Lecture 13 - Alternative splicing

Question 1

why is pol II particular

Answer 1

because of the CTD tail

Question 2

When can you recruit spliceosome parts

Answer 2

as soon as the 5’ end of the mRNA transcript is out of pol II

Question 3

Enzymatic activity associated to CTD tail is ______________ dependent. What are the 3 types of enzymes recruited.

Answer 3

phosphorylation. Capping factors. Poly(A) factors. Splicing factors

Question 4

Why alternative splicing is advantageous and how

Answer 4

More exons shuffling and few gene mutants. Since alternative splicing happens with most genes, less DNA is required (less DNA to replicate, maintain, etc.)

Question 5

Alternative splicing is all about __________ splice sites. How does it do that/principle ?

Answer 5

defining splice site. RNA binding proteins (with rna binding motifs) promote or block recognition of a splice site by components of the spliceosome.

Question 6

Exemple of alternative splicing

Answer 6

Fibroblast fibronectin mRNA contains EIIIB and EIIIA part of the fibronectin gene whereas hepatocyte fibronectin doesn’t contain EIIIA and EIIIB part of the fibronectin gene.

Question 7

In figure with the fibronectin gene, what does each colour and each square correspond to ?

Answer 7

each square is an exon. each color is a biochemical particularity that will be obtained in the protein.

Question 8

what is the advantage of shuffling exons

Answer 8

each exon is a particular function in the protein so shuffling them creates a diversity of proteins with different functions

Question 9

Are all exons spliced ? explain

Answer 9

No. You can have exons that are not spliced w/ other exons and that go out w/ introns.

Question 10

What proteins help define true splice sites

Answer 10

SR (Ser-Arg) rich proteins that bind to exonic splicing enhancers (ESE -> sequences in exons) … bind with their motifs.

Question 11

Alternative splicing controls ____________ in Drosophila. How does it work ?

Answer 11

sex determination. Genes in X and Y chromosomes recruit different splicing factors -> different proteins -> sexually dimorphic characteristics

Question 12

Because females in drosophila have 2 __________, __________ gene is expressed only in ______ in early ____________. 2 __________ = activation of ______________. _____ gene is under ___________.

Answer 12

X chromosomes, sex-lethal (Sxl), females, early embryogenesis, X chromosomes. early promoter. Sxl gene is under transcriptional control

Question 13

What happens later in development concerning promoters of Sxl gene in female drosophila

Answer 13

Early promoter is repressed and different Sxl promoter is activated IN BOTH SEXES

Question 14

Why there is a difference in late Sxl expression in male and female drosophila

Answer 14

Sxl pre-mRNA is alternatively spliced depending upon PRESENCE OF SXL PROTEIN. (already present in females !)

Question 15

Sxl gene (exons) in drosophila. Particularity of one of them

Answer 15

3 exons. From 5’ to 3’ : exon 2, exon 3, exon 4. Exon 3 has a premature termination codon.

Question 16

Sxl protein in females and males

Answer 16

F: exons 2 and 4. –> functional protein.
M: exons 2,3,4. Because exon 3 has premature terminating codon, no functional sxl protein is obtained (no sxl prot. in males)

Question 17

How does sxl protein (exists in females only) regulate splicing ? First step

Answer 17

Binds to sequence at 3’ end of intron between exons 2 and 3 and blocks association of U2AF and U2 snRNP. (Thus Sxl represses a particular splice site)

Question 18

How does sxl protein (exists in females only) regulate splicing ? Second step

Answer 18

U1 snRNP binds properly to 5’ end of intron between exons 2-3 and assembles spliceosome with U2 snRNP that is bound to branch point at the 3’ end of intron between exons 3-4.

Question 19

How does Sxl protein (exists in females only) regulate splicing ? 3rd step

Answer 19

Exon 2 is spliced to exon 4. Exon 3 goes out as part of a larger intron.

Question 20

Transfromer gene (Tra) in drosophila (exons). Particularity of one of them

Answer 20

3 exons : From 5’ to 3’ : Exon 1, exon 2, exon 3. Exon 2 has a premature terminating codon.

Question 21

Transformer protein in male and female drosophila

Answer 21

F : exons 1 and 3

M: exons 1,2,3 -> no functional protein because exon 2 has premature terminating codon.

