pre-mRNA splicing

Question 1

pre mRNA splicing is a series of 2 _________

Answer 1

transesterification reactions

Question 2

Is ATP required for splicing?

Answer 2

no

Question 3

What is the splicesome composed of?

Answer 3

5 distinct snurps (U1,2,4,5,6) and other proteins.

Question 4

What are snurps composed of?

Answer 4

1 snRNA and 6-10 other protiens.

Question 5

How do U1 and U2 bind at the splice site?

Answer 5

U1 binds to 5’ splice site. U2 binds to branchpoint adenosine kinking out of mRNA/U2 complex.

Question 6

Which are the catalytic and non catalytic snuRPS in the splicesome

Answer 6

U6 and U2 are catalytically active. U1 and U4 dissociate. U5 remains bound.

Question 7

How is ATP involved in the spliceosome cycle?

Answer 7

RNA helicases involved in the assembly and disassembly of spliceosome require ATP hydrolysis. (action of splicing does not require ATP)

Question 8

How does decreased ATP hydrolysis in Prp16 mutants (yeast) affect splicing?

Answer 8

decreased ATP hydrolysis activity leads to higher efficiency of splicing of sub optimal substrates

Question 9

How does the decreased ATP hydrolysis of Prp16 mutants lead to sup optimal splicing?

Answer 9

slowed hydrolysis time allows further time for incorrect exon alignment.

Question 10

How can mRNA at sites of splicing be analysed?

Answer 10

Resolve 32P labelled RNA substrates and nuclear extracts by PAGE. looped extracts will migrate slower. Substrates with altered sequence or extracts with depleted proteins can be assayed.

Question 11

Briefly describe analysis of yeast splicing mutants

Answer 11

yeast mutants generated by random mutagenesis. Temperature sensitive growth phenotypes selected. RNA isolated during growth at permissive and restrictive temperature. Analysed by northern blottings.

Question 12

What does the C terminal domain of RNApol2 contain?

Answer 12

tandem repeats of serine rich heptapeptide (YSPTSPS)

Question 13

How does CTD code impact RNA processing

Answer 13

CTD is differentially phosphorylated. Different phosphorylation patters allow coupling of transcription to RNA processing events.

Question 14

What is the purpose of U2AF protein

Answer 14

Aids U2 binding to branchpoint adenosine.

Question 15

What are the components in the molecular bridge between U1 and U2

Answer 15

SR proteins, Exonic splicing enhancers.

Question 16

What is recursive splicing?

Answer 16

Splicing event within a large intron. The intron is cut within itself and the cut 3’ end becomes new 5’ end for more splicing, as intron is too large to be cut at once.

Question 17

What are key features of SR proteins?

Answer 17

Rich in Arginine and serine residues. Connect 5’ and 3’ of exons tp U1 and U2 snurps.

Question 18

How can an altered splicing pattern affect the mRNA read

Answer 18

Can generate functional protein isoforms, or can cause loss of function

Question 19

How is alternative splicing regulated?

Answer 19

Regulated by alternative splicing factors, enhance or silence splicing.

Question 20

What is the well characterized mutation in Spinal Muscular Atrophy?

Answer 20

Mutation affecting exon definition in exon 7 of SMN gene, (homozygous mutation in SMN1)

Question 21

How does the exonic mutation in Spinal muscular atrophy affect the function of the gene?

Answer 21

Mutation lies in splicing enhancer, blocks 3’ splice site. Exon skipped = truncated protein.

Question 22

How are sex lethal genes transcribed differently in female and male Drosohpila?

Answer 22

In Females, sex lethal genes are transcribed from PE promoter at early transcription and PL at late transcription. Only transcribed from PL in males.

Question 23

What is Tra in Drosophila?

Answer 23

Tra is a splice site activator. Together with sr protein Tra2 regulates sex specific expression of double sex (dsx). DSX acts as transcription repressor for genes required in sexual differentiation in opposite sex.

Question 24

What is sex lethal (SXL) in drosophila?

Answer 24

SXL is a splice site repressor, promotes own expression and expression of tra in female flies vie EXON EXCLUSION. Blocks U2Af binding, avoids incorporation of premature termination signals in females.

Question 25

What is the function of DSX in gender determination?

Answer 25

encodes both male and female polypeptides, alternatively spliced, regulated by tra and tra2.

Question 26

How do group 2 introns splice?

Answer 26

autocatalytic self splicing introns. use same mechanism as pre-mRNA splicing. Intron has branch point adenosine and lariat intron is released.

Question 27

How do group 1 introns splice?

Answer 27

dependent on guanosine cofactor. Linear intron is released.

Question 28

How are self splicing intron structures restricted?

Answer 28

Must allow for juxtaposition between guanosine nucleotide or branch point adenosine with co factor.

Question 29

How is pre mRNA splicing though to have evolve?

Answer 29

Thought to have evolved from self splicing introns recruiting snRNAs to splice sites.

Question 30

Are you going to pass this exam?

Answer 30

yes