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Chapter 21
RNA Splicing and Processing
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21.1 Introduction
RNA splicing Theprocess of excising intronsfrom RNA and connecting
the exons into a continuousmRNA. It is mediated by
large RNA-protein complexcalled spliceosome.
pre-mRNA The nuclearprimary transcript that is
processed by modificationand splicing to give an
mRNA.
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21.1 Introduction
hnRNP The ribonucleoprotein form of hnRNA(heterogeneous nuclear RNA), in which the hnRNA iscomplexed with proteins.
Pre-mRNAs are not exported until processing iscomplete; thus they are found only in the nucleus.
heterogeneous nuclear RNA (hnRNA) RNA thatcomprises transcripts of nuclear genes made by RNA
polymerase II; it has a wide size distribution.
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21.2 The 5!End of Eukaryotic mRNA Is Capped
A 5!capis formed by adding aGto the terminal base of thetranscript via a 5!5!link. This G
is always methylated (7-methyl
guanosine).
Guanylyl transferase catalyzesaddition of G.
5 cap is methylated.
5 cap stabilizes RNA and bindsto ribosome.
5 capping occurs soon aftertranscription elongation.
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21.2 The 5!End of Eukaryotic mRNA Is
Capped
The 5!cap of most mRNA is monomethylated (in thenucleus; cap 0), but some small noncoding RNAs are
further methylated in the cytoplasm (di- or tri-metylated;
cap 1 or cap 2).
The cap structure is recognized by protein factors toinfluence mRNA stability, splicing, export, and
translation.
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21.3 Nuclear Splice Junctions Are Short Sequences
Splice sites are the sequences immediately surroundingthe exonintron boundaries. They are named for theirpositions relative to the intron.
The 5!splice site (donor site)at the 5!(left) end of theintron includes the consensus sequence GU.
The 3!splice site (acceptor site)at the 3!(right) end ofthe intron includes the consensus sequence AG.
Because two sites have different sequences, intronshave directionality.
The GU-AG rule (originally called the GT-AG rule interms of DNA sequence) describes the requirement forthese constant dinucleotides at the first two and last two
positions of introns in pre-mRNAs.
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21.3 Nuclear Splice Junctions Are Short Sequences
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21.4 Splice Junctions Are Read in Pairs
Splicing depends on recognition of pairs of splice junctionswithin the same intron.
Splicing occurs as RNA is made; it is possible that one donorand one acceptor site are only available.
Additional conserved sequences at both 5!and 3!splice sitesdefine functional splice sites among numerous other potential
sites.
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21.5 Pre-mRNA SplicingProceeds through a Lariat
Splicing requires the 5!and 3!splice sites and a branch sitejust upstream of the 3!splice site
(they are all short consensus
sequences).
The branch sequence isconserved in yeast but less well
conserved in multicellular
eukaryotes.
A lariatis formed when theintron is cleaved at the 5!splicesite, and the 5!end is joined to a
2!position at an A at the branch
site in the intron (5-2
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21.5 Pre-mRNA
Splicing Proceeds
through a Lariat
Splicing occurs bytransesterifications, in
which a bond is transferred
from one location to another. The intron is released as a
lariat when it is cleaved at the
3!splice site, and the left and
right exons are then ligated
together.
Lariat is debranched anddegraded.!"#$%& ()*+K, .$89&
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21.6 snRNAs Are Required for Splicing
small nuclear RNAs (snRNAs; snurps) Small RNAspecies confined to the nucleus; several of them areinvolved in splicing or other RNA processing reactions.
Snurps are the ribonucleoprotein (snRNPs) particlesthat include a specific snRNA and its protein partners.
small cytoplasmic RNAs (scRNAs; scyrps) RNAs thatare present in the cytoplasm (and sometimes are also
found in the nucleus).
Scyrps are the ribonucleoprotein (scRNP) particles thatinclude an scRNA and its associated proteins.
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21.6 snRNAs Are Required for Splicing
The five snRNPs involved in splicing are U1, U2, U5, U4,and U6; each snRNP contains one snRNA and many(
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21.7 Commitment of Pre-
mRNA to the Splicing
Pathway
Base pairingbetweensnRNA in snRNP
(component of
spliceosome) and pre-mRNA, or between
snRNAs plays crucial rolein splicing.
U1 snRNP initiatessplicing by binding to the5!splice site by means of
an RNARNA pairing
reaction.
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21.7 Commitment of Pre-mRNA to the Splicing
Pathway
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21.8 The SpliceosomeAssembly Pathway
Binding of U5 and U4/U6snRNPs converts the Acomplexto the B1
spliceosome, which
contains all the
components necessary forsplicing.
