pre-mRNA splicing Flashcards
pre mRNA splicing is a series of 2 _________
transesterification reactions
Is ATP required for splicing?
no
What is the splicesome composed of?
5 distinct snurps (U1,2,4,5,6) and other proteins.
What are snurps composed of?
1 snRNA and 6-10 other protiens.
How do U1 and U2 bind at the splice site?
U1 binds to 5’ splice site. U2 binds to branchpoint adenosine kinking out of mRNA/U2 complex.
Which are the catalytic and non catalytic snuRPS in the splicesome
U6 and U2 are catalytically active. U1 and U4 dissociate. U5 remains bound.
How is ATP involved in the spliceosome cycle?
RNA helicases involved in the assembly and disassembly of spliceosome require ATP hydrolysis. (action of splicing does not require ATP)
How does decreased ATP hydrolysis in Prp16 mutants (yeast) affect splicing?
decreased ATP hydrolysis activity leads to higher efficiency of splicing of sub optimal substrates
How does the decreased ATP hydrolysis of Prp16 mutants lead to sup optimal splicing?
slowed hydrolysis time allows further time for incorrect exon alignment.
How can mRNA at sites of splicing be analysed?
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.
Briefly describe analysis of yeast splicing mutants
yeast mutants generated by random mutagenesis. Temperature sensitive growth phenotypes selected. RNA isolated during growth at permissive and restrictive temperature. Analysed by northern blottings.
What does the C terminal domain of RNApol2 contain?
tandem repeats of serine rich heptapeptide (YSPTSPS)
How does CTD code impact RNA processing
CTD is differentially phosphorylated. Different phosphorylation patters allow coupling of transcription to RNA processing events.
What is the purpose of U2AF protein
Aids U2 binding to branchpoint adenosine.
What are the components in the molecular bridge between U1 and U2
SR proteins, Exonic splicing enhancers.
What is recursive splicing?
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.
What are key features of SR proteins?
Rich in Arginine and serine residues. Connect 5’ and 3’ of exons tp U1 and U2 snurps.
How can an altered splicing pattern affect the mRNA read
Can generate functional protein isoforms, or can cause loss of function
How is alternative splicing regulated?
Regulated by alternative splicing factors, enhance or silence splicing.
What is the well characterized mutation in Spinal Muscular Atrophy?
Mutation affecting exon definition in exon 7 of SMN gene, (homozygous mutation in SMN1)
How does the exonic mutation in Spinal muscular atrophy affect the function of the gene?
Mutation lies in splicing enhancer, blocks 3’ splice site. Exon skipped = truncated protein.
How are sex lethal genes transcribed differently in female and male Drosohpila?
In Females, sex lethal genes are transcribed from PE promoter at early transcription and PL at late transcription. Only transcribed from PL in males.
What is Tra in Drosophila?
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.
What is sex lethal (SXL) in drosophila?
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.
