RNA Splicing

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CHAPTER 13

• RNA Splicing

Made by Ren Jun (?? ???? 200431060118)
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RNA splicing is the process of excising the
sequences in RNA that correspond to introns, so
that the sequences corresponding to exons are
connected into a continuous mRNA.
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RNA processing events include capping of the 5
end of the RNA splicing and polyadenylation of
the 3 end of the RNA.
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The coding sequence of a gene is contiguous in
the vast majority of cases in bacteria and their
phage.
However, many eukaryotic genes are mosaics,
consisting of blocks of coding sequences (exons)
separated from each other by blocks of noncoding
sequences (introns).
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• The primary transcript for a typical eukaryotic
  gene contains introns as well as exons.
• Introns must be removed before translation.
• The process that introns are removed from the
  pre-mRNA is called RNA splicing, occurring with
  great precision.
• Some pre-mRNAs can be spliced in more than one
  way, generating alternative mRNAs. This is called
  alternative splicing.

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OUTLINE

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

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TOPIC 1

• The Chemistry of RNA Splicing

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Sequences within the RNA Determine Where Splicing
Occurs

• 5 splice site
• 3 splice site
• branch point site

Splice sites are the sequences immediately
surrounding the exon-intron boundaries.
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GT-AG rule describes the presence of these
constant dinucleotides at the first two and last
two positions of introns of nuclear genes.
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The Intron Is Removed in a Form Called a Lariat
as the Flanking Exons Are Joined

• two successive transesterification reactions
• The 2OH of the conserved A at the branch site
  attack the phosphoryl group of the conserved G in
  the 5 splice site
• three-way junction
• The newly liberated 3OH of the 5 exon attacks
  the phosphoryl group at the 3 splice site
  intron lariat

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the first
three-way junction
the second
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Features of pre-mRNA splicing

• It takes place in the nucleus, before the mature
  mRNA can be exported to the cytoplasm.
• It requires a set of specific sequences.
• It takes places in a two-step reaction, snRNPs
  are involved.
• The final step is methylation on the N6 position
  of A residues particularly in the sequence
  5-RRACX-3.

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Exons from Different RNA Molecules Can Be Fused
by Trans-Splicing

• In alternative splicing, exons can be skipped
• Trans-splicing gets two exons carried on
  different RNA molecules spliced together forming
  a Y-shaped branch structure (not a lariat!)

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TOPIC 2

• The Spliceosome Machinery

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The transesterification reactions are mediated by
the spliceosome

• comprise about 150 proteins and 5 RNAs
• The five RNAs U1,U2,U4,U5,U6 small
  nuclear RNAs (snRNAs)
• The RNA-protein complexes small nuclear
  ribonuclear proteins (snRNPs)

rich in uracil
Many of the functions of the spliceosome are
carried out by its RNA components
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The snRNPs have three roles in splicing

• recognize the 5 splice site and the branch site
• bring those sites together as required
• catalyze (or help to catalyze) the RNA cleavage
  and joining reactions
• Interactions are important to perform these
  functions!

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TOPIC 3

• Splicing Pathways

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Assembly, Rearrangement, and Catalysis Within the
Spliceosome

• The 5 splice site is recognized by the U1 snRNP.

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• One subunit of U2AF binds to the Py tract and
  helps BBP (branch-point binding protein) bind to
  the branch site, and the other to the 3 splice
  site.
• The Early (E) complex is formed.

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• U2 snRNP binds to the branch site, aided by U2AF
  and displacing BBP. A complex

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• The U4 and U6 snRNPs, along with the U5 snRNP
  (the tri-snRNP particle), join the A complex and
  convert it into the B complex.

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• U6 replaces U1 at the 5 splice site.
• The above steps complete the assembly pathway.

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• U4 is released from the complex, allowing U6 to
  interact with U2.
• This arrangement forms the C complex and produces
  the active site.
• The 5 splice site and the branch site are
  juxtaposed. the first
  transesterification reaction
• The U5 snRNP helps to bring the two exons
  together, triggering the second reaction.
• Finally, the mRNA product and the snRNPs are
  released.

