Chapter 21

RNA Splicing and Processing

21.1 Introduction
RNA splicing The
process of excising introns
from RNA and connecting
the exons into a continuous
mRNA. It is mediated by
large RNA-protein complex
called spliceosome.
pre-mRNA The nuclear
primary transcript that is
processed by modification
and splicing to give an
mRNA.

llgure 21.01: 8nA ls modled ln Lhe
nucleus by addluons Lo Lhe 3' and 3' ends
and by spllclng Lo remove Lhe lnLrons.
21.1 Introduction
hnRNP The ribonucleoprotein form of hnRNA
(heterogeneous nuclear RNA), in which the hnRNA is
complexed with proteins.
Pre-mRNAs are not exported until processing is
complete; thus they are found only in the nucleus.
heterogeneous nuclear RNA (hnRNA) RNA that
comprises transcripts of nuclear genes made by RNA
polymerase II; it has a wide size distribution.
21.2 The 5! End of Eukaryotic mRNA Is Capped
A 5! cap is formed by adding a
G to the terminal base of the
transcript via a 5!5! link. This G
is always methylated (7-methyl
guanosine).
Guanylyl transferase catalyzes
addition of G.
5 cap is methylated.
5 cap stabilizes RNA and binds
to ribosome.
5 capping occurs soon after
transcription elongation.
llgure 21.02: 1he cap blocks Lhe 3' end
of m8nA and can be meLhylaLed aL
several posluons.
21.2 The 5! End of Eukaryotic mRNA Is
Capped
The 5! cap of most mRNA is monomethylated (in the
nucleus; 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 to
influence mRNA stability, splicing, export, and
translation.
21.3 Nuclear Splice Junctions Are Short Sequences
Splice sites are the sequences immediately surrounding
the exonintron boundaries. They are named for their
positions relative to the intron.
The 5! splice site (donor site) at the 5! (left) end of the
intron includes the consensus sequence GU.
The 3! splice site (acceptor site) at the 3! (right) end of
the intron includes the consensus sequence AG.
Because two sites have different sequences, introns
have directionality.
The GU-AG rule (originally called the GT-AG rule in
terms of DNA sequence) describes the requirement for
these constant dinucleotides at the first two and last two
positions of introns in pre-mRNAs.
21.3 Nuclear Splice Junctions Are Short Sequences
llgure 21.03: 1he ends of nuclear lnLrons are dened by Lhe Cu-AC rule.
!"#$% "#'%$#( )%* +*,#*+ -. +"/*%*#' 0$#(*#(1( (*21*#0*( )' '3* 45
(67"0* ("'*8 -%)#036$"#'8 )#+ 95 (67"0* ("'*:
21.4 Splice Junctions Are Read in Pairs
Splicing depends on recognition of pairs of splice junctions
within the same intron.
Splicing occurs as RNA is made; it is possible that one donor
and one acceptor site are only available.
Additional conserved sequences at both 5! and 3! splice sites
define functional splice sites among numerous other potential
sites.
llgure 21.04
21.5 Pre-mRNA Splicing
Proceeds through a Lariat
Splicing requires the 5! and 3!
splice sites and a branch site
just upstream of the 3! splice site
(they are all short consensus
sequences).
The branch sequence is
conserved in yeast but less well
conserved in multicellular
eukaryotes.
A lariat is formed when the
intron is cleaved at the 5! splice
site, and the 5! end is joined to a
2! position at an A at the branch
site in the intron (5-2
phosphodiester bond).
llgure 21.03
21.5 Pre-mRNA
Splicing Proceeds
through a Lariat
Splicing occurs by
transesterifications, 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 and
degraded.
llgure 21.06: nuclear spllclng occurs
by Lwo LransesLerlcauon reacuons ln
whlch an CP group auacks a
phosphodlesLer bond.
21.6 snRNAs Are Required for Splicing
small nuclear RNAs (snRNAs; snurps) Small RNA
species confined to the nucleus; several of them are
involved in splicing or other RNA processing reactions.