U4 base pairs with U6; inconsequence, U6 cannot
base pair with U2 until U4
is released from
spliceosome.
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21.8 The Spliceosome Assembly Pathway
Release of U1 snRNP allowsU6 snRNA to interact with the
5!splice site and converts the
B1 spliceosome to the B2
spliceosome.
When U4 dissociates from U6snRNP, U6 snRNA can pair
with U2 snRNA to form the
catalytic active site.
Base pairings during splicing:U1::5 splice site; U2::branch
site; U6::5 splice site; U2::U6;
U4::U6
The catalytic center resemblesgroup II self-splicing introns
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21.8 The Spliceosome Assembly Pathway
An alternative splicing pathway uses another set ofsnRNPs that comprise the U12spliceosome (U11, U12,
U5 variant, U4atac, and U6atac.
The target introns are defined by longer consensussequences at the splice junctions rather than strictly
following the GU-AG or AU-AC rules.
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21.9 Splicing Is Temporally and FunctionallyCoupled with Multiple Steps in Gene Expression
The REF proteins bind to splicingjunctions by associating with the
spliceosome.
After splicing, they remainattached to the RNA at the exon-
exon junction. They interact with the transport
protein TAP/Mex that exports the
RNA through the nuclear pore.
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21.9 Splicing Is
Temporally and
Functionally Coupled with
Multiple Steps in GeneExpression
The REF proteins are part of EJCcomplex (exon junction complex).
After splicing, they remainattached to the RNA at the exon-
exon junction.
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21.11 Alternative Splicing Is a Rule, Rather Than
an Exception, in Multicellular Eukaryotes
Specific exons or exonic sequences may beexcluded or included in the mRNA products by
using alternative splicing sites.
Alternative splicing contributes to structural andfunctional diversity of gene products.
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21.11 Alternative Splicing Is a Rule, Rather Than
an Exception, in Multicellular Eukaryotes
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21.12 trans-Splicing Reactions Use Small RNAs
Splicing reactions usually occuronly in cisbetween splice junctionson the same molecule of RNA.
trans-splicing occurs intrypanosomes and worms where a
spliced leader RNA (SL RNA) isspliced to the 5!ends of many
precursor mRNAs.
SL RNAs have a structureresembling the Sm-binding site of UsnRNAs.
Trypanosomes do not have U1 orU5 snRNA (U2, U4 and U6 are
present).
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21.13 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
tRNA splicing occurs by successive cleavage and ligationreactions.
An endonuclease cleaves the tRNA precursors at bothends of the intron.
Common secondary structure of tRNA precursor isimportant rather than common sequence of intron.
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21.13 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
Release of the intron generates two half-tRNAs that pair toform the mature tRNA-like structure.
The halves have the unusual ends: 5!-OH and 2!-3!cyclicphosphate at 3.
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21.13 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
The 5!OH end is phosphorylated by a polynucleotide kinase. The cyclic phosphate group is opened by phosphodiesterase to
generate a 2!phosphate terminus and 3!OH group.
Exon ends are joined by an RNA ligase, and the 2!phosphate isremoved by a phosphatase.
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21.14 The 3!Ends of mRNAs Are Generated byCleavage and Polyadenylation
Transcription terminator ineukaryotes is poorly defined.
The sequence AAUAAA(polyadenylation signal) is a signal
for cleavage to generate a 3!end
of mRNA that is polyadenylated.
The reaction requires a proteincomplex that contains a specificity
factor, an endonuclease, and poly
(A) polymerase (PAP). The specificity factor and
endonuclease cleave RNA
downstream of AAUAAA.
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21.14 The 3!Ends of mRNAs Are Generated by
Cleavage and Polyadenylation
The specificity factor and poly(A) polymerase add ~200A residues processively to the 3!end.
The poly(A) tail facilitates mRNA stability, nuclear export,and translation.
The site of cleavage/polyadenylation is flanked by twocis-acting elements:
AAUAAA motif located 11-30 nucleotides upstream Downstream U-rich or GU-rich element
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21.15 Production of rRNA Requires Cleavage
Events and Involves Small RNAs
Eukaryotic rRNAs are generated by cleavage andtrimming.
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21.15 Production of rRNA Requires CleavageEvents and Involves Small RNAs
Bacterial rRNAs are generated by cleavage (no trimming).
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21.15 Production of rRNA Requires CleavageEvents and Involves Small RNAs
Processing and modification ofrRNA requires a class of small
RNAs calledsnoRNAs (small
nucleolar RNAs).
Hundreds of snoRNAs in S.cerevisiae: they are encoded byindividual genes, polycistrons, and
introns.
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