Question 22

How does Sxl protein regulate splicing in Tra gene. (in females)

Answer 22

Blocks splicing at 3’ end of intron between exons 1,2. (blocks binding of U2AF w/ U2 snRNP) so spliceosome formed between splice site at 3’ end of exon 1 and 5’ end of exon 3

Question 23

which protein stimulates dimorphicity in drosophila and what proteins regulate that (only ______ have _______gene)

Answer 23

Dsx protein. Tra protein + Tra2 and Rbp1 (SR rich proteins) regulate that. only females have transformer gene

Question 24

Dsx gene (exons) and particularities

Answer 24

from 5’ to 3’ : 1,2,3,4,5,6. Exons 4 and 6 have a Poly(A) signal at their 3’ end

Question 25

Spliced exons of dsx gene in males and females and where cleavage and polyadenylation occurs

Answer 25

M: exons 3 and 5. cleavage and Poly(A) occur downstream of exon 6.
F: exons 3 and 4. cleavage and Poly(A) occur at 3’ end of exon 4.

Question 26

How does Tra regulate splicing of dsx gene in females ?

Answer 26

Rbp1 and Tra2 (SR rich prots) bind to exonic splicing enhancers (ESE) on exon 4 and activate splice site at 3’ end of intron between exons 3 and 4. (recruits U2AF and U2 snRNP to this splice site)

Question 27

Dsx is a ___________________ (what kind of protein). Different _______ are produced in _______ and _________ embryos

Answer 27

transcriptional repressor. forms. male. female. embryos.

Question 28

what do we call the different forms proteins that come from same gene (but that are variants)

Answer 28

isoforms

Question 29

chicken cochlea (organ that produces nerve impulses in response to sound vibrations) form and description.

Answer 29

5 mm long tube that contains epithelium of auditory cells hair cells. Apical hair cell (left of image/goes to the bottom) responds to 50 Hz frequencies and Basal hair cell (right of image/goes to the top) responds to 5000 Hz frequencies.

Question 30

What is the channel in epithelial cells of chicken cochlea that respond to different frequencies. What it is made of

Answer 30

Ca 2+ activated K+ channel. 7 transmembrane helices (S0 to S6) that form K+ channel and 4 hydrophobic regions in the cytosol that regulate opening of channel; in response to Ca 2+.

Question 31

How does the chicken cochlea produce auditory hair cells (endothelium of cochlea) that respond to different frequencies

Answer 31

By alternative splicing of the Ca 2+ activated K+ channel mRNA primary transcript, isoforms of the channel are produced. These OPEN AT DIFFERENT Ca 2+ CONCENTRATIONS so respond to different frequencies.

Question 32

what is contained by each mRNA of the Dscam gene in drosophila

Answer 32

One of the 12 Ig2 exons, One of the 48 Ig3 exons, one of the 33 Ig7 exons and one of the two transmembrane exons.

Question 33

In Dscam protein, what is the region that contains the Ig2, Ig3 and Ig7 exons

Answer 33

Homophilic binding region (region that binds to antigen)

Question 34

example of consequence of uncorrect splicing junctions and what this shows

Answer 34

Neurological disorders (some have link with abnormality in alternative RNA splicing). This shows that splicing is very specific and must be correct.

Question 35

Process that alters sequence of pre-mRNAs and what it consists of

Answer 35

RNA editing. Sequence of coding region in pre-mRNA is altered. Usually single base change (C to U)

Question 36

Comparison of edited mRNA with genomic DNA sequence

Answer 36

Sequence of the mature mRNA differs from exons encoding it in genomic DNA.

Question 37

Frequency of RNA editing in eukaryotes and when RNA editing happens

Answer 37

Rare in eukaryotes. Happens after processing. (AFTER SPLICING)

Question 38

Exemple of RNA editing

Answer 38

APOB gene. 1) In liver, no RNA editing and approx. 4500 a.a. of APOB protein are transcribed. (prot is called apoB-100). 2) In intestine, CAA sequence in 26th exon of transcript of APOB gene is edited to UAA (which is a stop codon). Troncated protein is formed (approx. 2100 a.a. it is called apoB-48)