55 143 SelfSplicing RNAs

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427
10-mer
P
GF
(a)
1
Test exon
No ESS activity
No GFP produced
White cells
GF
P
(b)
2
ESS activity
GFP produced
Green cells
Figure 14.40 A reporter construct to detect ESS activity. Burge
and colleagues constructed a plasmid containing the two exons of the
GFP gene, separated by an intron that held a test exon (red) into
which random 10-mers (yellow) had been placed. They transfected
cells with collections of these plasmids, and then screened for green
color. (a) If the 10-mer has no ESS activity, splicing of the test exon
will not be silenced, so it will be included in the middle of the GFP
mRNA, disrupting its activity, and producing white cells. (b) If the
10-mer does have ESS activity, the test exon will not be recognized,
so it will be spliced out along with the surrounding intron. Thus, a
normal GFP mRNA will be produced and the cells will be green.
14.3 Self-Splicing RNAs
circular introns (plus a linear intron missing 15 nt), which
they had observed in previous studies. This suggested that
the RNA was being spliced, and that the excised intron
was circularizing.
Was this splicing carried out by the RNA itself, or was
the RNA polymerase somehow involved? To answer this
question, Cech and coworkers ran the RNA polymerase
reaction in the presence of polyamines (spermine, spermidine, and putrescine) that inhibit splicing. Then they electrophoresed the products, excised all four RNA bands plus
the material that remained at the origin, and purified the
RNAs. Next they incubated these RNAs under splicing
conditions (no polyamines) and reelectrophoresed them.
When they autoradiographed the electrophoretic gel, they
could see the intron in the lanes containing RNA from
three of the bands. Thus, these bands appear to be 26S rRNA
precursors that can splice themselves without any protein,
even RNA polymerase.
The band we are calling the intron is the right size, but is
it really what we think it is? Cech and coworkers sequenced
the first 39 nt of this RNA and showed that they corresponded exactly to the first 39 nt of the intron. Therefore, it
seemed clear that this RNA really was the intron.
Cech’s group also discovered that the linear intron—the
RNA we have been discussing so far—can cyclize by itself.
One of the most stunning discoveries in molecular biology
in the 1980s was that some RNAs could splice themselves
without aid from a spliceosome or any other proteins.
Thomas Cech (pronounced “Check”) and his coworkers
made this discovery in their study of the 26S rRNA gene of
the ciliated protozoan, Tetrahymena. This rRNA gene is a
bit unusual in that it has an intron, but the thing that really
attracted attention when this work was published in 1982
was that the purified 26S rRNA precursor spliced itself in
vitro. In fact, this was just the first example of self-splicing
RNAs containing introns called group I introns. Subsequent work revealed another class of RNAs containing introns called group II introns, some of whose members are
also self-splicing.
Group I Introns
To make the self-splicing RNA, Cech and coworkers cloned
part of the 26S rRNA gene containing the intron, and transcribed it in vitro with E. coli RNA polymerase. When they
electrophoresed the labeled products of these transcription
reactions, they observed four large RNA products, plus
three smaller RNAs corresponding in size to the linear and
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Chapter 14 / RNA Processing I: Splicing
All they had to do was raise the temperature, and the Mg21
and salt concentrations, and at least some of the purified
linear intron would convert to circular intron.
So far, we have seen that the rRNA precursor can remove its intron, but can it splice its exons together? Cech
and coworkers used a model splicing reaction to show that
it can (Figure 14.41a). They began by cloning a part of the
Tetrahymena 26S rRNA gene including 303 bp of the first
exon, the whole intron, and 624 bp of the second exon into
a vector with a promoter for phage SP6 polymerase. To
generate the labeled splicing substrate, they transcribed this
DNA in vitro with SP6 polymerase in the presence of
[a-32P]ATP. Then they incubated this RNA under splicing
conditions with and without GTP and electrophoresed the
products. Lane 1 displays the products of the reaction with
GTP. The familiar linear intron is present, as well as a small
amount of circular intron. In addition, a prominent band
(a)
(b)
E
PSP6
E
Transcription
with [α-32P]ATP
+GTP
–GTP
1
2
Substrate
Ligated
exons
Circular
intron
Linear
intron
Exon1 Intron Exon 2
Splicing substrate
GTP
+
Ligated exons
Intron
Figure 14.41 Demonstration of exon ligation. (a) Experimental
scheme. Cech and coworkers constructed a plasmid containing part
of the Tetrahymena 26S rRNA gene: 303 bp of exon 1 (blue); the
413-bp intron (red); and 624 bp of exon 2 (yellow). They linearized the
plasmid by cutting it with EcoRI, creating EcoRI ends (E), then
transcribed the plasmid in vitro with phage SP6 RNA polymerase and
[a-32P]ATP. This yielded the labeled splicing substrate. They incubated
this substrate under splicing conditions in the presence or absence of
GTP, then electrophoresed the splicing reactions and detected the
labeled RNAs by autoradiography. (b) Experimental results. In the
presence of GTP (lane 1), a prominent band representing the ligated
exons appeared, in addition to bands representing the linear and
circular intron. In the absence of GTP (lane 2), only the substrate band
appeared. Thus, exon ligation appears to be a part of the self-splicing
reaction catalyzed by this RNA. (Source: (b) Inane, T., F.X. Sullivan, and
T.R. Cech, Intermolecular exon ligation of the rRNA precursor of Tetrahymena:
Oligonucleotides can function as 59-exons. Cell 43 (Dec 1985) f. 1a, p. 432.
