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 the second three-way junction
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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) Many of the
functions of the spliceosome are carried out by its RNA components
rich in uracil
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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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assembly catalysis E complex A complex B complex C complex
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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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ensure the accuracy and efficiency of constitutive splicing
regulate alternative splicing come in many varieties SR proteins
are essential for splicing
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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