Topic 10 Flashcards
(84 cards)
1
Q
Cis splicing
A
Exons contained within the same mRNA transcript are merged together
2
Q
Trans splicing
A
Exons contained within different mRNA transcripts are merged together
3
Q
As organism complexity increases, what happens to the average number of introns per gene?
A
Increases
4
Q
Having a large repertoire of non-coding genes (e.g. introns) is important in which organisms?
A
Plants and vertebrates
5
Q
True or false: transcription and splicing are coupled very tightly
A
True
- As soon as transcription begins, splicing can also occur
6
Q
RNA processing enzymes are recruited to the…
A
Pol II CTD tail
7
Q
The factors recruited to the Pol II CTD tail depend on…
A
The phosphorylation state of the CTD tail
8
Q
Essentially all introns begin with __ and end with __
A
Begin with GU and end with AG
9
Q
What 2 sequences are absolutely required for splicing?
A
- GU-AG rule
- Branch site
10
Q
What makes up the 5’ splice site?
A
AG in 5’ exon preceding intron and A/G AGU sequence following GU
11
Q
The 5’ splice site is also known as the…
A
Donor
12
Q
What makes up the 3’ splice site?
A
The polypyrimidine tract preceding the AG and the G in the 3’ exon following the AG
13
Q
The 3’ splice site is also known as the…
A
Acceptor
14
Q
What is the branch site in an intron?
A
An adenine residue part of 7 nucleotides
15
Q
Transesterification
A
A reaction that breaks and makes chemical bonds (in this case, phosphodiester bonds) in a coordinated transfer so that energy isn’t required
16
Q
What 3 steps make up the first transesterification of cis splicing?
A
- 2’OH of adenine branch point acts like a nucleophile and attacks 5’ phosphate on G of GU at 5’ splice site
- Breakage of phosphodiester linkage in the pre-mRNA
- 3-way junction is formed at branch point, and 5’ exon with G is separated
17
Q
__ to __ bonds connect the branch A to its neighbours in the polynucleotide chain (in the lariat intermediate)
A
3’ to 5’
18
Q
__ to __ bond is formed during the first transesterification to create the lariat
A
2’ to 5’
19
Q
What 2 steps make up the second transesterification?
A
- 3’ OH on 5 exon acts as a nucleophile to attack 5’ phosphate on G in 3’ exon
- Intron lariat is isolated and rapidly degraded, while the 5’ and 3’ exons are joined
20
Q
True or false: cis-splicing requires energy
A
False
- Cis-splicing is energetically neutral because the number of phosphate bonds is conserved
21
Q
Describe the trans-splicing chemistry
A
The chemistry is the same as normal cis-splicing, but the intron ends in a Y-shaped branch arrangement rather than a lariat
22
Q
What organisms does trans-splicing occur in? What are the applications of trans-splicing?
A
Known only in trypanosomes and C. elegans (ie. not universal in eukaryotes)
- Can be used for molecular therapy to address mutated gene products
23
Q
Define snRNA
A
Small nuclear RNA of 100-300 bp long, such as U1-U6
24
Q
Define snRNP
A
Small nuclear ribonucleoprotein
25
Each snRNP consists of...
One RNA (snRNP) and many proteins
26
What are the catalytic agents in snRNPs?
The snRNA are the catalytic agents through their ability to locate the sequence elements at the intron-exon borders
27
The splicesome that removes introns during the transesterification reaction in made up of...
snRNP particles
28
What are the major roles of snRNPs?
1. Recognize the 5' and 3' splice sites, and the branch site
2. Catalyze the 5' splice site cleavage and joining with the branch site
29
Base pairing occurs with which component of the splicesome at the 5' splice site?
Base pairing of U1 snRNA at the 5' splice site, which is also recognized by U6 snRNA at a later stage (i.e. U1 and U6 can both recognize the border of the introns)
30
What component of the splicesome recognizes the branch site in introns?
U2 snRNA
31
What interaction brings the 5' splice site and the branch site together?
Interaction (base pairing) between U2 and U6 snRNA
32
U2AF function in splicing
U2AF (U2 Auxillary Factor) recognizes the Py tract/3' splice site and helps the BBP (Branch-Binding Protein) bind to the branch site
- BBP is displaced by U2 snRNP during splicing
33
What are the three steps for the formation of the E (early) complex in splicesome assembly?
1. U1 snRNP recognizes and binds to the 5' splice site
2. U2AF65 subunits bind to the Py tract and interacts with the BBP
3. U2AF35 subunit binds to the 3' splice site
34
What are the two steps for the formation of the A complex in splicesome assembly?
1. U2 snRNP recruits to the branch site and displaces the BBP
2. The unpaired A residue can react with the 5' splice site
A complex= U2 + U2AF65 + U2AF35
35
What are the three steps for the formation of the B complex in splicesome assembly?
1. Rearrangement of the A complex
2. Association of U4, U5 and U6 tri-snRNP particle
3. Displacement of the U1-snRNP
36
What steps completes the splicesome assembly step?
U6 replaces U1 at the overlapping site
37
What are the two steps for the formation of the C (Catalytic) complex in intron splicing?
