Alternative splicing and Introns

Question 1

alternative splicing

Answer 1

more than one type of transcript is made form single type of pre-mRNA by differential splicing

• a way of creating more than one type of protein from a single gene
• detected by comparing cDNA sequences with genomic DNA sequences
• major transcripts and minor transcripts
• ~95% of human intron containing genes
• only 50-60% in plants

Question 2

detect alternative splicing

Answer 2

compare cDNA to genomic DNA sequences
also by 2nd/3rd generation sequencing
- illumina or 454
- identification of new AS, comparison of AS between tissue types, biotic or abiotic stress treatments, wt. vs mutant, etc.

Question 3

types of alternative splicing events

Answer 3

1-Exon skipping (common in mammals, least common in plants)
2-alternative 5’ splice sites
3- alternative 3’ splice sites
4- intron retention (least common in animals, most common plants)
- mutually exclusive exon - exon skipping variation, not a major type
- also alternative position

Question 4

most common type of AS event

Answer 4

exon skipping in mammals (least common in plants)

intron retention in plants (least common in animals)

Question 5

effects of AS

Answer 5

alternative initiation, or alternative polyadenylation

Question 6

functions of AS

Answer 6

can create multiple protein isoforms

• “internal paralogs” of a gene
• Regulate gene expression (lowering expression)
• • down regulation by nonsense-mediated RNA decay
• – some transcripts with premature stop codons are degraded
• • AS in UTRs can have Post Translational effects (transport, stability, translation regulation) — down regulate gene expression

Question 7

patterns of AS

Answer 7

complex

• vary in different tissue and organ types
• affected by abiotic stresses, especially heat and cold in plants
• can be sex-specific in animals
• • ex Transformer protein, truncated by premature stop in males, in females functional — regulates female sexual differentiation

Question 8

transformer gene

Answer 8

sex specific AS in drosophila

• regulates female sex differentiation
• non functional due to premature stop in males
• females sex-lethal binds and blocks splicing at premature stop site– full protein

Question 9

Evolutionary conservation of AS

Answer 9

• conserved in some genes but not others or tissue specific
  1. non conserved exon with AS
• ex human AS in exon, mice no exon at all
  2. conserved exon, non conserved splicing
• differential splicing btw species ( ex humans 2 forms, mice 1)
  3. conserved AS
• same between species

Question 10

sequence evolution in AS exons

Answer 10

• ka/ks ratio is higher in AS exons
• • less selection in amino acid sequences
• Ks in regions (where splicing factors are binding) of AS exons important for splicing is low
• • selection preservation of sequence so that proteins involved in AS can bind

Question 11

examples of alternative splicing in plants

Answer 11

• transition from vegetative to reproductive phase

- N-gene - AS = resistance to virus (need both genes)

Question 12

alternative splicing in muscles

Answer 12

different splicing in different muscle types all from same gene

Question 13

AS of TF in bees

Answer 13

subspecies of honeybees with different reproductive behavior due to splicing of gemini TF

Question 14

AS in Dscam

Answer 14

mechanism:
DSCAM- exon guidance receptor
-docking sequence in introns pair with selector sequence from intron in one of the 48 alternative exons

Question 15

AS mechanisms

Answer 15

• 1. SR proteins: activate splicing by binding exons (introns in plants) and recruiting splicing machinery
• 2. Transcription rate: faster RNA pol may lead to exon skipping
• 3. Chromatin structure: can effect transcription rate, differences in histone methylation between constitutive and alternative exons

Question 16

ESEs

Answer 16

exon splicing enhancers- SR PROTEIN BINDING SITES

• sites where splicing factors bind
• functional constraint and reduced Ks
• • SR binding sites have constraints on them
• mammals exon definition
• animals intron definition

Question 17

intron evolution

Answer 17

-gains and losses

Question 18

How are new introns identified

Answer 18

detected by id on basis of restricted phylogenetic distribution - compare introns in subgroups

• sources include other introns, exons, and transposons
• RNA intermediate step likely for introns to insert in new locations

Question 19

intron loss mechanism

Answer 19

genomic deletion
- recombination events
integration of intronless cDNA from genes transcript back into genome by homologous recombination with original gene

Question 20

intron gain mechanisms

Answer 20

1- intron transposition
-- reverse splicing or reverse transcribed and inserted into new gene
2- transposon insertion
3- tandem duplication of part of gene
-- new splice site formation
4- intron transfer by recombination

Question 21

splicing mutations

Answer 21

changes in splicing (not AS) and phenotypic changes

• ex sticky rice – lacks amylose synthase gene that disrupts protein function - due to intron splicing
• cystic fibrosis and CFTR gene - 20% of disease causing mutations affect splicing
• • exon 9 loss

Question 22

detecting AS by sequencing

Answer 22

illumina and 454