Reprinted by permission by Elsevier Science.)
representing the ligated exons appeared. By contrast, lane 2
shows that no such products appeared in the absence
of GTP; only the substrate was present. This is what we
expect because splicing of group I introns is dependent on
GTP, and it reinforces the conclusion that these products
are all the result of splicing. In summary, these data argue
strongly for true splicing, including the joining of exons.
Cech’s group had already shown that splicing of the
26S rRNA precursor involved addition of a guanine
nucleotide at the 59-end of the intron. To verify that selfsplicing in the absence of protein used the same mechanism, they performed a two-part experiment. In the
first part, they incubated the splicing precursor with
[a-32P]GTP under splicing and nonsplicing conditions,
then electrophoresed the products to see if the intron had
become labeled. Figure 14.42a shows that it had, and a
similar experiment with [g-32P]GTP gave the same results.
In the second part, these workers 59-end-labeled the intron
with [a-32P]GTP in the same way and sequenced the product. It gave exactly the sequence expected for the linear
intron, with an extra G at the 59-end (Figure 14.42b). This
G could be removed by RNase T1, demonstrating that it is
attached to the end of the intron by a normal 59-39phosphodiester bond. Figure 14.43 presents a model for
the splicing of the Tetrahymena 26S rRNA precursor, up to
the point of ligating the two exons together and formation
of the linear intron.
We have seen that the excised intron can cyclize itself.
Cech and his coworkers showed that this cyclization actually involves the loss of 15 nt from the 59-end of the linear
intron. Three lines of evidence led to this conclusion:
(1) When the 59-end of the linear intron is labeled, none of
this label appears in the circularized intron. (2) At least two
RNase T1 products (actually three) found at the 59-end of
the linear intron are missing from the circular intron.
(3) Cyclization of the intron is accompanied by the accumulation of an RNA 15-mer that contains the missing
RNase T1 products.
But this is not the end of the process. After cyclization,
the circular intron opens up again at the very same phosphodiester bond that formed the circle in the first place.
Then the intron recyclizes by removing four more nucleotides from the 59-end. Finally, the intron opens up at the
same bond that just formed, yielding a shortened linear
intron.
Figure 14.44 presents a detailed mechanism of the cyclization and relinearization of the excised intron. Notice that
throughout the splicing process, for every phosphodiester
bond that breaks a new one forms. Thus, the free energy
change of each step is near zero, so no exogenous source of
energy, such as ATP, is required. Another general feature of
the process is that the bonds that form to make the circular
introns are the same ones that break when the circle opens
up again. This tells us that these bonds are special; the threedimensional shape of the RNA must strain these bonds to
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(a)
(b)
1
2
3
4
Origin
L IVS
5
G
A
C
U
A
U
U
G
A
(30)A
A
A
G
G
G
A
G
G
U
(20)N
U
C
C
A
U
U
U
A
U
(10)A
A
C
G
A
U
A
429
Enzyme
OH–
Phy M
U2
T1
OH–
14.3 Self-Splicing RNAs
A
A
G
Figure 14.42 Addition of GMP to the 59-end of the excised
intron. (a) Radioactive GTP labels the intron during splicing. Cech
and coworkers transcribed plasmid pIVS11 under nonsplicing
conditions with no labeled nucleotides. They isolated this unlabeled
26S rRNA precursor and incubated it under splicing conditions in the
presence of [a-32P]GTP. Then they chromatographed the products on
Sephadex G-50, electrophoresed the column fractions, and
autoradiographed the gel. Lanes 1–4 are successive fractions from the
Sephadex column. Lane 5 is a linear intron marker. Lanes 2 and 3
contain the bulk of the linear intron, and it is labeled, indicating that it
had incorporated a labeled guanine nucleotide. (b) Sequence of the
make them easiest to break during relinearization. This
strain would help to explain the catalytic power of the RNA.
