Topic 10 (Post-transcriptional Regulation) Flashcards
What is cis splicing?
The synthesis of a mature RNA from combining exons that were on the same pre-RNA
What is trans splicing?
The synthesis of a mature RNA from combining exons that originated from different pre-RNAs
What is the relationship between organism complexity and number of introns per gene?
Positive linear relationship
Where in the cell does splicing occur?
Nucleus
When does splicing occur? How is the spliceosome recruited?
While RNA is still transcribing the pre-RNA; spliceosome recruited to pre-RNA by phosphorylated RNA C-term tail
Describe the GU-AG rule
All introns start with 5’-GU and end in AG-3’, which is an absolute requirement
What are the absolute requirements for the splicing consensus sequence?
- GU/AG
- branch site
Is the sequence of the branch site well-conserved? How long is it?
No, it’s highly variable; 7 nt
Which nucleotide is key within the branch sequence?
A
The 5’ splice site is referred to as the:
Donor
The 3’ splice site is referred to as the:
Acceptor
What is the polypyrimidine tract? Where is it located?
Length of consecutive pyrimidines before the AG of the 3’ splice site
Describe the steps of splicing
First transesterification
1. 5’ end of the G (in 5’-GU) is attacked by the 2’ OH of the adenine in the branch site (nucleophilic attack)
2. Existing phosphodiester linkage is broken and a 3-way junction is formed
Second transesterification
3. 3’-OH of the spliced exon attacks the 5’ phosphate of the other exon
4. Intron lariat and spliced exons are released
Why is the intron lariat rapidly degrated?
It lacks a 5’ cap and poly A tail
What is a transesterification?
A reaction that breaks and makes chemical bonds in a coordinated transfer so that it is energetically neutral
True/False? Splicing requires energy
False. Energetically neutral
Why can’t DNA be spliced?
It lacks the 2’OH RNA has
What bond is formed when the three-way junction is made?
2’-5’ bond between the 5’ end of the intron and 2’ end of the A in the branch site
Describe the ways in which trans-splicing differs from cis-splicing
Doesn’t differ except for the formation of a Y-shaped branch instead of a 3-way junction
What is snRNA?
Small nuclear RNA 100-300nt long. Locates the sequence elements at the intron-exon borders
What is snRNP?
Small nuclear ribonucleoprotein (catalytic subunit). Contains one strand of snRNA and multiple proteins
What is the spliceosome made of?
snRNP particles
What are the major roles of snRNPs?
- recognize the 5’ and 3’ spice sites and branch site
- catalyze the 5’ splice site cleavage and joining with the branch site
What is the function of U1?
Base pairs its snRNA fragment with the 5’ splice site
What is the function of U2?
Base pairs its snRNA fragment with the branch site, but creates a bulge at the A
What is the function of U6?
Base pairs its snRNA fragment with the 5’ splice site at a later time than U1
What is the function of BBP?
Binds to branch site and is later displaced by U2
Describe the assembly of the spliceosome
E (early) Complex
1. U1 snRNP recognizes and binds 5’ splice site
2. U2AF65 subunits bind to the polypyrimidine tract and interact with BBP, which is bound to the branch site
3. U2AF35 subunit binds to the 3’ splice site
A Complex
4. U2 snRNP recruits to the branch site and displaces BBP
5. Unpaired A can interact with the 5’ splice site
B Complex
6. Association of U4, U5, U6, and tri-snRNP particle (bridge U1 and U2, forming a U-shaped structure with the RNA)
7. Displacement of the U1-snRNP
8. U6 replaces U1 at the 5’ splice site
Describe the formation of the catalytic complex
- U4 is released
- U2 and U6 pair to form an active site juxtaposed to the 5’ splice site and the branch site, facilitating the first transesterification
- U5-snRNP facilitates the second transesterification
- Release of the mRNA and snRNPs
- Degradation of the lariat RNA and recycling of the snRNPs
What are the three types of intron splicing?
- Pre-mRNA spliceosome
- Group II self-splicing
- Group I self-splicing
What is the main difference between using the spliceosome vs. group I and II splicing?
Spliceosome must be in-vivo. Group I and II may be in-vivo or in-vitro
Group II self-splicing
See card 13 (same process) (forms a lariat)
Group I self-splicing
3’OH of G within branch site attacks 3’ end of donor site, 3’OH end of donor site attacks 5’ end of acceptor site. No lariat is formed
What are the common properties between all three types of introns?
- use two transesterifications (energetically neutral)
- RNAs are catalytic
What are group I and II splicing found in?
Some bacteria, not higher eukaryotes
Why is group II splicing believed to be ancestral of the spliceosomal pathway?
- use the same splicing pathway and both generate a lariat intermediate
- have equivalent domains (D5 and U2/U6. D6 and U2-branch site pairing
What is common between group I and II splicing?
Both self-splicing
What is exon skipping?
One or more exons are spliced out with the introns
What is pseudo splice-site selection?
Mistakenly chosen due to the loose consensus sequence of the splice site. The end of an exon is spliced out
What are the methods used by spliceosomes to ensure accurate splice site recognition?
- formation of an active site by sequential recognition of snRNPs
- co-transcriptional loading process via the RNA Pol II C-terminal tail to facilitate the recognition of the 3’ splice site
- SR (serine arginine-rich) proteins bind to ESE sequence to recruit the splicing machinery to nearby correct sites (set the boundaries of splicing)
What does ESE stand for?
Exonic splicing enhancer
Binding of ____ proteins to the ESEs has what function?
