Alternative Splicing

Question 1

alternative splicing (3)

• definition
• purpose
• common in…

Answer 1

• a gene can be spliced in different ways to produce variants of the same protein
• don’t need one gene for every single protein
• common feature in eukaryotes

Question 2

why does alternative splicing occur (3)

Answer 2

• generates more diversity in proteins

- many mutations in splice donor and acceptor sites

Question 3

transcript processing: mRNA splicing (3)

Answer 3

• special recognition sequences on the pre-mRNA are located at the intron-exon junctions and within the intron
• spliceosome complex facilitates process
• carried out by interaction of 5 small snRNPs and 100+ proteins using ATP

Question 4

alternative promoter and first exon (2)

Answer 4

• two promoter sites for the same gene

- allows for different first exon in alternatively spliced proteins

Question 5

alternative poly A site and terminal exon (2)

Answer 5

• two poly A sites for the same gene

- allows for different last exon in alternatively spliced proteins

Question 6

splicing control elements

Answer 6

• sequences within introns and exons that recruit factors that can promote or suppress recognition of splice sites

Question 7

what are the components of regulation of alternative splicing (2)

Answer 7

• cis-elements: enhancers and silencers located on introns and exons
• trans-acting splicing factors: proteins, etc

Question 8

four classes of splicing regulatory elements (4)

Answer 8

• exonic splicing enhancers
• exonic splicing silencers
• intronic splicing enhancers
• intronic splicing silencers

Question 9

exonic/intronic splicing enhancers (2)

Answer 9

• stabilization: the splice site is recognized more often and the exon is included
• can enhance the splicing events at weak splice sites

Question 10

exonic/intronic splicing silencers

Answer 10

• destabilization: splice site is suppressed and the exon is skipped

Question 11

how can you detect alternative splicing regulatory sequences and events: large-scale genome-wide approaches (4)

Answer 11

• RNA-sequencing to identify transcript variants and their relative amounts
• compare cDNA sequences to reference genome to exon-exon junctions
• DNA microarrays using exons instead of entire gene sequences
• cross-linking and immunoprecipitation

Question 12

how can you detect alternative splicing regulatory sequences and events: smaller-scale approaches (3)

Answer 12

• RT-PCR and gel electrophoresis to detect alternative splice variants through differences in size
• electrophoretic mobility shift assays (EMSA) to establish binding of spliceosome facters to RNA transcripts
• use of splicing reporter genes

Question 13

bi-chromatic fluorescent splicing reporters

Answer 13

• allows for tissue and temporal specificity of alternate isoforms
• method 1: reporter constructs expressing distinct fluorescent markers (blue vs red) are inserted and are dependent on the expressed isoform
• method 2: reporter genes expressing distinct fluorescent markers in different frames