At first glance, there appears to be a major difference
between the splicing mechanisms of spliceosomal introns
and group I introns: Whereas the group I introns use an
exogenous nucleotide in the first step of splicing, spliceosomal introns use a nucleotide that is integral to the intron
itself. However, on closer examination we see that the difference might not be as great as it seems. Michael Yarus and
his colleagues used molecular modeling techniques to predict the lowest energy conformation of the Tetrahymena
26S rRNA intron as it associates with GMP. They proposed
that part of the intron folds into a double helix with a
pocket that holds the guanine nucleotide through hydrogen
bonds (Figure 14.45). This guanine, held fast to the intron,
labeled intron. Cech and coworkers used an enzymatic method to
sequence the 59-end of the RNA. They cut it with base (OH2 ), which
cuts after every nucleotide; RNase Phy M, which cuts after A and U;
RNase U2, which cuts after A; and RNase T1, which cuts after G.
Treatment of each RNA sample is indicated at top. The deduced
sequence is given at left. Note the 59-G at bottom. (Source: Kruger K.,
P.J. Grabowski, A.J. Zaug, J. Sands, D.E. Gottschling and T.R. Cech, Self-splicing
RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence
of Tetrahymena. Cell 31 (Nov 1982) f. 4, p. 151. Reprinted by permission of Elsevier
Science.)
behaves in essentially the same way as the adenine in spliceosomal introns. Of course, it cannot form a lariat because
it is not covalently linked to the intron.
Until the discovery of self-splicing RNAs, biochemists
thought that the catalytic parts of enzymes were made only
of protein. Sidney Altman had shown a few years earlier that
RNase P, which cleaves extra nucleotides off the 59-ends of
tRNA precursors, has an RNA component called M1. But
RNase P also has a protein component, which could have
held the catalytic activity of the enzyme. In 1983, Altman
confirmed that the M1 RNA is the catalytic component of
RNase P (Chapter 16). This enzyme and self-splicing RNAs
are examples of catalytic RNAs, which we call ribozymes.
Actually, the reactions we have seen so far, in which
group I introns participate, are not enzymatic in the strict
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Chapter 14 / RNA Processing I: Splicing
(a)
Pre-rRNA
5.8S
begins with an attack by a guanine nucleotide on
the 59-splice site, adding the G to the 59-end of the
intron, and releasing the first exon. In the second
step, the first exon attacks the 39-splice site, ligating
the two exons together, and releasing the linear intron. The intron cyclizes twice, losing nucleotides
each time, then linearizes for the last time.
26S
}
17S
Cloned
Intron
(b)
Group II Introns
Exon 1
5′
Exon 2
3′
GpU
UpA
GOH
Intron
Exon 1
5′
Exon 1
GpA
UOH
Exon 2
GpU
Exon 2
UpU
+ GpA
3′
Intron
GOH
Figure 14.43 Self-splicing of Tetrahymena rRNA precursor.
(a) Structure of the rRNA precursor, containing the 17S, 5.8S, and 26S
sequences. Note the intron within the 26S region (red). The cloned
segment used in subsequent experiments is indicated by a bracket.
(b) Self-splicing scheme. In the first step (top), a guanine nucleotide
attacks the adenine nucleotide at the 59-end of the intron, releasing
exon 1 (blue) from the rest of the molecule and generating the
hypothetical intermediates shown in brackets. In the second step,
exon 1 (blue) attacks exon 2 (yellow), performing the splicing reaction
that releases a linear intron (red), and joins the two exons together.
Finally, in a series of reactions not shown here, the linear intron loses
19 nt from its 59-end.
sense, because the RNA itself changes. A true enzyme is
supposed to emerge unchanged at the end of the reaction.
But the final linearized group I intron from the Tetrahymena 26S rRNA precursor can act as a true enzyme by
adding nucleotides to, and subtracting them from, an oligonucleotide. We should also make another qualification
about ribozymes. They can operate on their own in vitro.
But many, including many group I introns, are aided by
proteins in vivo. These proteins have no catalytic activity of
their own, but they can stabilize the catalytically active
structure of the ribozyme. As such, these ribonucleoprotein
complexes can be called RNPzymes.
SUMMARY Group I introns, such as the one in the
Tetrahymena 26S rRNA precursor, can be removed
in vitro with no help from protein. The reaction
The introns of fungal mitochondrial genes were originally
classified as group I or group II according to certain conserved sequences they contained. Later, it became clear that
mitochondrial and chloroplast genes from many species
contained group I and II introns, and that RNAs containing both classes of intron have members that are selfsplicing. However, the mechanisms of splicing used by
RNAs with group I and group II introns are different.
Whereas the initiating event in group I splicing is attack by
an independent guanine nucleotide, the initiating event in
group II splicing involves intramolecular attack by an A
residue in the intron to form a lariat.
