Splicing

Question 1

When were introns discovered, and by whom?

Answer 1

In 1977 by Richard J Roberts, and Phillip A Sharp

Question 2

Roberts and Sharp discovered introns through their examination of the genetic material in which virus? Why?

Answer 2

Adenovirus (causes common cold)

Because it infect the cells of higher organisms, and its genome has many properties resembling those of the host cell.
ALSO has a simple structure

Question 3

Why were Roberts and Sharp studying adenovirus specifically?

Answer 3

It infects the cells of higher organisms, and its genome has many similar properties to the host cell

It has a simple structure, so more readily studied

Question 4

Why did Sharp and Roberts suspect that introns existed, when it was widely assumed that nuclear DNA was directly transferred to RNA?

Answer 4

Because the genome of many animals contained such large amounts of DNA that the possibility that it all encoded simple, bacteria-type genes seemed unlikely.

In addition, unusually long RNA was detected in the nucleus compared to the shorter mRNA that emerged in the cytoplasm

Question 5

How many introns do vertebrates typically have per gene?

Answer 5

10

Question 6

About ? of all mutations in the globin gees causing Beta-thalassemia in humans results from defects in?

Answer 6

1/4 …. splicing

Question 7

Defects in splicing result in mutations in which genes that cause B-thalassemia?

Answer 7

Globin genes

Question 8

When was alternative splicing first discovered?

Answer 8

1977

Question 9

How do primary transcripts (ie pre-mRNA) differ from mRNA?

Answer 9

They are bigger

Question 10

Why is pre-mRNA bigger than mRNA?

Answer 10

It contains introns

Question 11

When was the first example of alternative splicing in a transcript from a normal, endogenous gene characterised?

Answer 11

1981

Question 12

What was the first normal endogenous gene to have its alternative splicing characterised?

Answer 12

The mammalian gene encoding the thyroid hormone calcitonin

Question 13

The primary transcript from the calcitonin-encoding gene contains how many exons?

Answer 13

6

Question 14

The calcitonin-encoding gene produces a primary transcript containing 6 exons. It can be alternatively spliced to produce which mRNA products, and which exons do these contain?

Answer 14

Calcitonin mRNA: exons 1-4 (terminates after a polyA site in exon 4)

CGRP mRNA: exons 1-3 and 5-6 (SKIPS exon 4)

Question 15

Calcitonin gene related peptide (CGRP) is synthesised from the calcitonin-encoding gene. How?

Answer 15

Alternative splicing: It skips exon 4 in the primary transcript to produce an mRNA containing exons 1-3 and 5-6.

Question 16

The D.melanogaster gene, Dscam, is the ‘record holder’ for alternative splicing. How many splice variants may it have?

Answer 16

38,016 (approx. 38,000)

Question 17

Which gene is thought to have the most splice variants? How many splice variants is it thought to have?

Answer 17

Dscam (a D.melanogaster gene) … 38,016 splice variants

Question 18

Which 4 main techniques were used in the discovery of splicing?

Answer 18

1) Southern blot with cDNA probe
2) Electron microscopy of (viral) cDNA with genome
3) Nuclease S1 mapping of boundaries relative to restriction enzyme sites with probes from cloned genomic DNA
4) Northern blot probed with cDNA

Question 19

As a result of alternative splicing, different mRNAs producing different proteins may be generated from the same adenovirus primary transcript. How are these mRNAs similar?

Answer 19

They share the same 5’ sequence, which is stitched together from three short non-protein-coding sequences known as the ‘tripartite leader’

Question 20

What was R-loop mapping used for? What does it involve?

Answer 20

An experiment that elucidated that mRNA is formed from RNAs arising from several regions of the genome.

It involves incubating RNA with dsDNA containing a sequence complementary to the RNA. The RNA anneals to its complement, displacing a stretch of the non-complementary strand in the form of a loop.
Following the staining procedure used to visualise nucleic acids, the R-loop can be observed in the electron microscope.

Question 21

Why can the R-loop be visualised by electron microscopy?

Answer 21

Because RNA-DNA and DNA-DNA duplexes appear thicker than single-stranded nucleic acids

Question 22

In identifying the splice sites on pre-mRNA, why did mutating conserved sequences not work well with transfected genes?

