Questions Study Lectures 15/16 RNA processing and splicing
Question 1. Radiolabeled pre-mRNA has been incubated with nuclear extract and ATP to carry
out an in vitro splicing reaction. Three different precursors are used. The first (1)
contains a mutation that abolishes interaction of the 5 splice site with U1, the second
(2) contains all sequences sufficient to promote splicing. And the third (3), contains a mutation at
the 3 splice site.
a) (5) For letters a-e identify the name of the molecule that best describes the
structure to the right:
__d__ spliced product
__a__ lariat intermediate
__c__ unspliced precursor
__b__ lariat intron
__e__ free exon 1
b) (3) Indicate which step(s) of splicing, if any (i.e. step 1 only; step 1 and step 2; or
no splicing) has successfully occurred using the three different precursor substrates.
1. ___no splicing_____
2. ___Step1 and Step 2___
3. ___only step 1______
Question 2. Provide a brief statement of why each of the statements below is false:
a) Addit ion of the 7-me G cap and loading of splicing factors occurs co -transcriptionally.
Following transcription terminat ion, the transcript is exported from the nucleus and then
polyadenylated.
Poly-adenlyation occurs in the nucleus prior to the transcript being exported
b) The first step of splicing involves the nucleophilic attack of the 2 OH of the branch site
nucleotide at the 5 splice site to give two products: a free exon 1 terminating with a 3
phosphate and a branched lariat structure containing the unusual 2-5 phosphodiester linkage
involving the first nucleotide of the intron and the branch site nucleotide.
The free exon 1 that follows the first step of splicing terminates in a 3OH
d) The term snRNP stands for small nuclear RNA Particle.
SnRNP stands for small nuclear ribonucleo-protein (particle or complex)
Question 3. Mark the single most correct answer to the questions below.
Which of the following cannot be co -transcriptional (i. e. cannot occur on an RNA
prior to termination of transcription of that RNA) for a eukaryotic nuclear gene?
_________Recognition by polyadenylation factors.
_________Capping.
_____X___ Translation.
________Splicing.
________Intron recognition by splicing factors
Which of the following is not encoded in the gene sequence and not transcribed into the premRNA?
________ The AAUAAA signal for 3 cleavage of the mRNA transcript and addition of polyA
____X____ The poly-A tail of ~150 residues
________ The branch-point A residue that is involved in the first transesterification reaction
________ The 5 splice site GU and the 3 splice site AG nucleotides
Question 4. You have been gazing at the nucleotide sequences of the U2 and U6 snRNAs and
notice a region of complementary sequences between the two which has not been described to
date. You wonder if this potential base pairing interaction might play a role in the process of
mRNA splicing. To address this problem, you make a C to G mutation in U2 (called U2CG) and
a G to C mutation in U6 (called U6GC) at nucleotides that would presumably destabilize the
putative base pair interaction. The two mutations combined (U2CG/U6GC ) would re store base
pairing interaction. You make nuclear extracts containing mutated forms of U2 and/or U6 and
add radiolabeled pre-mRNA and ATP. After 20 minutes incubation you isolate RNA and run
these samples on a polyacrylamide gel. As a control you include a reaction using extract with
nonmutated forms of the snRNAs (U2/U6). Below is a n autoradiogram of what you see.
a) Indicate the products and/or intermediates of the splicing reaction using a diagram to the left
of the gel. The pre-mRNA substrate is given. See above.
b) Which step of splicing appears to be affected by U2CG and the U6GC mutations?
Second step: The mutations have allowed for the first step of splicing since the lariat
intermediate and unligated exon 1 are present. Since no spliced product or lariat intron
product is seen we can conclude that the mutations affect the 2nd step of splicing
c) From these results do you think your hypothesis is correct? Explain briefly.
Yes, since the combined mutations in U2 and U6 allow for the completion of splicing we
can infer that this potential basepair interaction plays a role during the second step of
splicing
Question 7. (6) Indicate if the following processes take place in the nucleus (N) or the cytosol
(C) in a eukaryotic cell.
___N___ Splicing
__N____ mRNA Capping
__N____ Alternative splicing
___C___ Translation
____N____ Addition of the Poly-A tail
Question 8. You are studying RNA splicing, which occurs via two transesterification reactions.
Below is a schematic showing an intron and two exons.
ApG
pGpU ------------pAp---------------Yn---ApGp G
(Y = pyrimidine; p = phosphate of phosphodieser backbone)
a) On the above diagram, circle all region(s) of the pre -mRNA where there is basepairing
with U snRNA(s).
U1 snRNA basepairs with the 5 splice site initially, and is then displaced by U6 pairing in
the same region. U2 snRNA basepairs with the branchpoint. These are the major base
pairing interactions. U5 can interact with sequences in the 5 and 3 exon junctions to
facilitate their interaction in the second step of splicing, but there is not a strong consensus
for the binding interaction)
b) Draw out the product(s) of the first transesterification reaction below. Only indicate
the RNA, but be sure to include all of the bases and phosphate groups shown above; use
boxes to indicate the exons.
