determine whether a particular splice site is
  used in particular cell type ,or at a particular
  stage of development.

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Regulated alternative splicing
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• RNA-recognition motif RRM
  
• RS domain rich in Arg and

Binding RNA
RS protein
Ser. Found at C-terminal end of protein ,mediates
interactions between SR and the pro. within the
splicing machinery, recruiting them to a nearby
splice site.
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An example of repressors inhibition of splicing
by hnRNPI

• Repressor are hnRNPs . They can bind RNAs
  sliencer and repress the use of those sites.
• They dont have RS domain ,so cant recruit
  spliceosome. By binding sites, this blocking can
  repress splice.

Coat exon entirely to avoid splicing
Conceal exon as a loop
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• The second way alternative splicing can be used
  as an on/off switch is by regulating the use of
  an intron, which, when retained in the mRNA ,the
  mRNA will be never transported out of nucleous
  and be never translated.

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Three A small group of introns are spliced by
an spliceosome composed of a different set of
snRNPs

• Higher eukaryotes use the major splicing
  machinery we have discussed before, and some
  pre-mRNAs are spliced by a low-aboundance form of
  spliceosome.
• The rare form contains some components common to
  major spliceosome but other unique. U11 and U12
  have the same roles in the splicing reaction as
  U1 and U2, but they recognize distinct sequences.
  U4 and U6 share the same names but their snRNPs
  are distinct. U5 is identical.

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• The minor spliceosome recognizes rarely occurring
  introns having consensus sequences distinct from
  the sequences of most pre-mRNA. This intron
  contains 5 AT and 3AC ( AT-AC low ), so the
  new form is known as the AT-ACspliceosome.
• Later ,we discover that it also recognizes GT-AG.

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• The ability of the snRNAs and splice site
  sequences to base-pair is conserved, not any
  sequences within either.
• Although the different splice sites and branch
  site ,the two forms of spliceosome share the same
  chemical pathway to remove introns.

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Topic 5 Exon Shuffling

• Exons are shuffled by recombination to produce
  genes encoding new proteins.
• All eukaryotes have introns which rare in the
  bacteria. Two model
• intron early model all organism have
  introns, and bacteria lost them.
• intron late model introns were inserted
  into genes by a transposon-like machanism

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• Why introns can exist in eukaryotes??
• First the presence of introns and the need to
  remove them ,allow for alternative splicing which
  can generate multiple proteins from a gene.
• Second having the coding sequence of genes
  divided into several exons allows new gene to be
  created by reshuffling exons.

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Three observations

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

?Exons have been reused in genes encoding
different proteins
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Topic 6 RNA editing

• RNA editing is another way of changing the
  sequence of an mRNA.
• Two mechanisms mediate editing
• Site-specific deamination
• Guide RNA-directed uridine insertion or
  deletion

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Site-specific deamination

• The process occurs only in certain tissues or
  cell types and in a regulated manner.

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RNA editing by deamination of human
apolipoprotein gene
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• 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.
• ? An ion channel expressed in mammalian brains is
  the target of Adenosine deamination.

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Guide RNA-directed uridine insertion or deletion.
directing the gRNAs to the region of mRNAs it
will edit
determining where the Us will be inserted
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Topic 7 mRNA transport

• Once processed (capped, intron-free and
  polyadenylated ), mRNA is packaged and exported
  from the nucleus into the cytoplasm for
  translation.

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How are RNA selection and transport achieved?

• RNA associates with proteins as soon as
  transcription Initially proteins involved in
  capping, then splicing factors, and finally the
  proteins that mediate polyadenylation .
• Some proteins are replaced at various steps
  during the processing path ,but some such as SR
  are not, moreover ,additional proteins join.

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• As a result, a typical mature mRNA carries a
  collection of proteins that identifies it as
  being mRNA destined for transport.
• Others not only lack the particular signature
  collection required for transport, also have
  their collection that block transport.

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Examples

• The excised introns carry hnRNPs which mark such
  an RNA for nuclear retention and destruction.
• Mature mRNA carry residual SR proteins , even
  another group of proteins binding specifically to
  exon-exon junctions.
• Conclusion the set of proteins, not any
  individual kind of protein, marks RNAs for either
  export or retention in the nucleus.

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• Nuclear pore complex a specific structure in
  the nuclear membrane. Small molecules under 50Kd
  can pass through it unaided. And large
  molecules and complexes require active transport.
  
• mRNAs and their associated proteins can actively
  transport through NPC

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Transport of mRNAs out of the nucleus
GTPase
NPC
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• The end !!