Alternative Splicing

Question 1

What does alternative splicing result in?

Answer 1

Alternative splicing generates multiple RNAs and multiple proteins.

A single genes codes for multiple proteins

Question 2

Types of alternative splicing

Answer 2

1. Exon skipping/inclusion
2. Alternative 5’ splice sites
3. Alternative 3’ splice sites
4. Mutually exclusive exons
5. Intron retention

Question 3

Do UNTRANSLATED exons code for proteins?

Answer 3

NO

Question 4

How does alternative splicing affect gene expression?

Answer 4

Proteins created with different target locales.

Proteins created with different FUNCTIONS

MRNA half life can be affected

Question 5

At what rate does splicing take place and what can you do for this?

Answer 5

Splicing happens quickly and because RNA is unstable, it’s best to flash freeze a sample in the moment.

Question 6

How can we measure the extent of alternative splicing?

Answer 6

Using a variety of methods:

RT-PCR - measure alternative splicing. Extract RNA, convert RNA to cDNA, PCR

RT-qPCR - measure relevant abundance

Microarray - how much cDNA product is found on one spot?

RNA-Seq - sequence lots and lots of transcripts. Reads of transcripts that have exon vs reads of transcripts that don’t have exon.

Question 7

RT-PCR large PCR product vs smaller PCR product?

Answer 7

Large PCR product - includes exon

Smaller PCR product - excludes exon

Question 8

In what area is alternative splicing more common to happen in?

Answer 8

The human BRAIN

Question 9

How many human encoding genes are subject to alternative splicing?

Answer 9

About 94%

Question 10

Explain the function and outcome of including/excluding these 2 different exons together or separately in the human brain. 18A and/or 18N in the SCN8A that encodes sodium channel protein (abundant in neurons).

Answer 10

1. Exon 18N contains coding sequence with a STOP CODON - included in nonneuron cells and fetal cells (and mRNA is degraded via NMD (nonsense mediated decay) OR skipped altogether and we get a Non active encoded protein.
2. Alternate Exon 18A included in specific neural tissues and a FUNCTIONAL SODIUM CHANNEL is produced - requires RbFOX1
3. If neither is included, don’t get an active tissue.

Question 11

Many Alternatively splice INTRONS have “weak 5’ and 3’ splice sites consensus sequences”. What does this imply?

Answer 11

Implies that other signals are needed for their recognition (or nonrecognition in place of a slipped exon). Eg: SF1/BBP