2 - Alternative Splicing and 3' End Formation - Wilson Flashcards
why is alternative splicing necessary?
needed for complexity generation - humans only have around 20,000 genes
give the 6 types of alternative splicing, draw diagrams for these
325 - 2 word
how is intron retention splicing useful?
all of the introns contained within the pre-mRNA can be spliced out apart from one. upon environmental signal, this intron can be quickly spliced out and functional protein made
give an example of a gene that uses alternative splicing to generate many mRNA isoforms. state a clever way it uses to create these isoforms
- Drosophila Dscam gene
- contains clusters of mutually exclusive exons that can generate over 40,000 mRNA isoforms from single gene
- RNA;RNA interactions can drive a certain pathway for splicing
describe the proteins and draw diagrams that allow certain splice sites to be used or ignored
ENHANCERS;
- SR proteins bind to certain exonic sequences
- enhance the use of the splice sites flanking the exon leading to inclusion of this exon within the mRNA
- enhancers make it more likely for the 5’/3’ splice sites to be recognised by the splicing factors
REPRESSION;
- binding of silencer proteins to sites on the exon (can also be bound directly to introns)can repress the use of certain splice sites by preventing the binding of the U1/U2snRNP splicing factors and therefore stops spliceosome assembly
- resulting in exclusion of the exon
325 - 2 word
give an example of alternative splicing in flies. DAG
- sex determination in flies
MALE; - inclusion of Exon3, causing nonsense mediated decay of the mRNA therefore no Sxl protein made -> male
FEMALE;
- binding of repressor proteins to intronic sequences
- repress binding of splicing factors therefore Exon3 not included in protein
- functional protein made (Sxl) -> female
- NO NMD
give an example of cell-type specific alternative splicing
DAG
- neuronal and normal cells regulated by 2 different forms of polypyrimidine tract binding protein (PTB)
- SRC gene has 2 different ways it can be spliced
NORMAL (HeLa); - the PTB form that binds to polypyrimidine tract prevents splicing factor recruitment therefore no splicing of Exon N1 therefore exclusion
- mRNA jus contains Exons3/4
NEURONAL (WERI-1);
- nPTB binds at polypyrimidine tract. allows splicing factor recruitment
- splicing and inclusion of Exon N1 in mRNA
- mRNA = Exon 3 - Exon N1 - Exon 4
- neurons do lots of alternative splicing
draw a diagram showing how a high/low elongation rate of RNAP affects alternative splicing. include splice sites that are either weak or strong in your diagram
- also, state what else determines which splice sites can be used
- Ser5P affects which splice sites used
325 - 2 word
draw a diagram showing the 5’/3’ UTRs of pre-mRNA and mRNA. in this highlight which sites the 3’ UTRs hold and state why this is important.
- basically showing that the 3’ UTRs contain a number of poly(A) signal sequence sites that can be chosen or not giving rise to a number of different mRNAs which all differ in the length of their 3’ UTR
325 - 2 word
give the polyA site signal sequence
- AAUAAA
give an example of alternative polyadenylation. DAG.
describe how we get differential choosing of these sites
- alternative polyadenylation in IgM mRNA
- 2 different polyA signal sequence sites, the first one being weaker than the second
- conc of CstF64 levels determine which site is chosen
325 - 2 word
what are the functions of 3’UTRs?
in this case, 3’ UTRs involved in regulation
- miRNAs can bind, trigger RNA interference regulation -> suppression of the mRNA
- therefore UTR length influences mRNA stability
- longer 3’UTRs have > miRNA binding sites therefore less stable
- shorter 3’ UTRs have < miRNA binding sites therefore more stable because less likely to be repressed/degraded
how do cancer cells differ to normal cells in terms of their 3’ UTR regions? how does this difference occur?
widespread 3’UTR shortening in cancer cells
- cancer cells will choose the first polyA site they come across in the 3’ UTR hence leading to shorter 3’ UTR therefore much less miRNA binding therefore escapes regulation by RNAi. evade normal control in cells
what do U1 snRNP concentrations determine? DAG
what is this process called?
- U1 snRNP is 6x > concentrated than any other snRNP in the cell, showing it has an additional function
- because the polyA sequence = AAUAAA it can arise randomly in the genome (internal to genes). probability of it arising = every 2kb.
- if we were to use the pA sites then we would get shorter, potentially non functioning genes
- U1 snRNP binds to sites near these pA sequences and block their recognition by RNApII (more specifically blocks activity of CPSF therefore blocks recognition)
- the pA sites therefore will not be chosen therefore no cleavage and production of normal length gene
- control of the RNA length is known as telescripting