The lariat formation by group II introns sounds very
similar to the situation in spliceosomal splicing of nuclear
mRNA precursors, and the similarity extends to the overall
shapes of the RNAs in the spliceosomal complex and of the
group II introns, as we saw in Figure 14.20. This implies a
similarity in function between the spliceosomal snRNPs
and the catalytic part of the group II introns. It may even
point to a common evolutionary origin of these RNA species. In fact, it has been proposed that nuclear pre-mRNA
introns descended from bacterial group II introns. These
bacterial introns presumably got into eukaryotic cells
because they inhabited the bacteria that invaded the precursors of modern eukaryotic cells and evolved into mitochondria. This hypothesis has become even more attractive
since the discovery of group II introns in archaea, as well as
in two classes of bacteria: cyanobacteria and purple bacteria.
If we assume that the group II introns are older than the
common ancestor of these two bacterial lineages, then they
are old enough to have inhabited the bacteria that were the
ancestors of modern eukaryotic organelles. Nevertheless,
convergent evolution to a common mechanism also remains a possibility.
SUMMARY RNAs containing group II introns self-
splice by a pathway that uses an A-branched lariat
intermediate, just like the spliceosomal lariats. The
secondary structures of the splicing complexes involving spliceosomal systems and group II introns
are also strikingly similar.
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Summary
Linear intron:
13 nt
5′G
431
UpACCUpUUG
OH
G
15 nt
(1)
(2)
GpACCUpUUG
19 nt
GpUUG
C-15
C-19
H2O
(3)
H2O
(5)
(4)
pACCU
pACCUpUUG
L-15
OH
L-19
OH
G
Figure 14.44 Fate of the linear intron. We begin with the linear
intron originally excised from the 26S rRNA precursor. This can be
cyclized in two ways: In reaction 1 (green arrows), the 39-terminal G
attacks the bond between U-15 and A-16, removing a 15-nt fragment
and giving a circular intron (C-15). In the alternative reaction (2, blue
arrows), the terminal G attacks 4 nt farther into the intron, removing a
pUUG
G
19-nt fragment and leaving a smaller circular intron (C-19). Reaction 3,
C-15 can open up at the same bond that closed the circle, yielding a
linear intron (L-15). Reaction 4, the terminal G of L-15 can attack the
bond between the first two U’s, yielding the circular intron C-19.
Reaction 5, C-19 opens up to yield the linear intron L-19.
A263
C262
G312
C311
U310
3′-OH
GMP
5′-OH
(a)
(b)
Figure 14.45 Two views of GMP held in a pocket of the 26S rRNA
intron. (a) A cross-eyed stereogram that can be viewed in three
dimensions by crossing the eyes until the two images merge. Carbon
atoms of RNA, green; carbon atoms of G, yellow; phosphorus, lavender.
Other atoms are standard colors. The GMP is at lower left. (b) Spacefilling model. Colors are as in part (a). (Source: Yarus, M., I. Illangesekare,
S U M M A RY
Nuclear mRNA precursors are spliced via a lariat-shaped,
or branched, intermediate. In addition to the consensus
sequences at the 59- and 39-ends of nuclear introns,
and E. Christian, An axial binding site in the Tetrahymena precursor RNA. Journal of
Molecular Biology. 222 (1991) f. 7c–d, p. 1005, by permission of Elsevier.)
branchpoint consensus sequences also occur. In yeast, this
sequence is nearly invariant: UACUAAC. In higher
eukaryotes, the consensus sequence is more variable:
YNCURAC. In all cases, the branched nucleotide is the
final A in the sequence. The yeast branchpoint sequence
also determines which downstream AG is the 39-splice site.
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Chapter 14 / RNA Processing I: Splicing
Splicing appears to take place on a particle called a
spliceosome. Yeast and mammalian spliceosomes have
sedimentation coefficients of about 40S and 60S,
respectively. Genetic experiments have shown that base
pairing between U1 snRNA and the 59-splice site of an
mRNA precursor is necessary, but not sufficient, for
splicing. The U6 snRNP also associates with the 59-end of
the intron by base pairing. This association first occurs
prior to formation of the lariat intermediate, but its
character may change after this first step in splicing. The
association between U6 and the splicing substrate is
essential for the splicing process. U6 also associates with
U2 during splicing.
The U2 snRNA base-pairs with the conserved
sequence at the splicing branchpoint. This base pairing is
essential for splicing. U2 also forms vital base pairs with
U6, forming a region called helix I, which apparently
helps orient these snRNPs for splicing. The U4 snRNA
base-pairs with U6, and its role seems to be to bind U6
until U6 is needed in the splicing reaction. The U5 snRNP
associates with the last nucleotide in one exon and the
first nucleotide of the next. This presumably lines the
59- and 39-splice sites up for splicing.