1. Release of U4, and U2:U6 paired to form an active site juxtaposed to the 5' splice site and the branch site, facilitating the first transesterification
2. U5 snRNP facilitates the second transesterification
38
True or false: snRNPs are degraded after each intron splicing
False
- snRNPs are recycled after splicing
39
What are the three types of intron splicing?
1. pre-mRNA splicesome
2. group II self-splicing
3. group I self-splicing
40
Describe group II intron splicing
Uses the same mechanism as the pre-mRNA splicesome, but catalytic machinery is RNA enzyme encoded by intron (ribozyme) rather than splicesome
41
Describe group I intron splicing
Two transesterification reactions; branch site G
- RNA enzyme encoded by intron (ribozyme)
- No true lariat intermediate is formed
42
True or false: all 3 types of intron splicing undergo two transesterification reactions, which is energetically neutral and doesn't require ATP
True
43
Where are group II introns found?
Rare; some eukaryotic genes from organelles and prokaryotes
- Found in some bacteria, and in mitochondria and chloroplasts, not found in humans or higher eukaryotes
44
Where are group I introns found?
Rare; nuclear rRNA in some eukaryotes, organelle genes and a few prokaryotic genes
- Found in some bacteria, and in mitochondria and chloroplasts, not found in humans or higher eukaryotes
45
Where is nuclear pre-mRNA splicing found?
Very common; used for most eukaryotic genes
46
True or false: the RNAs are thought to be catalytic in all 3 types of introns
True
47
True or false: group I and group II introns are self splicing (i.e. under certain conditions the RNAs themselves can catalyze splicing without protein)
True
48
____ and ___ introns use the same splicing pathway, with a lariat intermediate
Nuclear pre-mRNAs (i.e. spliceosomal introns) and group II introns
49
Which introns are thought to be the ancestors of the spliceosomal introns and why?
Group II introns, because of the parallels of group II introns and spliceosomal introns
50
What are 5 structural similarities between spliceosomal snRNAs and group II introns?
1. 2 snRNAs and the 5' exon from form pairings in spliceosomal introns (U2/U6/5' exon) similar to group II introns
2. The U2-U6 pairing is thought to be catalytic
3. Domain 5 in group II introns is thought to be catalytic, and equivalent to U2/U6
4. Domain 6 in group II introns is equivalent to U2-UACUAAC box pairing
5. 2'OH of bulged A attacks 5' junction
51
True or false: formation of an active site by sequential recognition of snRNPs is enough to ensure accurate splicing
False
52
What is the main problem of appropriate splice site recognition?
Often, genes have many exons to choose from
- Errors lead to mistaken splice site selection
53
What are two errors in splicing that may occur?
1. Exon skipping
2. Pseudo splice-site selection (some of an exon is spliced out): the pseudo splice site is mistakenly chosen due to the loose consensus sequence of the splice site
54
What 2 methods, other than the formation of an active site by sequential recognition of snRNPs, is used by the spliceosome to ensure accurate splice site recognition?
1. Co-transcriptional loading process via the RNA Pol II C-terminal tail to facilitate the recognition of the 3' splice site
2. SR (serine arginine- rich) proteins bind to ESEs (Exonic Splicing Enhancers) sequence to recruit the splicing machinery to the nearby correct sites
55
Binding of SR proteins to the ESEs (Exonic Splicing Enhancers) marks the _____, and facilitates the recruitment of __________
Binding of SR proteins to the ESEs (Exonic Splicing Enhancers) marks the correct splice site, and facilitates the recruitment of U1 snRNP to the 5' site and U2AF to the 3' site
56
What are two possible ways in which SR protein binding to ESEs marks the correct splice site, and facilitates the recruitment of U1 snRNP to the 5' site and U2AF to the 3' site?
1. Direct interaction between SR and spliceosomal subunits
2. SR-spliceosome interaction may stabilize the RNA:RNA hybrids formed during spliceosome assembly
57
What is the minor spliceosome?
Minor (AT-AC) spliceosome uses the same chemical pathway and some components (e.g. U5 snRNP) of the typical spliceosome machinery
- Catalyzes the removal (splicing) of an atypical class of spliceosomal introns (AU-AC introns)
58
Explain how the minor spliceosome recognizes introns and how this compares to the major spliceosome
Minor spliceosome recognizes introns that contain AU at the 5' site (by U11 snRNP) and AC at the 3' site (by U12 snRNP), whereas the major spliceosome recognizes GU at the 5' site (by U1 snRNP) and AG at the 3' site (by U2 snRNP)
59
True or false: AU-AC introns which are spliced by the minor spliceosome are very common
False
- AU-AC introns are very rare (e.g. ~1 in 1,000 introns in humans)
60
True or false: There are a lot of snRNPs in the human because the dinucleotide sequences (GU-AG, for example) are so degenerate
True
61
What does alternative splicing produce?
Isoforms of a mRNA that contains different selections of exons from a given pre-mRNA
62
How can alternative splicing control gene regulation using stop codons?
Often, there is a stop codon in an alternatively spliced exon to produce a truncated, nonfunctional product (mechanism of turning off gene function)
63
What's an example of alternative splicing involving troponin?
