Chapter 21

RNA Splicing and Processing

 21.1 Introduction

pre-mRNA The nuclear transcript that is processed by

modification and splicing to give an mRNA.
RNA splicing The process of excising introns from
RNA and connecting the exons into a continuous mRNA.
FIGURE 01: Eukaryotic mRNA is modified, processed, and transported
 21.1 Introduction

heterogeneous nuclear RNA (hnRNA) RNA

that comprises transcripts of nuclear genes
made by RNA polymerase II; it has a wide size
distribution and low stability.
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.
 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 55 link.
The capping process takes place during the
transcription, which may be important for transcription
reinitiation.

FIGURE 02: Eukaryotic mRNA

has a methylated 5 cap
 21.2 The 5 End of Eukaryotic mRNA Is
Capped
The 5 cap of most mRNA is monomethylated, but some
small noncoding RNAs are trimethylated.
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 at the 5 (left) end of the intron includes
the consensus sequence GU.
The 3 splice site at the 3 (right) end of the intron
includes the consensus sequence AG.
21.3 Nuclear Splice Junctions Are Short Sequences
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.

FIGURE 03: The ends of

nuclear introns are defined
by the GU-AG rule
 21.3 Nuclear Splice Junctions Are Short
Sequences
There exist minor introns relative to the major introns
that follow the GU-AG rule.
Minor introns follow a general AU-AC rule with a different
set of consensus sequences at the exonintron
boundaries.
 21.4 Splice Junctions Are Read in Pairs

Splicing depends only on recognition of pairs of splice

junctions.
All 5 splice sites are functionally equivalent, and all 3
splice sites are functionally equivalent.
Additional conserved sequences at both 5 and 3 splice
sites define functional splice sites among numerous
other potential sites in the pre-mRNA.
FIGURE 04: Correct splicing removes three introns by pairwise recognition
of the junctions
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.
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.
21.5 Pre-mRNA Splicing Proceeds through
a Lariat

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.

FIGURE 05: Splicing proceeds

through a lariat
 21.6 snRNAs Are Required for Splicing

small cytoplasmic RNAs (scRNA; scyrps) RNAs

that are present in the cytoplasm (and sometimes are
also found in the nucleus).
small nuclear RNA (snRNA; snurps) One of many
small RNA species confined to the nucleus; several of
them are involved in splicing or other RNA processing
reactions.
small nucleolar RNA (snoRNA) A small nuclear RNA
that is localized in the nucleolus.
 21.6 snRNAs Are Required for Splicing

The five snRNPs involved in splicing are U1, U2, U5, U4,
and U6.
Together with some additional proteins, the snRNPs form
the spliceosome.

FIGURE 07: The spliceosome

is a large particle
 21.6 snRNAs Are Required for Splicing

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 factor A protein component of the
spliceosome that is not part of one of the snRNPs.
transesterification A reaction that breaks and makes
chemical bonds in a coordinated transfer so that no
energy is required.
 21.7 Commitment of Pre-mRNA to the
Splicing Pathway
U1 snRNP initiates splicing by binding to the 5 splice site
by means of an RNARNA pairing reaction.
The commitment complex (or E complex) contains U1
snRNP bound at the 5 splice site and the protein U2AF
bound to a pyrimidine tract between the branch site and
the 3 splice site.
FIGURE 10: Formation of the commitment complex
 21.7 Commitment of Pre-mRNA to the
Splicing Pathway
In multicellular eukaryotic cells, SR proteins play an
essential role in initiating the formation of the
commitment complex.
Pairing splice sites can be accomplished by intron
definition or exon definition.
FIGURE 11: Two route for initial recognition of 5 and 3 splice sites
 21.8 The Spliceosome Assembly Pathway

The commitment complex progresses to pre-

spliceosome (the A complex) in the presence of ATP.
Recruitment of U5 and U4/U6 snRNPs converts the pre-
spliceosome to the mature spliceosome (the B1
complex).
The B1 complex is next converted to the B2 complex in
which U1 snRNP is released to allow U6 snRNA to
interact with the 5 splice site.
21.8 The Spliceosome Assembly Pathway
U4 dissociates from U6
snRNP to allow U6 snRNA
to pair with U2 snRNA to
form the catalytic center
for splicing.
Both transesterification
reactions take place in the
activated spliceosome (the
C complex).
The splicing reaction is
reversible at all steps.

FIGURE 12: Splicing reaction proceeds through discrete stages

 21.9 An Alternative Spliceosome Uses
Different snRNPs to Process the Minor
Class of Introns
An alternative splicing pathway uses another set of
snRNPs with only U5 snRNP in common with the major
spliceosome.
The target introns are defined by longer consensus
sequences at the splice junctions, rather than strictly
according to the GU-AG or AU-AC rules.
Major and minor spliceosomes share critical protein
factors, including SR proteins.
21.10 Pre-mRNA Splicing Likely Shares the
Mechanism with Group II Autocatalytic
Introns
Group II introns excise themselves from RNA by an
autocatalytic splicing event (autosplicing or self-
splicing).
The splice junctions and mechanism of splicing of group
II introns are similar to splicing of nuclear introns.
A group II intron folds into a secondary structure that
generates a catalytic site resembling the structure of U6-
U2-nuclear intron.
FIGURE 15: Splicing uses transesterification
 21.11 Splicing Is Temporally and
Functionally Coupled with Multiple Steps in
Gene Expression
Splicing can occur during or after transcription.
The transcription and splicing machineries are physically
and functionally integrated.
Splicing is connected to mRNA export and stability
control.
exon junction complex (EJC) A protein complex that
assembles at exonexon junctions during splicing and
assists in RNA transport, localization, and degradation.
FIGURE 18: Splicing is required for mRNA export
 21.11 Splicing Is Temporally and
Functionally Coupled with Multiple Steps in
Gene Expression
Splicing in the nucleus can
influence mRNA translation in
the cytoplasm.
nonsense-mediated mRNA
decay (NMD) A pathway
that degrades an mRNA that
has a nonsense mutation
prior to the last exon.