SR; marks the correct splice site and facilitates the recruitment of U1 to the 5’ site and U2AF to the 3’ site
What are the hypotheses for when SR protein binding to ESE aids in splicing?
- direct interaction btw SR and spliceosomal subunits
- SR-spliceosome interaction may stabilize the RNA:RNA hybrids formed during spliceosome assembly
Explain how the minor spliceosome works
Uses typical spliceosome machinery, except it recognizes 5’-AU/AC-3’ intron sequences using U11 and U12, respectively. (5’-GU/AG-3’ recognized by U1 and U2AF65/35 in typical cases)
What does alternative splicing produce?
Isoforms of mRNA that contain different selections of exons given from a pre-mRNA
What is the exon makeup of alpha-troponin T?
1, 2, 3, 5
What is the exon makeup of beta-troponin T?
1, 2, 4, 5
Explain splicing in human troponin T gene
May be alternatively spliced via exon skipping to produce alpha or beta troponin T
What is commonly found in an alternatively spliced exon? Why?
Stop codon; produces a truncated, nonfunctional product (for turning off gene function)
List the five ways alternative splicing may occur
- normal splicing
- exon skipping
- exon extension (part of an intron)
- intron retention (full intron)
- alternative exons (type of exon skipping)
What determines quantity of different splicing products?
Competition between splicing factors
What are the different protein products of SV40 splicing? What kind of alternative splicing is used?
Small “t” antigen and large “T” antigen; exon extension
Where is the 5’ SST splice site found in SV40?
3’ end of exon 1 (5’ end of the entire intron)
Where is the 5’ sst splice site found in SV40?
Within the intron
Where is the 3’ SST splice site found in SV40?
5’ end of exon 2 (3’ end of the entire intron)
Describe what happens in SV40 when the 5’ sst and 3’ SST are used in splicing
A larger transcript is synthesized (exon extension) that has a stop codon embedded within it. Translation ends at this stop codon (exon 2 is not translated) and produces the small “t” antibody
Small “t’ antigen causes:
Apoptosis is blocked
Describe what happens in SV40 when the 5’ SST and 3’ SST are used in splicing
Intron is fully spliced out, so a smaller transcript is synthesized. Translation of the 2 exons results in the large “T” antibody
Large “T” antibody causes:
Transformation and cell cycle re-entry
The ratio of t to T antibodies is regulated by:
Splicing regulators (SR) SF2/ASF
Describe how a repressor prevents splicing
Binds to RNA using an RNA-recognition motif (RRM) and physically blocks the spliceosome from binding
Describe how an activator allows splicing
Binds to RNA using an RNA-recognition motif (RRM) and interacts with the spliceosome with an arginine and serine rich domain (RS domain), which facilitates spliceosome binding
Expression if Sxl is _______ in females and _______ in males
Activated; repressed
If two X chromosomes are present (Drosophila), what happens to Sxl expression?
Sis-a and Sis-b are transcribed and translated into SisA and SisB, which turn the Pe promoter on (Sxl transcription)
If one X chromosome is present (Drosophila), what happens to Sxl expression?
Dpn inhibits Pe, so Sxl is not transcribed
Is Dpn only present in male Drosophila? If not, why do we see Sxl expression in females?
No. It is transcribed from an autosome that males and females share; SisA and SisB outcompete Dpn for Pe
What kind of splicing does Sxl undergo?
Alternative splicing through autoregulation (Sxl protein regulates pre-mRNA splicing)
Explain what happens in the presence/absence of Sxl in D. melanogaster
Presence: Sxl alternatively splices tra gene. Tra splices dsx gene to make the female Dsx (represses male genes and promotes female development)
Absence: no transcription and translation of sxl and tra, so no functional proteins emerge. dsx is not spliced by Tra, so male Dsx is made (represses female genes)
What are iPS cells?
Induced pluripotent stem cells
How are iPS cells made?
Terminally differentiated cells are reprogrammed (dedifferentiated) and differentiated into new cell types
Alternative splicing of FOXP1 gene may result in:
FOXP1-ES (stimulates pluripotency) or FOXP1 (stimulates differentiation)
What is the early intron model?
Initially, introns were present in all organisms, but primitive organisms had selective pressure to increase the rate of chromosome replication and cell division, so lost them
What is the late intron model?
Introns were inserted into genes that previously had no introns (derived character)
Why are so many introns found in multicellular organisms?
- generate multiple protein products from a single pre-RNA
- create “new genes” by reshuffling exons
Each exon encodes:
A protein domain with an independent function
What is the hypothesis for the creation of the LDL receptor gene?
C9 and EGF precursor genes were shuffled together to create a new gene with properties from both
What is RNA editing?
Changes the RNA sequence post-transcriptionally, so the protein translated is different than what the gene sequence originally encoded for
In what three ways may RNA be edited?
- deamination of C into U by cytidine deaminase
- deamination of A into I by ADAR
- uridine insertion
Deamination of ____ in APOB occurs in ________ cells, causing a _________ protein due to the encoding of a __________
C; intestinal; truncated; stop codon (CAA to UAA)
Is RNA editing in APOB random?
No, it occurs at a specific C
Inosine is interpreted as ____ by translational machinery
G
Where does A deamination via RNA editing most commonly occur?
Nervous system
Describe uridine insertion in trypanosome coxII
Multiple U’s are inserted into specific regions of mRNAs after transcription, changing the codon and therefore the protein product
Describe nuclear export as a post-transcriptional modification
Some proteins coating mRNA are shed and recycled into the nucleus upon export. Export is regulated so that RNAS are not exported prematurely
