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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.

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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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