The spliceosomal complex (substrate, U2, U5, and U6)
poised for the second step in splicing can be drawn in the
same way as a group II intron at the same stage of
splicing. Thus, the spliceosomal snRNPs seem to
substitute for elements at the center of catalytic activity of
the group II introns, and probably have the spliceosome’s
catalytic activity.
Indeed, the catalytic center of the spliceosome appears
to include Mg21 and a base-paired complex of three
RNAs: U2 and U6 snRNAs, and the branchpoint region
of the intron. Protein-free fragments of these three RNAs
can catalyze a reaction related to the first splicing step.
The spliceosome cycle includes the assembly, splicing
activity, and disassembly of the spliceosome. Assembly
begins with the binding of U1 to the splicing substrate to
form a commitment complex. U2 is the next snRNP to
join the complex, followed by the others. The binding of
U2 requires ATP. U6 dissociates from U4, then displaces
U1 at the 59-splice site. This ATP-dependent step activates
the spliceosome and allows release of U1 and U4. The five
snRNPs that participate in splicing all contain a common
set of seven Sm proteins and several other proteins that
are specific to each snRNP. The structure of U1 snRNP
reveals that the Sm proteins form a doughnut-shaped
structure to which the other proteins are attached. A
minor class of introns with 59-splice sites and
branchpoints can be spliced with the help of a minor
spliceosome containing a variant class of snRNAs,
including U11, U12, U4atac, and U6atac.
The splicing factor Slu7 is required for correct
39-splice site selection. In its absence, splicing to the
correct 39-splice site AG is specifically suppressed and
splicing to aberrant AG’s within about 30 nt of the
branchpoint is activated. U2AF is also required for
39-splice site recognition. The 65-kD U2AF subunit binds
to the polypyrimidine tract upstream of the 39-splice site,
and the 35-kD subunit binds to the 39-splice site AG.
Commitment to splice at a given site is determined by
an RNA-binding protein, which presumably binds to the
splicing substrate and recruits other spliceosomal
components, starting with U1. For example, the SR
proteins SC35 and SF2/ASF commit splicing on human
b-globin pre-mRNA and HIV tat pre-mRNA, respectively.
In the yeast commitment complex, the branchpoint
bridging protein (BBP) binds to a U1 snRNP protein at
the 59-end of the intron, and to Mud2p near the 39-end of
the intron. It also binds to the RNA near the 39-end of the
intron. Thus, it bridges the intron and could play a role in
defining the intron prior to splicing. The mammalian
counterpart of BBP, SF1, may serve the a similar function, but
in exon definition, in the mammalian commitment complex.
The CTD of the Rpb1 subunit of RNA polymerase II
stimulates splicing of substrates that use exon definition,
but not those that use intron definition, to prepare the
substrate for splicing. The CTD binds to splicing factors
and could therefore assemble the factors at the ends of
exons to set them off for splicing.
The transcripts of many eukaryotic genes are subject to
alternative splicing. This can have profound effects on the
protein products of a gene. For example, it can make the
difference between a secreted or a membrane-bound
protein; it can even make the difference between activity
and inactivity. In the fruit fly, the products of three genes in
the sex determination pathway are subject to alternative
splicing. Female-specific splicing of the tra transcript gives
an active product that causes female-specific splicing of the
dsx pre-mRNA, which produces a female fly. Male-specific
splicing of the tra transcript gives an inactive product that
allows default, or male-specific, splicing of the dsx premRNA, producing a male fly. Tra and its partner Tra-2 act
in conjuction with one or more other SR proteins to
commit splicing at the female-specific splice site on the dsx
pre-mRNA. Such commitment is undoubtedly the basis of
most, if not all, alternative splicing schemes.
Alternative splicing is a very common phenomenon in
higher eukaryotes. It represents a way to get more than
one protein product out of the same gene, and a way to
control gene expression in cells. Such control is exerted
by splicing factors that bind to the splice sites and
branchpoint, and also by proteins that interact with
exonic splicing enhancers (ESEs), exonic splicing silencers
(ESSs), and intronic silencing elements. SR proteins
tend to bind to ESEs, while hnRNP proteins, such as
hnRNP A1, bind to ESSs and intronic silencing elements.
Group I introns, such as the one in the Tetrahymena
26S rRNA precursor, can be removed with no help from
protein in vitro. The reaction begins with an attack by a
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Review Questions
guanine nucleotide on the 59-splice site, adding the G to
the 59-end of the intron and releasing the first exon. In the
second step, the first exon attacks the 39-splice site,
ligating the two exons together and releasing the linear
intron. The intron cyclizes twice, losing nucleotides each
time, then linearizes for the last time.