FIGURE 20: The EJC complex

couples splicing with NMD
21.12 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.
FIGURE 21: Different modes of alternative splicing.
21.12 Alternative Splicing Is a Rule, Rather
Than an Exception, in Multicellular
Eukaryotes
Sex determination in Drosophila involves a series of
alternative splicing events in genes coding for
successive products of a pathway.
FIGURE 23: Sex determination in D. melanogaster
21.13 Splicing Can Be Regulated by Exonic
and Intronic Splicing Enhancers and
Silencers
Alternative splicing is often associated with weak splice
sites.
Sequences surrounding alternative exons are often more
evolutionarily conserved than sequences flanking
constitutive exons.
Specific exonic and intronic sequences can enhance or
suppress splice site selection.
FIGURE 24: Exonic and intronic sequences can modulate the splice site
selection
 21.13 Splicing Can Be Regulated by Exonic
and Intronic Splicing Enhancers and Silencers
The effect of splicing enhancers and silencers is
mediated by sequence-specific RNA binding proteins,
many of which may be developmentally regulated and/or
expressed in a tissue-specific manner.
The rate of transcription can directly affect the outcome
of alternative splicing.

FIGURE 25: The Nova

and Fox families of RNA
binding proteins
 21.14 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 short sequence (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.
FIGURE 26: Trans-splicing can occur
when special constructs are made
 21.15 The 3 Ends of mRNAs Are
Generated by Cleavage and
Polyadenylation
The sequence AAUAAA 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.
The specificity factor and
endonuclease cleave RNA
FIGURE 29: The 3 end of
downstream of AAUAAA.
mRNA is generated by cleavage
21.15 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 controls
mRNA stability and
influences translation.
Cytoplasmic polyadenylation
plays a role in Xenopus
embryonic development.

FIGURE 30: There is a single 3 end-processing complex

21.16 The 3 mRNA End Processing Is
Critical for Transcriptional Termination
There are various ways to
end transcription by
different RNA
polymerases.
The mRNA 3 end
formation signals
termination of Pol II
transcription.

FIGURE 31: Transcription by Pol I

and Pol III
 21.17 The 3 End Formation of Histone
mRNA Requires U7 snRNA
The expression of histone mRNAs is replication
dependent and is regulated during the cell cycle.
Histone mRNAs are not polyadenylated; their 3 ends are
generated by a cleavage reaction that depends on the
structure of the mRNA.
 21.17 The 3 End Formation of Histone
mRNA Requires U7 snRNA
The cleavage reaction requires the SLBP to bind to a
stem-loop structure and the U7 snRNA to pair with an
adjacent single-stranded region.
The cleavage reaction is catalyzed by a factor shared
with the polyadenylation complex.
FIGURE 33: Generation of the 3 end of histone H3 mRNA
 21.18 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
RNA polymerase III terminates transcription in a poly(U)4
sequence embedded in a GC-rich sequence.
tRNA splicing occurs by successive cleavage and
ligation reactions.

FIGURE 34: tRNA splicing

recognized a specific structure
 21.18 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
An endonuclease cleaves the tRNA precursors at both
ends of the intron.
Release of the intron generates two half-tRNAs with
unusual ends that contain 5 hydroxyl and 23 cyclic
phosphate.

FIGURE 36: The endonuclease

complex has four proteins
 21.18 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
The 5OH end is phosphorylated by a polynucleotide
kinase, the cyclic phosphate group is opened by
phosphodiesterase to generate a 2phosphate terminus
and 3OH group, exon ends are joined by an RNA
ligase, and the 2phosphate is removed by a
phosphatase.
FIGURE 38: tRNA splicing has separate cleavage and ligation stages
 21.19 The Unfolded Protein Response Is
Related to tRNA Splicing
Ire1 is an inner nuclear membrane protein with its N-
terminal domain in the ER lumen and its C-terminal
domain in the nucleus; the C-terminal domain exhibits
both kinase and endonuclease activities.
Binding of an unfolded protein to the N-terminal domain
activates the C-terminal endonuclease by
autophosphorylation.
FIGURE 39: Unfolded proteins activate a TF
 21.19 The Unfolded Protein Response Is
Related to tRNA Splicing
The activated endonuclease cleaves HAC1 (Xbp1 in
vertebrates) mRNA to release an intron and generate
exons that are ligated by a tRNA ligase.
Only spliced HAC1 mRNA can be translated to a
transcription factor that activates genes coding for
chaperones that help to fold unfolded proteins.
Activated Ire1 induces apoptosis when the cell is over
stressed by unfolded proteins.
 21.20 Production of rRNA Requires
Cleavage Events and Involves Small RNAs
RNA polymerase I terminates transcription at an 18-base
terminator sequence.
The large and small rRNAs are released by cleavage
from a common precursor rRNA; the 5S rRNA is
separately transcribed.

FIGURE 40: Generation of mature eukaryotic rRNAs

 21.20 Production of rRNA Requires
Cleavage Events and Involves Small RNAs
The C/D group of snoRNAs is
required for modifying the 2
position of ribose with a methyl
group.
The H/ACA group of snoRNAs is
required for converting uridine to
pseudouridine.
In each case the snoRNA base
pairs with a sequence of rRNA
FIGURE 42: A snoRNA base
that contains the target base to
pairs with a region of rRNA that generate a typical structure that
is to be methylated is the substrate for modification.