Snurps are the ribonucleoprotein (snRNPs) particles
that include a specific snRNA and its protein partners.
small cytoplasmic RNAs (scRNAs; scyrps) RNAs that
are present in the cytoplasm (and sometimes are also
found in the nucleus).
Scyrps are the ribonucleoprotein (scRNP) particles that
include an scRNA and its associated proteins.
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
(<20) proteins (some are common and some are unique).
Together with some additional proteins, the snRNPs form
the spliceosome.
All the snRNPs except U6 contain a conserved sequence
that binds the Sm proteins that are recognized by
antibodies (anti-SM) generated in autoimmune disease.
splicing factors protein components of the spliceosome
that is not part of one of the snRNPs.
21.7 Commitment of Pre-
mRNA to the Splicing
Pathway
Base pairing between
snRNA in snRNP
(component of
spliceosome) and pre-
mRNA, or between
snRNAs plays crucial role
in splicing.
U1 snRNP initiates
splicing by binding to the
5! splice site by means of
an RNARNA pairing
reaction.
llgure 21.08: u1 sn8nA has a base-palred
sLrucLure LhaL creaLes several +$;)"#(. 1he
3' end remalns slngle sLranded and can base
palr wlLh Lhe 3' spllclng slLe.
8lnds Lo common
sn8n proLelns (Sm)
21.7 Commitment of Pre-mRNA to the Splicing
Pathway
E complex (early complex = commitment complex)
contains U1 snRNP, splicing factor U2AF, and SR
proteins (containing Ser-Arg rich region)
The direct way of forming an E complex is for U1 snRNP
to bind at the 5! splice site and U2AF to bind at a
pyrimidine tract between the branch site and the 3!
splice site. This is intron definition.
Two splice sites are recognized without requiring any sequences
outside the intron.
Another possibility is for the complex to form between
U2AF at the pyrimidine tract and U1 snRNP at a
downstream 5! splice site. This is exon definition.
It is common when introns are long and splicing sites are weak.
It requires sequence in next intron (5 splice site).
21.7 Commitment of Pre-mRNA to the Splicing
Pathway
llgure 21.11: 1here are Lwo rouLes for lnlual recognluon of 3' and 3' spllce slLes by elLher
lnLron denluon or exon denluon.
u1 sn8n u1 sn8n on
nexL lnLron
noL unlversal
21.8 The Spliceosome
Assembly Pathway
Binding of U5 and U4/U6
snRNPs converts the A
complex to the B1
spliceosome, which
contains all the
components necessary for
splicing.
U4 base pairs with U6; in
consequence, U6 cannot
base pair with U2 until U4
is released from
spliceosome.
llgure 21.12
<67"0*$($;*
21.8 The Spliceosome Assembly Pathway
Release of U1 snRNP allows
U6 snRNA to interact with the
5! splice site and converts the
B1 spliceosome to the B2
spliceosome.
When U4 dissociates from U6
snRNP, 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 resembles
group II self-splicing introns
llgure 21.13
21.8 The Spliceosome Assembly Pathway
An alternative splicing pathway uses another set of
snRNPs that comprise the U12 spliceosome (U11, U12,
U5 variant, U4
atac
, and U6
atac
.
The target introns are defined by longer consensus
sequences at the splice junctions rather than strictly
following the GU-AG or AU-AC rules.
21.9 Splicing Is Temporally and Functionally
Coupled with Multiple Steps in Gene Expression
The REF proteins bind to splicing
junctions by associating with the
spliceosome.
After splicing, they remain
attached to the RNA at the exon-
exon junction.
They interact with the transport
protein TAP/Mex that exports the
RNA through the nuclear pore.
llgure 21.16: A 8Ll proLeln (shown ln green)
blnds Lo a spllclng facLor and remalns wlLh Lhe
spllced 8nA producL. 8Ll blnds Lo a LransporL
proLeln LhaL blnds Lo Lhe nuclear pore.
