2. 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.
3. 21.1 Introduction
Figure 21.01: RNA is modified in the nucleus by additions to the 5’ and 3’ ends and by
splicing to remove the introns.
4. 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.
5. 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.
• The capping process takes place during transcription
and may be important for release from pausing of
transcription.
Figure 21.02: The cap blocks the 5’ end
of mRNA and is methylated at several
positions.
6. 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.
7. 21.3 Nuclear Splice Sites Are Short
Sequences
• Splice sites are the sequences immediately surrounding
the exon–intron 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.
8. 21.3 Nuclear Splice Sites 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 21.03: The ends of nuclear introns are defined by the GU-AG rule.
9. 21.3 Nuclear Splice Sites 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 exon–intron
boundaries.
10. 21.4 Splice Sites Are Read in Pairs
• Splicing depends only on recognition of pairs of splice
sites.
• 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.
11. 21.4 Splice Sites Are Read in Pairs
Figure 21.04: Splicing junctions are recognized only in the correct pairwise combinations.
12. 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.
13. 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 21.05: Splicing occurs in two stages. First
the 5’ exon is cleaved off, and then it is joined to
the 3’ exon.
14. 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.
15. 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 21.07: The spliceosome is
~12 MDa. Five snRNPs account for
almost half of the mass.
16. 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.
17. 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 RNA–RNA 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.
18. 21.7 Commitment of Pre-mRNA to the
Splicing Pathway
Figure 21.10: The commitment (E) complex formation.
19. 21.7 Commitment of Pre-mRNA to the
Splicing Pathway
• In cells of multicellular eukaryotes, 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.
20. 21.7 Commitment of Pre-mRNA to the
Splicing Pathway
Figure 21.11: There are two routes for initial recognition of 5’ and 3’ splice sites by either
intron definition or exon definition.
21. 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 A
complex 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.
22. 21.8 The Spliceosome Assembly Pathway
• When U4 dissociates
from U6 snRNP, U6
snRNA can pair with U2
snRNA to form the
catalytic active site.
• Both transesterification
reactions take place in
the activated spliceosome
(the C complex).
• The splicing reaction is
reversible at all steps.
Figure 21.12: The splicing reaction proceeds
through discrete stages.
23. 21.9 An Alternative Spliceosome Uses
Different snRNPs to Process the Minor
Class of Introns
• An alternative splicing pathway uses another set of
snRNPs that comprise the U12 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.
24. 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.
25. 21.10 Pre-mRNA Splicing Likely Shares the
Mechanism with Group II Autocatalytic
Introns
Figure 21.15: Three classes of splicing reactions
proceed by two transesterifications.
26. 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 exon–exon junctions during splicing and
assists in RNA transport, localization, and degradation.
27. 21.11 Splicing Is Temporally and
Functionally Coupled with Multiple Steps in
Gene Expression
Figure 21.18: The EJC (exon junction complex) is
deposited near the splice junction as a
consequence of the splicing reaction.
28. 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 21.20: The EJC complex couples splicing with
NMD.
29. 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.
30. 21.12 Alternative Splicing Is a Rule, Rather
Than an Exception, in Multicellular
Eukaryotes
Figure 21.21: Different modes of alternative splicing.
31. 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 encoding successive
products of a pathway.
32. 21.12 Alternative Splicing Is a Rule, Rather
Than an Exception, in Multicellular
Eukaryotes
Figure 21.23: Sex determination in D. melanogaster involves a pathway in
which different splicing events occur in females.
33. 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.
34. 21.13 Splicing Can Be Regulated by Exonic
and Intronic Splicing Enhancers and
Silencers
Figure 21.24: Exonic and intronic sequences can modulate the splice site selection by
functioning as splicing enhancers or silencers.
35. 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 21.25: The Nova and Fox families
of RNA binding proteins can promote or
suppress splice site selection in a
context dependent fashion.
36. 21.14 trans-Splicing Reactions Use Small
RNAs
• Splicing reactions usually
occur only in cis between
splice sites 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 21.26: Splicing usually occurs
only in cis between exons carried on
the same physical RNA molecule.
37. 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
downstream of AAUAAA.
Figure 21.29: The sequence AAUAAA
is necessary for cleavage to generate
a 3’ end for polyadenylation.
38. 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 21.30: The 3’ processing
complex consists of several activities.
39. 21.16 The 3′ mRNA End Processing Is
Critical for Termination of Transcription
• There are various ways to
end transcription by
different RNA
polymerases.
• The mRNA 3′ end
formation signals
termination of Pol II
transcription.
Figure 21.31: Transcription by Pol I and
Pol III uses specific terminators to end
transcription.
40. 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.
41. 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.
42. 21.17 The 3′ End Formation of Histone
mRNA Requires U7 snRNA
Figure 21.33: Generation of the 3’ end of histone H3 mRNA depends on a conserved
hairpin and a sequence that base pairs with U7 snRNA.
43. 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 21.34: The intron in yeast
tRNAPhe base pairs with the anticodon
to change the structure of the anticodon
arm.
44. 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 2′–3′ cyclic
phosphate.
Figure 21.36: The 3’ and 5’ cleavages in
S. cerevisiae pre-tRNA are catalyzed by
different subunits of the endonuclease.
45. 21.18 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.
46. 21.18 tRNA Splicing Involves Cutting and
Rejoining in Separate Reactions
Figure 21.38: Splicing of tRNA requires separate nuclease and ligase activities.
47. 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.
48. 21.19 The Unfolded Protein Response Is
Related to tRNA Splicing
Figure 21.39: The unfolded protein response
occurs by activating special splicing of HAC1
mRNA to produce a transcription factor
recognizing the UPRE.
49. 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 encoding
chaperones that help to fold unfolded proteins.
• Activated Ire1 induces apoptosis when the cell is
overstressed by unfolded proteins.
50. 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 21.40: Mature
eukaryotic rRNAs are
generated by cleavage and
trimming events from a
primary transcript.
51. 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
that contains the target base to
generate a typical structure that
is the substrate for modification.
Figure 21.42: A snoRNA base pairs
with a region of rRNA that is to be
methylated.