Answer 22

It was hard to prove that low levels of expression were caused by a failure to splice, because:

1) the pre-mRNA degraded rather than accumulate
2) many conserved nucleotides had little effect.

Question 23

Why might it be that mutating conserved nucleotides not work well in determining splice sites in pre-mRNA?

Answer 23

Perhaps because, in the absence of other choices, slow splicing at poor sites didn’t affect expression overall because there were other rate-limiting steps

Question 24

Mutating conserved sequences (or sequences identified by deletions) did not work well with transfected genes when trying to determine the splice site. What experiments were carried out instead?

Answer 24

Splice site sequences were duplicated, so that they were in competition. Then one could be mutated systematically

Question 25

Where is the 5’SS located on pre-mRNA?

Answer 25

At the exon-intron boundary (3’-end of the 5’exon; 5’-end of intron)

Question 26

Where is the 3’SS on pre-mRNA?

Answer 26

At the intron-exon boundary (ie the 3’-end of the intron, and the 5’-end of the 3’-exon)

Question 27

What are the names of the three sequences required for splicing?

Answer 27

5’SS, 3’SS and the branchpoint site

Question 28

Where is the branchpoint site located?

Answer 28

In the middle of the intron, towards the 3’-end, followed by a polypyrimidine (Py) tract

Question 29

What are the consensus sequences for the 5’SS and 3’SS, and what is the conserved residue found in the branchpoint site?

Answer 29

5’SS: C/A, A, G, G, U, A/G, A, G, U
Branchpoint: Y, N, Y, U, R, A, Y (A is the conserved residue)
3’SS: Y, N, C, A, G, G

Question 30

Which residues are conserved at the 5’ end of the intron, and which are conserved at the 3’ end?

Answer 30

5’: GU

3’: AG

Question 31

Who discovered the presence of introns within genes?

Answer 31

Richard J Roberts (Cold Spring Harbor Laboratory)

Phillip A Sharp (Massachusetts Institute of Technology)

Question 32

Approximately how big are exons, and how big can introns be?

Answer 32

exons: 150 nucleotides
introns: up to 800,000 nucleotides

Question 33

What are two types of multi-step splicing in large introns?

Answer 33

Recursive splicing

Nested splicing

Question 34

What is the predominant reason behind the great variation of sizes in human genes?

Answer 34

The variation in intron sizes

Question 35

How has the mechanism of splicing so far been studied, and what does this mean?

Answer 35

Mainly in small introns (100-250 nt). These splice efficiently in vitro & in vivo. Co-transcriptional splicing mechanisms of larger introns remain to be confirmed

Question 36

Multi-step splicing may take place in large introns. Give an example of an organism containing genes with large introns.

Answer 36

The fruit fly (Drosophila Melanogaster)

Question 37

For larger introns, multi-step splicing may take place. Recursive splicing is an example of this. What does recursive splicing involve?

Answer 37

The stepwise removal of introns by sequential re-splicing at composite 3’/5’SSs

Question 38

The human dystrophin (DMD) gene is one of the largest annotated genes. How big is it? What size primary transcript does it generate, and how many coding exons does it contain?

Answer 38

More than 2.5 Mbp
Generates primary transcript ~14kb
Containing 79 coding exons

Question 39

Mutations in the human dystrophin (DMD) gene often results in which conditions? Why?

Answer 39

the Duchenne and Becker muscular dystrophies

..because it encodes the essential 427 kDa dystrophin protein in skeletal and cardiac muscles

Question 40

What percentage of the human dystrophin (DMD) gene is made up of introns?

Answer 40

More than 99%

Question 41

A type of multi-step splicing known as ‘Nested Splicing’ is thought to occur in large intron 7 (~110k nt in size) of the DMD pre-mRNA. What does ‘nested splicing’ involve? What does it do?

Answer 41

‘intrasplicing’ - i.e. splicing events within the authentic 5’SS and 3’SS of the intron

..this brings the authentic splice sites together into close proximity to facilitate the final splicing, removing the huge intron eventually

Question 42

Give an example of an intron of a gene in which nested splicing is thought to occur

Answer 42

Large intron 7 of the human dystrophin (DMD) gene

Question 43

What is the purpose of nested splicing?

Answer 43

Brings the ‘authentic’ 5’SS and 3’SS together to facilitate splicing of the whole intron