Question 9. You have isolated a protein called PATCH (PCH) that you believe is involved in
RNA splicing. To test its ability to bind to pre-mRNAs, you use the following vectors for
recombinant protein expression in E. coli:
1) an pET empty vector that doesnt make any protein
2) pET-PCH a vector that will produce the PCH protein
You perform electrophoretic mobility shift assays using crude extracts made from bacterial cells
containing the above vectors (bacterial proteins plus proteins t hat would be made from the
expression vector). Your probe is a radioactively labeled pre-mRNA (pictured below, labeled
wild-type). In a few reactions, you add a 100x excess of unlabeled (cold) competitor premRNA; this competitor is either wild type or one of two mutant sequences at either the 5 or 3
splice site (pictured below).
Below is an autoradiogram showing the results of your gel-shift assays. Use this information to
answer the questions below.
a) (4) What is the likely molecular nature of the band labeled A?
__X____ pre-mRNA complexed with a bacterial protein It is present in all lanes, even the lane
with the vector alone which would not produce any recombinant protein.
______ free pre-mRNA
______ pre-mRNA splicing intermediate
______ a labeled bacterial protein
b) Does PCH bind to the pre-mRNA in a sequence-specific manner?
Yes
No
(circle one)
If yes, to which region of the pre-mRNA does it bind?
The 5 splice site; the pre-mRNA with a mutant 5 splice site does not compete for PCH
binding, but the wild-type and mutant 3splice site do (both have a wild-type 5 ss sequence).
Question 10. A student has identified a new plant gene by insertiona l mutagenesis (getting a
mutant by randomly inserting transposons or transfo rming DNA in the genome). The student was
then able to isolate the gene with the insert contained within the coding region (shown below).
The student wants to express the gene in transgenic plants but the presence of the insert would
interrupt the coding region. In an attempt to directly use the currently cloned interrupted gene the
student engineers a "GT" at the 5'- end (relative to transcription) of the insert and an "AG" at the
3'-end of the insert.
a) Why were these sequences engineered at the ends of the insert, and what did the
student expect the sequence changes to accomplish?
These are the consensus sequences for 5' and 3' ends of an intron. The student
appears to hope that the introduction of these sequences will cause the insert to be
treated as an intron so that that it will be spliced from the transcript of the
introduced gene. This would reconstitute the intact coding region in the resulting
mRNA allowing the gene to be expressed.
b) Do you think this approach is likely to work? Why or why not?
Probably not. While these consensus sequences are usually essential for splicing,
they are not sufficient. Other nucleotides around the splice junctions are necessary
for proper recognition by the splicing apparatus. In addition, there must be a
proper branch site near the 3'-splice site, and other sequences which aid in
recruitment of "RS" domain proteins are likely also necessary
Question 11. The diagram below illustrates the alternative splicing of a newly discovered mouse
gene called JAVA. Alternative splicing of the JAVA transcript produces one mRNA in the
stomach and another in the brain as indicated by the lines linking exons (boxes, each
numbered below). Sequence elements in the introns that have been shown to be involved in
regulation of this alternative splicing are indicated by U8C (the sequence UUUUUUUUC) and
Pu-rich (a purine rich region). The U8C region is a sequence that binds well to U2AF (it is
similar to the typical pyrimidine-rich consensus sequence in that region). On the other hand, the
Pu-rich sequence does not bind well to U2AF. Each of these sequences have been shown to bind
a protein that regulates the alternative splicing of those exons. You have data that indicates that
these regulatory proteins are both expressed in t he brain. Answer the questions below.
(A.) Proteins which regulate splicing of JAVA are most likely to reside in (circle one):
a) the cytosol
b) the nucleus
e) the plasma membrane
c) the telomeres
d) mitochondria
f) nucleosomes
(B.) Circle the correct answer for each statement :
The default splicing pattern is represented by the BRAIN / STOMACH
pattern.
The regulatory protein that binds to U8C is probably BLOCKING / ACTIVATING
splice of exon 1 to exon 2.
The protein that binds to the Pu-rich element is probably
splice of exon 4 to exon 5.
BLOCKING / ACTIVATING
the
the
Explain your reasoning.
U2AF binding helps to recruit the U2 snRNP to the branchpoint regi on to identify the 3
splice site. The U8C sequence binds well to U2AF, and would be likely to be used in the default
splicing pattern, which would be expected to join Exon 1 to Exon 2. The protein which
controls the alternative splicing pattern is known to be expressed in the brain. This would be
consistent with the brain splice being the alternative pattern, directed by binding of a protein
to the U8C sequence to block its normal default interaction with U2AF, forcing the use of the
next 3 splice site in front of exon 3.
The Pu-rich sequence was described as not binding well to U2AF. It would therefore not be
likely to be used in a default splicing pattern, making the splice between Exon 4 and Exon 6
the most likely default pattern. The brain-specific protein that controls alternative splicing
would therefore be expected to encode a protein that would activate the splice to Exon 5,
perhaps by being able to bind to that sequence and attract the U2snRNP, replacing the
function of U2AF.
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