21.9 Splicing Is
Temporally and
Functionally Coupled with
Multiple Steps in Gene
Expression
The REF proteins are part of EJC
complex (exon junction complex).
After splicing, they remain
attached to the RNA at the exon-
exon junction.
llgure 21.13: 1he L!C (exon [uncuon complex) ls
deposlLed near Lhe spllce [uncuon as a
consequence of Lhe spllclng reacuon.
21.11 Alternative Splicing Is a Rule, Rather Than
an Exception, in Multicellular Eukaryotes
Specific exons or exonic sequences may be
excluded or included in the mRNA products by
using alternative splicing sites.
Alternative splicing contributes to structural and
functional diversity of gene products.
21.11 Alternative Splicing Is a Rule, Rather Than
an Exception, in Multicellular Eukaryotes
llgure 21.19: ulerenL modes of alLernauve spllclng
21.12 trans-Splicing Reactions Use Small RNAs
Splicing reactions usually occur
only in cis between splice junctions
on the same molecule of RNA.
trans-splicing occurs in
trypanosomes and worms where a
spliced leader RNA (SL RNA) is
spliced to the 5! ends of many
precursor mRNAs.
SL RNAs have a structure
resembling the Sm-binding site of U
snRNAs.
Trypanosomes do not have U1 or
U5 snRNA (U2, U4 and U6 are
present).
llgure 21.21: Spllclng usually
occurs only ln cls beLween
exons
carrled on Lhe same physlcal
8nA molecule.
21.13 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
tRNA splicing occurs by successive cleavage and ligation
reactions.
An endonuclease cleaves the tRNA precursors at both
ends of the intron.
Common secondary structure of tRNA precursor is
important rather than common sequence of intron.
llgure 21.24: 1he lnLron ln yeasL L8nAhe base
palrs wlLh Lhe anucodon Lo change Lhe
sLrucLure of Lhe anucodon arm.
21.13 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
Release of the intron generates two half-tRNAs that pair to
form the mature tRNA-like structure.
The halves have the unusual ends: 5!-OH and 2!-3! cyclic
phosphate at 3.
llgure 21.23
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 is
removed by a phosphatase.
llgure 21.23
21.14 The 3! Ends of mRNAs Are Generated by
Cleavage and Polyadenylation
Transcription terminator in
eukaryotes 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 protein
complex that contains a specificity
factor, an endonuclease, and poly
(A) polymerase (PAP).
The specificity factor and
endonuclease cleave RNA
downstream of AAUAAA.
llgure 21.28
21.14 The 3! Ends of mRNAs Are Generated by
Cleavage and Polyadenylation
The specificity factor and poly(A) polymerase add ~200
A 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 two
cis-acting elements:
AAUAAA motif located 11-30 nucleotides upstream
Downstream U-rich or GU-rich element
21.15 Production of rRNA Requires Cleavage
Events and Involves Small RNAs
Eukaryotic rRNAs are generated by cleavage and
trimming.
llgure 21.30: MaLure eukaryouc r8nAs are generaLed by 07*)=)>* )#+ '%";;"#>
evenLs from a prlmary LranscrlpL.
21.15 Production of rRNA Requires Cleavage
Events and Involves Small RNAs
Bacterial rRNAs are generated by cleavage (no trimming).
llgure 21.31: 1he rrn operons ln L. coll conLaln genes for boLh r8nA and L8nA.
21.15 Production of rRNA Requires Cleavage
Events and Involves Small RNAs
Processing and modification of
rRNA requires a class of small
RNAs called snoRNAs (small
nucleolar RNAs).
Hundreds of snoRNAs in S.
cerevisiae: they are encoded by
individual genes, polycistrons, and
introns.
llgure 21.34: P/ACA sno8nAs have
Lwo shorL conserved sequences and
Lwo halrpln sLrucLures, each of whlch
has reglons ln Lhe sLem LhaL are
complemenLary Lo r8nA.