Molecular Biology

Robert F. Weaver

Chapter 14 RNA Processing I: Splicing - all with Video Answers

Educators

Chapter Questions

Problem 1

Describe and show the results of an R-looping experiment that demonstrates that an intron is transcribed.

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02:57

You are investigating a gene with one large intron and two short exons. Show the results of R-looping experiments performed with:
a. $m R N A$ and single-stranded DNA
b. mRNA and double-stranded DNA
c. mRNA precursor and single-stranded DNA
d. mRNA precursor and double-stranded DNA

Joanna Quigley

Numerade Educator

Problem 2

Diagram the lariat mechanism of splicing.

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Problem 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 $7 \mathrm{nt}$ (CCUUGAG) complementary to the 3'-splice site. You suspect that base-pairing between V3 and the $3^{\prime}$-splice site is required for splicing. Design an experiment to test this hypothesis and show sample positive results.

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Problem 3

Present gel electrophoretic data that suggest that the excised intron is circular, or lariat-shaped.

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01:08

Problem 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?

Hailey Tomashek

Numerade Educator

Problem 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).

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02:32

Problem 4

You are studying a grave human disease called $\beta$-thalassemia in which no $\beta$-globin protein is produced. You find that the $\beta$-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 $\beta$-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 $\beta$-globin in these patients.

John Barone

Numerade Educator

Problem 5

The lariat model predicts an intermediate with a branched nucleotide. Describe and show the results of an experiment that confirms this prediction.

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02:53

Problem 5

Consider the gene illustrated in Figure 14.38, but remove $P_2$ and poly $(\mathrm{A})_1$, so there is only one promoter $\left(P_1\right)$ and one polyadenylation site [poly $\left.(\mathrm{A})_2\right]$. How many different spliced mRNAs can now be produced by this gene?

Mikayla Stephens

Numerade Educator

05:26

Problem 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 13 th, 14 th, and 15 th bands from the bottom represent U's?

Eric Goldman

Numerade Educator

Problem 6

Describe and give the results of an experiment that shows that a sequence (UACUAAC) within a yeast intron is required for splicing.

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Problem 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.

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01:51

Problem 8

What role does the UACUAAC sequence play in the lariat model of splicing?

Rashmi Gondi

Numerade Educator

Problem 9

Describe and show the results of an experiment that demonstrates that yeast spliceosomes have a sedimentation coefficient of $40 S$.

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Problem 10

Describe and show the results of an experiment that demonstrates that base pairing between U1 snRNA and the $5^{\prime}$-splice site is required for splicing.

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Problem 11

Describe and show the results of an experiment that demonstrates that base pairing between $\mathrm{U} 1$ and the 5 '-splice site is not sufficient for splicing.

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Problem 12

What snRNP besides U1 and U5 must bind near the 5 '-splice site in order for splicing to occur? Present cross-linking data to support this conclusion.

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Problem 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 cell's only copy of the U2 gene?

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02:29

Problem 14

Besides base-pairing with the pre-mRNA, U6 base-pairs with two snRNAs. Which ones are they?

Ronald Prasad

Numerade Educator

Problem 15

Describe and show the results of an experiment that demonstrates that U5 contacts the 3'-end of the upstream exon and the 5 '-end of the downstream exon during splicing. Make sure your experiment(s) provide positive identification of the RNA species involved, not just electrophoretic mobilities.

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Problem 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.

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Problem 17

Summarize the evidence for a catalytic $\mathrm{Mg}^{2+}$ in spliceosomal splicing.

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Problem 18

Summarize the evidence that a mixture of spliceosonal RNA fragments can catalyze a reaction related to the first splicing step.

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01:16

Problem 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?

Joanna Quigley

Numerade Educator

Problem 20

Describe and show the results of an experiment that demonstrates that U1 is the first snRNP to bind to the splicing substrate.

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Problem 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.

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04:52

Problem 22

What are $\mathrm{Sm}$ proteins?

John Barone

Numerade Educator

03:08

Problem 23

How do the characteristics of minor spliceosomes help show the importance of base-pairing between snRNAs and pre-mRNA sites?

Shiksha Dutta

Numerade Educator

Problem 24

Describe and show the results of an experiment that demonstrates that Slu7 is required for selection of the proper AG at the 3 '-splice site.

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Problem 25

Describe a splicing commitment assay to screen for splicing factors involved in commitment. Show sample results.

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03:48

Problem 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?

Rabeya Zahid

Numerade Educator

Problem 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.

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00:04

Problem 28

Diagram the alternative splicing of the immunoglobulin $\mu$ 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?

Maxime Rossetti

Numerade Educator

01:05

Problem 29

Describe a computational and an experimental method to identify sequences that act as exonic splicing silencers (ESSs).

Mikayla Stephens

Numerade Educator

Problem 30

Describe and show the results of an experiment that demonstrates self-splicing by a group I intron.

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Problem 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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00:47

Problem 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.

Mikayla Stephens

Numerade Educator

Problem 33

Diagram the steps involved in forming the L-19 intron from the original excised linear intron product of the Tetralymena 26S pre-rRNA. Do not go through the C-15 intermediate.

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