Alternative mRNA processing

Question 1

What is alternative splicing?

Answer 1

A process that allows a single gene to produce multiple proteins via production of multiple splice variants

Question 2

What is a cassette exon? (2)

Answer 2

• A splicing event where an exon can be included or skipped to create 2 protein isoforms
• Often dependent on the tissue the mRNA is being expressed in

Question 3

What is mutually exclusive exon splicing?

Answer 3

2 (or more) possible exons to be included in the transcript but only 1 is included at a time, never both

Question 4

What are examples of alternative splicing? (6)

Answer 4

• Cassette exon
• Mutually exclusive exons
• Intron retention
• Alternative 5’ or 3’ splice sites
• Alternative promoters
• Alternative splicing and polyadenylation

Question 5

What is intron retention? (2)

Answer 5

• Intron isn’t removed in splicing
• RNA-binding proteins (RBPs) self-regulate by binding to RBP mRNA introns to suppress splicing, these transcripts are either degraded by NMD in cytoplasm or retained in the nucleus in nuclear speckles so they can be spliced later to make proteins e.g. under stress

Question 6

What are alternative 5’ or 3’ splice sites?

Answer 6

Choose between different splice sites to influence the size of the exon being made

Question 7

What are alternative promoters?

Answer 7

Use of alternative transcription start sites depending on the transcription factors that are bound to the promoters which can lead to alternative splicing by including/excluding exons

Question 8

What is alternative splicing and polyadenylation?

Answer 8

Different polyadenylation sites on the mRNA based on which exons have been included

Question 9

What is the drosophila Dscam gene? (2)

Answer 9

• Single gene with 40000 different mRNA isoforms containing many clusters of mutually exclusive exons
• Can be achieved through RNA:RNA interactions (base pairing within the RNA to steer the spliceosome in the desired direction)

Question 10

What are SR proteins? (3)

Answer 10

• Contain a region rich in serines and arginines
• Bind to exons via exonic splicing enhancer sequences
• Strengthens the adjacent splice sites by influencing recruitment of U2 to the 3’ acceptor splice site of the upstream intron and U1 to the 5’ donor splice site of the downstream intron) therefore encouraging inclusion of the exon they’re bound to

Question 11

How does a cell work out where the exons and introns are? (2)

Answer 11

• Intron definition: U1 bound at the 5’ end and U2 bound at the 3’ end
• Exon definition: SR proteins bind to exons causing recruitment of U2 to the upstream intron and U1 to the downstream intron, common because exons are much shorter than introns

Question 12

How do silencer proteins work? (3)

Answer 12

• Repress splice sites to cause exon exclusion
• Can bind to introns and block spliceosome recruitment
• Can bind to silencer sequences within exons to block recruitment of U1 snRNP to the downstream splice site and U2 snRNP to the upstream splice site

Question 13

What is an example of silencer proteins?

Answer 13

hnRNPs

Question 14

What are hnRNPs?

Answer 14

Heterogeneous nuclear ribonuclear proteins

Question 15

How is sex determined in flies? (3)

Answer 15

• Alternative splicing of the sex lethal (Sxl) gene which contains a poison exon 3 containing a stop codon
• Males: U2AF and U2 snRNP are recruited to the upstream splice site, U1 snRNP is recruited to the downstream splice site, resulting in exon inclusion which triggers NMD = no Sxl protein = male
• Females: silencing proteins trigger skipping of exon 3 = more Sxl protein = female

Question 16

How is alternative splicing used in muscle?

Answer 16

Different types of muscle contain different types of tropomyosin which are generated by alternative splicing

Question 17

How can alternative splicing cause disease? (3)

Answer 17

• Splicing mistakes is one of the most common causes of human disease
• Mistakes during splicing of the dystrophin gene can generate out-of-frame transcripts = truncated dystrophin or NMD = not enough dystrophin in the cell = Duchenne muscular dystrophy
• If mistakes generate in-frame transcripts, the resulting dystrophin protein may have partial function = Becker muscular dystrophy

Question 18

What is the average length of an intron?

Answer 18

~10kb (very long, exons are much smaller in comparison so not surprising that splicing can go wrong and cause disease)

Question 19

How does transcription speed affect splicing? (2)

Answer 19

• High elongation rate: the spliceosome doesn’t have enough time to assemble at weak splice sites which causes exon skipping, only strong splice sites are recognised
• Slow elongation rate: the spliceosome can recognise weak splice sites to include all the exons

Question 20

How can transcription speed of RNA polymerase II be influenced? (2)

Answer 20

• RNA polymerase II usually transcribes at 2.5kb per minute
• Speed can be changed by sequences within the DNA, phosphorylation of CTD e.g. CDK9 phosphorylates serine 2 driving transition of paused polymerase to a rapidly elongating polymerase

Question 21

How does polyadenylation happen? (3)

Answer 21

• mRNA is cleaved 20-30 nucleotides downstream from the AAUAAA polyadenylation site, just after a CA sequence
• GU/U-rich sequence downstream from this is cleaved off and degraded by Xrn2 in transcription termination
• PolyA modification added after CA

Question 22

How does alternative cleavage and polyadenylation (CPA) influence splicing? (6)

Answer 22

• Alternative CPA occurs in ~50% of transcripts
• IgM (antibody) RNA has 2 possible polyA sites, upstream is weak and downstream is strong
• Using the downstream strong polyA site causes inclusion of the M1 and M2 sequences, producing an mRNA for the membrane-bound form antibody used by pre-B/B cells
• Using the upstream weak polyA site excludes M1 and M2, producing an mRNA for the secreted form antibody used by plasma cells
• Lots of CstF64 protein is required for recognition of weak polyA site so needs lots in the cell, strong polyA sites have better CstF64 recruitment so less of it is required in the cell for the site to be recognised
• Plasma cells have high CstF64 so make secreted form, pre-B/B cells have low CstF64 so make membrane-bound form

Question 23

What happens to 3’ UTR of mRNAs in cancer cells? (4)

Answer 23

• Cancer cells use early polyA sites making the 3’ UTR shorter
• UTRs contain control sequences for micro RNAs which suppress mRNA expression
• Shortening of the UTR enables evasion of micro RNA regulation
• Common in cell cycle gene mRNAs

Question 24

How does UTR length influence mRNA stability? (2)

Answer 24

• UTR contains binding sites for micro RNAs which cause instability
• Short UTR makes mRNA more stable so produces more protein

Question 25

What is telescripting? (5)

Answer 25

- Every 2kb there will be a AAUAAA sequence and introns are generally approx 10kb so RNA introns contain premature polyA sites - Excess of U1 snRNP in the cell compared to other spliceosome elements - U1 snRNP complex binds to 5' splice site (GU) as well as sequences that look like the 5' splice site, therefore bind all the way down introns which prevents CPSF protein recognising early polyA sites, suppressing them - This is influenced by the concentration of U1 snRNA, blocking causes early termination and polyadenylation of mRNAs - Wouldn't be able to make full length mRNAs without this process

Question 26

What is the point of alternative splicing?

Answer 26

Enhance the protein-coding repertoire of cells and the stability of transcripts