RNAs containing group II introns self-splice by a
pathway that uses an A-branched lariat intermediate, just
like the spliceosomal lariats. The secondary structures of
the splicing complexes involving spliceosomal systems and
group II introns are also strikingly similar.
433
15. Describe and show the results of an experiment that
demonstrates that U5 contacts the 39-end of the upstream
exon and the 59-end of the downstream exon during
splicing. Make sure your experiment(s) provide positive
identification of the RNA species involved, not just
electrophoretic mobilities.
16. Describe and show the results of an experiment that
demonstrates which bases in U5 can be cross-linked to
bases in the pre-mRNA.
17. Summarize the evidence for a catalytic Mg21 in spliceosomal
splicing.
18. Summarize the evidence that a mixture of spliceosonal
RNA fragments can catalyze a reaction related to the first
splicing step.
REVIEW QUESTIONS
1. Describe and show the results of an R-looping experiment
that demonstrates that an intron is transcribed.
2. Diagram the lariat mechanism of splicing.
3. Present gel electrophoretic data that suggest that the excised
intron is circular, or lariat-shaped.
4. Present gel electrophoretic data that distinguish between a
lariat-shaped splicing intermediate (the intron—exon-2
intermediate) and a lariat-shaped product (the excised
intron).
5. The lariat model predicts an intermediate with a branched
nucleotide. Describe and show the results of an experiment
that confirms this prediction.
6. Describe and give the results of an experiment that shows
that a sequence (UACUAAC) within a yeast intron is
required for splicing.
7. Describe and show the results of an experiment that
demonstrates that the UACUAAC sequence within a yeast
intron dictates splicing to an AG downstream.
8. What role does the UACUAAC sequence play in the lariat
model of splicing?
9. Describe and show the results of an experiment that
demonstrates that yeast spliceosomes have a sedimentation
coefficient of 40S.
10. Describe and show the results of an experiment that
demonstrates that base pairing between U1 snRNA and the
59-splice site is required for splicing.
11. Describe and show the results of an experiment that
demonstrates that base pairing between U1 and the
59-splice site is not sufficient for splicing.
12. What snRNP besides U1 and U5 must bind near the 59-splice
site in order for splicing to occur? Present cross-linking data
to support this conclusion.
13. Describe and show the results of an experiment that
demonstrates that base pairing between U2 snRNA and the
branchpoint sequence is required for splicing. In this
experiment, why was it not possible to mutate the cell9s
only copy of the U2 gene?
14. Besides base-pairing with the pre-mRNA, U6 base-pairs
with two snRNAs. Which ones are they?
19. Draw a diagram of a pre-mRNA as it exists in a
spliceosome just before the second step in splicing. Show
the interactions with U2, U5, and U6 snRNPs. This scheme
resembles the intermediate stage for splicing of what kind
of self-splicing RNA?
20. Describe and show the results of an experiment that
demonstrates that U1 is the first snRNP to bind to the
splicing substrate.
21. Describe and show the results of an experiment that
demonstrates that binding of all other snRNPs to the
spliceosome depends on U1, and that binding of U2
requires ATP.
22. What are Sm proteins?
23. How do the characteristics of minor spliceosomes help
show the importance of base-pairing between snRNAs and
pre-mRNA sites?
24. Describe and show the results of an experiment that
demonstrates that Slu7 is required for selection of the
proper AG at the 39-splice site.
25. Describe a splicing commitment assay to screen for splicing
factors involved in commitment. Show sample results.
26. Describe and give the results of a yeast two-hybrid assay
that shows interaction between yeast branchpoint bridging
protein (BBP) and two other proteins. What are the two
other proteins, and where are they found with respect to the
ends of the intron in the commitment complex?
27. Describe and give the results of an experiment that shows
that the RNA polymerase II CTD stimulates splicing of
pre-mRNAs that use exon definition.
28. Diagram the alternative splicing of the immunoglobulin m
heavy-chain transcript. Focus on the exons that are involved
in one or the other of the alternative pathways, rather than
the ones that are involved in both. What difference in the
protein products is caused by the two pathways of splicing?
29. Describe a computational and an experimental method to
identify sequences that act as exonic splicing silencers (ESSs).
30. Describe and show the results of an experiment that
demonstrates self-splicing by a group I intron.
31. Describe and show the results of an experiment that
demonstrates that a guanine nucleotide is added to the end
of a spliced-out group I intron.
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Chapter 14 / RNA Processing I: Splicing
32. Draw a diagram of the steps involved in autosplicing of an
RNA containing a group I intron. You do not need to show
cyclization of the intron.
