RNA splicing

Question 1

Is splicing co or post-transcriptional?

Answer 1

Cotranscriptional

Question 2

What is the function of splicing?

Answer 2

To remove introns and join exons together

Question 3

What are the 3 key highly conserved sequences needed for splicing?

Answer 3

1. A 5’ GU splice site
2. A branch site (A)
3. A 3’ AG splice site

Question 4

What carries out splicing?

Answer 4

The spliceosome

Question 5

What is the spliceosome?

Answer 5

Complex of proteins and snRNA that assemble into ribonucleoprotein complexes (snRNP), then the snRNPs assemble into the spliceosome

Question 6

What is snRNA? What does it do?

Answer 6

Short nuclear RNA. Are catalytic ribozymes that complementary base pair to the pre-mRNA and do the cutting and pasting

Question 7

What are snRNP proteins? What do they do?

Answer 7

Proteins that assist the snRNA, but don’t do any splicing. They position the snRNA so it can catalyze splicing and will remodel the entire spliceosome during splicing

Question 8

What is alternative splicing?

Answer 8

Where a single gene can be spliced in different ways to produce multiple transcripts and multiple polypeptides from the same gene

Question 9

Is alternative splicing common?

Answer 9

Really common. 95% of mammalian genes

Question 10

How does alternative splicing occur?

Answer 10

Mediated by RNA binding proteins that enhance or suppress the selection of a particular splice site by the spliceosome

Question 11

What is the advantage of alternative splicing?

Answer 11

Allows multiple proteins to be encoded by the same gene, so the organism needs fewer genes

Question 12

What are the 3 genes involved in the sex determination pathway in Drosophila?

Answer 12

Sxl, tra, and dsx

Question 13

How does alternative splicing leads to the development of female Drosophila?

Answer 13

1. A TF early in the development of females activates expression of sxl
2. sxl gets activated again later, and the sxl that was already around changes splicing so that exon 3 gets spliced out and the protein is fully functional
3. SXL alters the splicing of tra mRNA to produce a fully functional protein
4. TRA will alter the splicing of dsx so that it contains exons 1,2,3 and 4. This version of the TF binds to male sex differentiation genes and represses them

Question 14

How does alternative splicing lead to the development of male Drosophila?

Answer 14

1. The early development TF doesn’t get produced, so theres no SXL
2. When sxl gets produced later on, exon 3 is not spliced out and its contains a premature stop codon. No functional SXL is produced
3. tra then gets spliced to also retain an exon with a premature stop codon, so no functional protein is produced
4. DSX gets produced to have exons 1,2,3 and 5, and it will bind to and repress female sex differentiation genes

Question 15

How does alternative splicing of dsx lead to vastly different functions?

Answer 15

The two isoforms have different DNA binding domains

Question 16

How does alternative splicing contribute to the complexity of gene regulation in eukaryotes?

Answer 16

It allows multiple transcripts encoding proteins with multiple functions to be produced from one gene

Question 17

What are the 3 possible results of alternative mRNAs?

Answer 17

1. different isoforms with different activities
2. a transcript that doesn’t get expressed even after transcription
3. a transcript becoming more highly expressed after transcription

Question 18

What are 4 mechanisms by which alternative mRNAs are produced from the same gene?

Answer 18

1. Alternative promoters and TSSs
2. Alternative polyadenylation sites
3. RNA editing
4. Alternative splicing

Question 19

How does the use of alternative promoters and TSSs create alternative mRNAs?

Answer 19

The produced transcripts have different first exons, which changes the proteins they encode. It also changes the 5’ UTR so that sequences targeted by RNA binding proteins or miRNAs are present or absent, which changes the stability and translational efficiency of the transcript

Question 20

How does the use of alternative polyadenylation sites create alternative mRNAs?

Answer 20

The transcripts have different C-term domains, which changes the protein’s functions. Changing the polyA tail and 3’ UTR changes protein binding site or miRNA target sequences, as well as transcript stability and translation

Question 21

How does the use of RNA editing create alternative mRNAs?

Answer 21

If the coding sequence is altered, the polypeptide produced is changed. Changes to other sequences can change the transcript’s stability by altering protein binding or miRNA target sites

Question 22

How does the use of alternative splicing create alternative mRNAs?

Answer 22

Polypeptides with different functions from a single gene. Inclusion or exclusion of protein binding or miRNA sites. Increase or repress gene expression

Question 23

What are the 4 mechanisms of alternative splicing?

Answer 23

1. Excluding an exon
2. Retaining an intron
3. Using a different 5’ or 3’ splice site
4. Changes through alternative polyadenylation