Primary RNA transcript can be alternatively spliced to yield either α-troponin T or β- troponin T.
64
What are the 5 ways to alternatively splice an mRNA?
1. Normal (all introns spliced out)
2. Exon skipped
3. Exon extended (part of an intron isn't spliced out)
4. Intron retained
5. Alternative exons
65
True or false: Any combination of alternative splice sites can occur in nature
True
66
What are different splicing products formed by?
Formed by competition between different splicing factors
67
Describe the alternative splicing of SV40 T-antigen by exon extension
- SV40 T-antigen pre-mRNA has different 5' splicing sites (i.e. 5'SST and 5'sst)
- There is an in-frame stop codon within the intron
- The ratio of the 2 forms produced depends on the levels of splicing regulator (SR) SF2/ASF
- Small "t" antigen due to presence of stop codon blocks apoptosis
- Large "t" antigen due to splicing of intron with the stop codon induces transformation and cell cycle re-entry
68
Alternative splicing can be regulated by...
Either activators or repressors of a particular splicing event
69
How do splicing regulator (SR) proteins bind to RNA?
Bind to RNA with an RNA-recognition motif (RRM) and interact with other splicing machinery proteins with an arginine and serine rich (RS)- domain
70
Describe the splicing of the sex determination gene in Drosophila (5)
- Females make twice as much of X chromosomes than males
- Expression of Sxl (sex lethal) gene is activated in females and repressed in males
- Sis-a and b genes are both found on the X chromosome and activate Sxl transcription while outcompeting the Dpn inhibitor (found on autosome) in females -> leads to transcription, and splicing of pre-mRNA to make early Sxl protein
- Dpn inhibitor outcompetes Sis-a and b genes in males
- Pm (promoter for maintenance) maintains Sxl expression in embryos, while Pe (promoter for establishment) is controlled by SisA and SisB
71
Describe the sex determination in flies using Sxl
- Sxl is a splicing factor that induces alternative splicing of its own transcript (i.e. autoregulation)
- Sxl is also an alternative splicing factor for the tra (transformer) gene
- Tra is a splicing regulator for Dsx (double sex)
- female gene undergoes alternative splicing -> female Dsx protein represses male genes and promotes female development
- Male Dsx represses gene that directs female development
72
How is iPS pluripotency activated? (4 steps)
1. Terminally differentiation cells are isolated from patient (skin or fibroblasts) and grown in a dish
2. Treat cells with "reprogramming" factors, aka transcription factors
3. Wait a few weeks
4. Pluripotent stem cells yay
73
Describe how the alternative splicing of FOXP1 contributes to iPS pluripotency activation
FOXP1, a transcription factor, stimulates the activation of other transcription factors such as OCT4 and NANOG depending on how it's spliced
- If it's spliced to include the 18b exon, pluripotency is induced and differentiation genes is inhibited
- If it's spliced to include the 18a exon, pluripotency is inhibited and differentiation genes are induced
74
Originally introns were present in all organisms. Why were introns lost in bacteria? Explain the intron early model
Primitive organisms have selective pressure to increase the rate of chromosome replication and cell division
- So a long genomic sequence, including all the introns, is not beneficial
75
Originally introns were present in all organisms. Why were introns lost in bacteria? Explain the intron late model
Introns were inserted into genes that previously had no introns
- posits that introns are not an ancient evolutionary product, and were more recently evolved
76
Why are two reasons why extensive introns are found in multi-cellular organisms?
1. To generate multiple protein products from a single gene through alternative splicing
2. To create new genes by reshuffling exons
77
Explain exon reshuffling
- Each exon encodes a protein domain with independent function
- Domains can exchange between proteins productively, by recombination events in the genomic DNA
78
Describe exon shuffling in the LDL receptor gene
The LDL (Low Density Lipoprotein) receptor gene is made up of exons shuffled between the C9 and EGF precursor genes
79
Exon domains can be reshuffled to make...
Families of proteins in different species
80
Describe RNA editing by deamination
The enzyme cytidine deaminase specifically recognizes the correct location to do the deamination
- Cytosine deaminated to uracil
- Cytosine deamination can change the function of the RNA, not always a harmful mutation
81
Describe how cytidine deamination affects the APOB gene encoding apolipoprotein B
Deamination of the mammalian gene occurs in a tissue-specific manner
- In the intenstine, but not in the liver cells, a specific C residue is edited to a U, changing a Gln codon to stop and producing a shorter protein
82
Describe adenosine deamination as a form of RNA editing
ADAR (Adenosine Deamination Act on RNA) deaminated A to I (inosine; translated as G)
- very prevalent in the nervous system
83
Describe an example of insertional editing
In trypanosome coxII (cytochrome oxidase subunit II) transcript, multiple Us (uridines) are inserted into specific regions of mRNAs after transcription -> codon changes, leads to change in amino acid sequence
84
Describe nuclear export
- mRNAs are coated with proteins in the nucleus during splicing and processing
- Upon export, some proteins are recycled to the nucleus (this edits the RNA)
- Export is regulated so that RNAs are not exported prematurely