33. Diagram the steps involved in forming the L-19 intron from
the original excised linear intron product of the Tetrahymena
26S pre-rRNA. Do not go through the C-15 intermediate.
A N A LY T I C A L Q U E S T I O N S
1. You are investigating a gene with one large intron and two
short exons. Show the results of R-looping experiments
performed with:
a. mRNA and single-stranded DNA
b. mRNA and double-stranded DNA
c. mRNA precursor and single-stranded DNA
d. mRNA precursor and double-stranded DNA
2. You have discovered a new class of introns that do not
require any proteins for splicing, but do require several
small RNAs. One of these small RNAs, V3, has a sequence
of 7 nt (CCUUGAG) complementary to the 39-splice site.
You suspect that base-pairing between V3 and the 39-splice
site is required for splicing. Design an experiment to test
this hypothesis and show sample positive results.
3. Diagram the mechanism of RNase T1 (or T2) action. Because
this is the same mechanism used in base hydrolysis, how does
this explain why DNA is not subject to base hydrolysis?
4. You are studying a grave human disease called
b-thalassemia in which no b-globin protein is produced.
You find that the b-globin gene’s coding region in people
with this disease is normal, but the mRNA is over a
hundred nucleotides longer than normal. You sequence the
b-globin gene in these people and find a single base change
within the gene’s first intron. Present a hypothesis to
explain the absence of b-globin in these patients.
5. Consider the gene illustrated in Figure 14.38, but remove
P2 and poly(A)1, so there is only one promoter (P1) and one
polyadenylation site [poly(A)2]. How many different spliced
mRNAs can now be produced by this gene?
6. Consider the RNA sequencing results in Figure 14.42b.
Knowing the cutting specificities of each enzyme, how do
we know (a) that the band at the bottom in the first lane
represents G? (b) that the next band represents A? (c) that
the eighth band from the bottom represents C? (d) that the
13th, 14th, and 15th bands from the bottom represent U’s?
(Hint: PhyM cut inefficiently after U’s in this experiment.)
SUGGESTED READINGS
General References and Reviews
Baker, B.S. 1989. Sex in flies: The splice of life. Nature
340:521–24.
Black, D.L. 2003. Mechanisms of alternative pre-messenger
RNA splicing. Annual Review of Biochemistry 72:291–336.
Fu, X.-D. 2004. Towards a splicing code. Cell 119:736–38.
Guthrie, C. 1991. Messenger RNA splicing in yeast: Clues to
why the spliceosome is a ribonucleoprotein. Science
253:157–63.
Hirose, Y. and J.L. Manley. 2000. RNA polymerase II and the
integration of nuclear events. Genes and Development
14:1415–29.
Lamm, G.M. and A.I. Lamond. 1993. Non-snRNP protein
splicing factors. Biochimica et Biophysica Acta 1173:247–65.
Manley, J. 1993. Question of commitment. Nature 365:14.
Murray, H.L. and K.A. Jarrell. 1999. Flipping the switch to an
active spliceosome. Cell 96:599–602.
Newman, A. 2002. RNA enzymes for RNA splicing. Nature
413:695–96.
Nilsen, T.W. 2000. The case for an RNA enzyme. Nature
408:782–83.
Orphanides, G. and D. Reinberg. 2002. A unified theory of gene
expression. Cell 108:439–51.
Proudfoot, N., A. Furger, and M.J. Dye. 2002. Integrating
mRNA processing with transcription. Cell 108:501–12.
Sharp, P.A. 1994. Split genes and RNA splicing. (Nobel Lecture.)
Cell 77:805–15.
Villa, T., J.A. Pleiss, and C. Guthrie. 2002. Spliceosomal
snRNAs: Mg21-dependent chemistry at the catalytic core?
Cell 109:149–52.
Weiner, A.M. 1993. mRNA splicing and autocatalytic introns:
Distant cousins or the products of chemical determinism?
Cell 72:161–64.
Wise, J.A. 1993. Guides to the heart of the spliceosome. Science
262:1978–79.
Research Articles
Abovich, N. and M. Rosbash. 1997. Cross-intron bridging
interactions in the yeast commitment complex are conserved
in mammals. Cell 89:403–12.
Berget, S.M., C. Moore, and P. Sharp. 1977. Spliced segments at
the 59 terminus of adenovirus 2 late mRNA. Proceedings of
the National Academy of Sciences USA 74:3171–75.
Berglund, J.A., K. Chua, N. Abovich, R. Reed, and M. Rosbash.
1997. The splicing factor BBP interacts specifically with the
pre-mRNA branchpoint sequence UACUAAC. Cell 89:781–87.
Brody, E. and J. Abelson. 1985. The spliceosome: Yeast premessenger RNA associated with a 40S complex in a
splicing-dependent reaction. Science 228:963–67.
