Lecture 13 - Alternative splicing Flashcards
why is pol II particular
because of the CTD tail
When can you recruit spliceosome parts
as soon as the 5’ end of the mRNA transcript is out of pol II
Enzymatic activity associated to CTD tail is ______________ dependent. What are the 3 types of enzymes recruited.
phosphorylation. Capping factors. Poly(A) factors. Splicing factors
Why alternative splicing is advantageous and how
More exons shuffling and few gene mutants. Since alternative splicing happens with most genes, less DNA is required (less DNA to replicate, maintain, etc.)
Alternative splicing is all about __________ splice sites. How does it do that/principle ?
defining splice site. RNA binding proteins (with rna binding motifs) promote or block recognition of a splice site by components of the spliceosome.
Exemple of alternative splicing
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.
In figure with the fibronectin gene, what does each colour and each square correspond to ?
each square is an exon. each color is a biochemical particularity that will be obtained in the protein.
what is the advantage of shuffling exons
each exon is a particular function in the protein so shuffling them creates a diversity of proteins with different functions
Are all exons spliced ? explain
No. You can have exons that are not spliced w/ other exons and that go out w/ introns.
What proteins help define true splice sites
SR (Ser-Arg) rich proteins that bind to exonic splicing enhancers (ESE -> sequences in exons) … bind with their motifs.
Alternative splicing controls ____________ in Drosophila. How does it work ?
sex determination. Genes in X and Y chromosomes recruit different splicing factors -> different proteins -> sexually dimorphic characteristics
Because females in drosophila have 2 __________, __________ gene is expressed only in ______ in early ____________. 2 __________ = activation of ______________. _____ gene is under ___________.
X chromosomes, sex-lethal (Sxl), females, early embryogenesis, X chromosomes. early promoter. Sxl gene is under transcriptional control
What happens later in development concerning promoters of Sxl gene in female drosophila
Early promoter is repressed and different Sxl promoter is activated IN BOTH SEXES
Why there is a difference in late Sxl expression in male and female drosophila
Sxl pre-mRNA is alternatively spliced depending upon PRESENCE OF SXL PROTEIN. (already present in females !)
Sxl gene (exons) in drosophila. Particularity of one of them
3 exons. From 5’ to 3’ : exon 2, exon 3, exon 4. Exon 3 has a premature termination codon.
Sxl protein in females and males
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)
How does sxl protein (exists in females only) regulate splicing ? First step
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)
How does sxl protein (exists in females only) regulate splicing ? Second step
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.
How does Sxl protein (exists in females only) regulate splicing ? 3rd step
Exon 2 is spliced to exon 4. Exon 3 goes out as part of a larger intron.
Transfromer gene (Tra) in drosophila (exons). Particularity of one of them
3 exons : From 5’ to 3’ : Exon 1, exon 2, exon 3. Exon 2 has a premature terminating codon.
Transformer protein in male and female drosophila
F : exons 1 and 3
M: exons 1,2,3 -> no functional protein because exon 2 has premature terminating codon.
How does Sxl protein regulate splicing in Tra gene. (in females)
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
which protein stimulates dimorphicity in drosophila and what proteins regulate that (only ______ have _______gene)
Dsx protein. Tra protein + Tra2 and Rbp1 (SR rich proteins) regulate that. only females have transformer gene
Dsx gene (exons) and particularities
from 5’ to 3’ : 1,2,3,4,5,6. Exons 4 and 6 have a Poly(A) signal at their 3’ end
Spliced exons of dsx gene in males and females and where cleavage and polyadenylation occurs
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.
How does Tra regulate splicing of dsx gene in females ?
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)
Dsx is a ___________________ (what kind of protein). Different _______ are produced in _______ and _________ embryos
transcriptional repressor. forms. male. female. embryos.
what do we call the different forms proteins that come from same gene (but that are variants)
isoforms
chicken cochlea (organ that produces nerve impulses in response to sound vibrations) form and description.
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.
What is the channel in epithelial cells of chicken cochlea that respond to different frequencies. What it is made of
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+.
How does the chicken cochlea produce auditory hair cells (endothelium of cochlea) that respond to different frequencies
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.
what is contained by each mRNA of the Dscam gene in drosophila
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.
In Dscam protein, what is the region that contains the Ig2, Ig3 and Ig7 exons
Homophilic binding region (region that binds to antigen)
example of consequence of uncorrect splicing junctions and what this shows
Neurological disorders (some have link with abnormality in alternative RNA splicing). This shows that splicing is very specific and must be correct.
Process that alters sequence of pre-mRNAs and what it consists of
RNA editing. Sequence of coding region in pre-mRNA is altered. Usually single base change (C to U)
Comparison of edited mRNA with genomic DNA sequence
Sequence of the mature mRNA differs from exons encoding it in genomic DNA.
Frequency of RNA editing in eukaryotes and when RNA editing happens
Rare in eukaryotes. Happens after processing. (AFTER SPLICING)
Exemple of RNA editing
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)