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U6-U4 pairing is incompatible with U6-U2 pairing.
When U6 joins the spliceosome it is paired with
U4. Release of U4 allows a conformational change
in U6 one part of the released sequence forms a
hairpin (dark grey), and the other part (black)
pairs with U2. Because an adjacent region of U2
is already paired with the branch site, this
brings U6 into juxtaposition with the branch.
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E complex
A complex
assembly
B complex
C complex
catalysis
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Two points should be noticed

• Some of the components of the splicing machinery
  do not arrive or leave precisely when described
  above. It is also not possible to be sure of the
  order of some changes.
• The tragedy of forming the active sited ensures
  the correct splicing

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Self-splicing Introns Reveal that RNA Can
Catalyze RNA Splicing

• Self-splicing introns are ones that fold into a
  specific conformation within the precursor RNA
  and catalyze the chemistry of its own release
• They can remove themselves from RNAs in the test
  tube in the absence of any proteins or other RNA
  molecules

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• grouped into two classes on the basis of
  structure and splicing mechanism
• Strictly speaking, self-splicing introns are not
  enzymes. (Why?)
• The mechanism of group ? introns is the same as
  nuclear pre-mRNAs. (a highly reactive Adenine
  within the intron)

Table 13-1 gives the comparison of three classes
of RNA splicing
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Group ? Introns Release a Linear Intron Rather
than a Lariat

• use a free G nucleotide or nucleoside instead of
  a branch point A residue
• This G species is bound by the RNA and its 3OH
  group is presented to the 5 splice site.
• Two steps of the reaction are similar to that of
  pre-mRNA. (But the result is a linear intron with
  the G fused to the 5 end of it.)

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Some characteristics of group ? introns

• smaller than group ? introns
• a conserved secondary structure
• a binding pocket that accommodates the G
  ribonucleotide or ribonucleoside
• an internal guide sequence that base-pairs with
  the 5 splice site sequence

Box 13-1 tells us how group ? introns can be
converted into true ribozymes
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Group ? introns have a common secondary structure
that is formed by 9 base paired regions. The
sequences of regions P4 and P7 are conserved, and
identify the individual sequence elements P, Q,
R, and S. P1 is created by pairing between the
end of the left exon and the IGS of the intron a
region between P7 and P9 pairs with the 3' end of
the intron.
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• Much of the sequence of a self-splicing intron is
  critical for the splicing reaction
• It must fold into a precise structure to perform
  the reaction chemistry
• In vivo, the intron is complexed with a number of
  proteins that help stabilize the correct
  structurepartly by shielding regions of the
  backbone from each other

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Some views related to evolution

• The similar chemistry seen in self- and
  spliceosome-mediated splicing is believes to
  reflect an evolutionary relationship
• Perhaps ancestral group ?-like self-splicing
  introns were the starting point for the evolution
  of modern pre-mRNA splicing

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How Does the Spliceosome Find the Splice Sites
Reliably?

• Although there exists one mechanism that guards
  against inappropriate splicing, errors may
  happen.
• First, splice sites can be skipped
• Second, other sites, close in sequence but not
  legitimate splice sites, could be mistakenly
  recognized (pseudo splice site)

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But we also have two ways in which the accuracy
of splice-site selection can be enhanced

• The co-transcriptional loading process greatly
  diminishes the likelihood of exon skipping.
  (While transcribing a gene to produce the RNA,
  RNA polymerase ? carries with it various proteins
  with roles in RNA processing)

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• A second mechanism ensures that splice sites
  close to exons are recognized preferentially
• Serine Argenine rich (SR) proteins bind to
  sequences called exonic splicing enhancers (ESEs)
  within the exons
• interact with components of the splicing
  machinery, recruiting them to the nearby splice
  sites
• specifically recruit the U2AF proteins to the 3
  splice site and U1 snRNP to the 5 site (These
  factors demarcate the splice sites for the rest
  of the machinery to assemble correctly)

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SR proteins are essential for splicing

• ensure the accuracy and efficiency of
  constitutive splicing
• regulate alternative splicing
• come in many varieties

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TOPIC 4

• Alternative Splicing

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Single Genes Can Produce Multiple Products by
Alternative Splicing

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

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• Alternative splicing can arise by a number of
  means

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Since we have mechanisms take ensure variations
of this sort do not take place, how does
alternative splicing occur so often?
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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Alternative splicing can be either constitutive
or regulated

• Constitutive alternative splicing always makes
  more than one product from the transcribed gene
• In the case of regulated splicing, different
  forms are generated at different times, under
  different conditions, or in different cell or
  tissue types

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constitutive alternative splicing T antigen of
the monkey virus SV40
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Alternative Splicing Is Regulated by Activators
and Repressors

• exonic splicing enhancer (ESE)
• intronic splicing enhancer (ISE)
• exonic splicing silencer (ESS)
• intronic splicing silencer (ISS)

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• important in directing the splicing machinery to
  many exons
• 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

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Structure of the SR proteins

• RNA-recognition motif (RRM)
• bind RNA
• RS domain (rich in arginine and serine, found at
  the C-terminal end)
• mediate interactions between the SR
  protein and proteins within the splicing
  machinery and recruit it to a nearby splice site