Chua, K. and R. Reed. 1999. The RNA splicing factor hSlu7 is
required for correct 39 splice-site choice. Nature
402:207–10.
Early, P., J. Rogers, M. Davis, K. Calame, M. Bond, R. Wall, and
L. Hood. 1980. Two mRNAs can be produced from a single
immunoglobulin g gene by alternative RNA processing
pathways. Cell 20:313–19.
Fu, X.-D. 1993. Specific commitment of different pre-mRNAs to
splicing by single SR proteins. Nature 365:82–85.
Grabowski, P., R.A. Padgett, and P.A. Sharp. 1984. Messenger
RNA splicing in vitro: An excised intervening sequence and a
potential intermediate. Cell 37:415–27.
Grabowski, P., S.R. Seiler, and P.A. Sharp. 1985. A
multicomponent complex is involved in the splicing of
messenger RNA precursors. Cell 42:345–53.
wea25324_ch14_394-435.indd Page 435
13/12/10
7:24 AM user-f467
/Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile
Suggested Readings
Kruger, K., P.J. Grabowski, A.J. Zaug, J. Sands,
D.E. Gottschling, and T.R. Cech. 1982. Self-splicing RNA:
Autoexcision and autocylization of the ribosomal RNA
intervening sequence of Tetrahymena. Cell 31:147–57.
Langford, C. and D. Gallwitz. 1983. Evidence for an introncontained sequence required for the splicing of yeast RNA
polymerase II transcripts. Cell 33:519–27.
Lesser, C.F. and C. Guthrie. 1993. Mutations in U6 snRNA that
alter splice site specificity: Implications for the active site.
Science 262:1982–88.
Liao, X.C., J. Tang, and M. Rosbash. 1993. An enhancer screen
identifies a gene that encodes the yeast U1 snRNP A protein:
Implications for snRNP protein function in pre-mRNA
splicing. Genes and Development 7:419–28.
Parker, R., P.G. Siliciano, and C. Guthrie. 1987. Recognition of
the TACTAAC box during mRNA splicing in yeast involves
base pairing to the U2-like snRNA. Cell 49:229–39.
Peebles, C.L., P. Gegenheimer, and J. Abelson. 1983. Precise
excision of intervening sequences from precursor tRNAs by a
membrane-associated yeast endonuclease. Cell 32:525–36.
Ruby, S.W. and J. Abelson. 1988. An early hierarchic role of U1
small nuclear ribonucleoprotein in spliceosome assembly.
Science 242:1028–35.
Sontheimer, E.J. and J.A. Steitz. 1993. The U5 and U6 small
nuclear RNAs as active site components of the spliceosome.
Science 262:1989–96.
Staley, J.P. and C. Guthrie. 1999. An RNA switch at the
59 splice site requires ATP and the DEAD box protein Prp28p.
Molecular Cell 3:55–64.
435
Stark, H., P. Dube, R. Lührmann and B. Kastner. 2001.
Arrangement of RNA and proteins in the spliceosomal U1
small nuclear ribonucleoprotein particle. Nature 409:539–42.
Tian, M. and T. Maniatis. 1993. A splicing enhancer complex
controls alternative splicing of doublesex pre-mRNA. Cell
74:105–14.
Tilghman, S.M., P. Curtis, D. Tiemeier, P. Leder, and
C. Weissmann. 1978. The intervening sequence of a mouse
b-globin gene is transcribed within the 15S b-globin mRNA
precursor. Proceedings of the National Academy of Sciences
USA 75:1309–13.
Valadkhan, S. and J.L. Manley. 2001. Splicing-related catalysis
by protein-free snRNAs. Nature 413:701–07.
Wang, Z., M.E. Rolish, G. Yeo, V. Tung, M. Mawson, and
C.B. Burge. 2004. Systematic identification and analysis of
exonic splicing silencers. Cell 119:831–45.
Yarus, M., I. Illangesekare, and E. Christian. 1991. An axial
binding site in the Tetrahymena precursor RNA. Journal of
Molecular Biology 222:995–1012.
Yean, S.-L., G. Wuenschell, J. Termini, and R.-J. Lin. 2001.
Metal-ion coordination by U6 small nuclear RNA contributes
to catalysis in the spliceosome. Nature 408:881–84.
Zeng, C. and S.M. Berget. 2000. Participation of the C-terminal
domain of RNA polymerase II in exon definition during
pre-mRNA splicing. Molecular and Cellular Biology
20:8290–8301.
Zhuang, Y. and A.M. Weiner. 1986. A compensatory base change
in U1 snRNA suppresses a 59-splice site mutation. Cell
46:827–35.