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Most silencers are recognized by members of the
heterogeneous nuclear ribonucleoprotein (hnRNP)
family

• lack the RS domains so cannot recruit the
  splicing machinery
• block specific splice sites and repress the use
  of these sites
• cooperative and competitive binding

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inhibition of splicing by hnRNPI
bind at each end of the exon and conceal it
within a loop
coat the entire exon
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The meaning of alternative splicing

• Multiple protein products can be produced from a
  single gene (isoforms)
• used simply as a way of switching expression of
  the gene that encodes only a single functional
  protein on and off
• determine whether or not an exon with the stop
  codon is included in a given mRNA
• regulate the use of an intron related to mRNA
  transport

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A Small Group Of Introns Are Spiced by an
Alternative Spliceosome Composed of a Different
Set of snRNPs

• In higher eukaryotes some pre-mRNAs are spliced
  by a low-abundance form of spliceosome
• The minor spliceosome recognizes rarely occurring
  introns having consensus sequences (AU-AC
  termini)
• the same chemical pathway

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• U11 and U12 components have the same roles as U1
  and U2 of the major form
• U4 and U6 share the same names but are distinct
• The U5 component is identical

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TOPIC 5

• Exon Shuffling

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Two likely explanations for the situation that
introns are almost nonexistent in bacteria

• introns early model introns existed in all
  organisms but have been lost from bacteria
• introns 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
and in the extensive form in multicellular
eukaryotes?

• The presence of introns, and the need to remove
  them, allows for alternative splicing
• Having the coding sequence of genes divided into
  several exons allows new genes to be created by
  reshuffling exons

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Three observations strongly suggest that the
reshuffling process actually occurs

• 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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• Many genes, and the proteins they encode, have
  apparently arisen during evolution in part via
  exon duplication and divergence
• Related exons are sometimes found in otherwise
  unrelated genes

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Exons Are Shuffled by Recombination to Produce
Genes Encoding New Proteins

• The size ratio ensures that recombination is more
  likely to occur within the introns than within
  the exons (Thus)
• The mechanism of splicing guarantees that almost
  all recombinant genes will be expressed

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TOPIC 6

• RNA Editing

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The sequence of the primary transcript is altered
by either changing, inserting or deleting
residues at the specific points along the
molecules is called RNA editing.
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RNA Editing Is Another Way of Altering the
Sequence of an mRNA

• two mechanisms that mediate editing
• site-specific deamination
• guide RNA-directed uridine insertion or deletion

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Site-specific deamination

• A specifically targeted cytosine residue within
  mRNA is converted into uridine by cytidine
  deaminase (occur only in certain tissues or cell
  types and in a regulated manner)

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The longer form of apolipoprotein B, found in the
liver, is involved in the transport of
endogenously synthesized cholesterol and
triglycerides. The smaller version, found in the
intestines, is involves in the transport of
dietary lipids to various tissues.
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• Adenosine deamination carried out by the enzyme
  ADAR (adenosine deaminase acting on RNA) produces
  Inosine that can base-pair with cytosine. This
  change can readily alter the sequence of the
  protein encoded by the mRNA

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Editing of mRNA occurs when a deaminase acts on
an adenine in an imperfectly paired RNA duplex
region.
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guide RNA-directed uridine insertion or deletion
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• Three regions of guide RNA (gRNA)
• At the 5 end is the anchor and directs the
  gRNA to the region of the mRNA it will edit
• The middle determines exactly where the Us will
  be inserted within the edited sequence
• At the 3 end is a poly-U stretch

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• an RNA-RNA duplex with looped out single-stranded
  regions
• endonuclease
• 3 terminal uridylyl transferase (TUTase)
• RNA ligase

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The biological significance of editing

• proofreading
• translation regulation
• expanded genetic information

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TOPIC 7

• mRNA Transport

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Once Processed, mRNA Is Packaged and Exported
from the Nucleus into the Cytoplasm for
Translation

• The movement is not a passive process, and must
  be carefully regulated
• A typical mature mRNA carries a collection of
  proteins that identifies it as being mRNA
  destined for transport
• It is the set of proteins, not any individual
  kind of protein, that marks RNAs for either
  export or retention in the nucleus

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• Export takes place through a special structure in
  the nuclear membrane called the nuclear pore
  complex
• mRNAs and their associated proteins require
  active transport
• Once in the cytoplasm, the proteins are
  discarded, and are then recognized for import
  back into the nucleus
• Export requires energy supplied by hydrolysis of
  GTP by a GTPase protein called Ran

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