Tony Southhall Flashcards
(109 cards)
1
Q
What is gene linkage? How can recombination helps us understand genetic distance?
A
2
Q
Overlapping theme in Dr. Tony Southhalls lecture series?
A
Common overlapping theme –> diversifying the proteome
How we can amplify the proteins we get from the genes we have?
3
Q
What is alternative splicing?
A
Alternative splicing refers to different combinations of exons that can be assembled together from the same gene transcript
Easy/one of the most power ways to generate different proteins
4
Q
Brief overview of the splicing process, not including the players, what happens?
A
5
Q
General information about splicing?
A
- Needs no free energy - except in more complex organisms – provides more control
- Splicing can occur over large distances (10’s of kilobases)
- Types of splicing can change depending on the cell type
- Process is coordinated by the spliceosome and spliceosome co-factors.
6
Q
What is Exon skipping and cryptic splicing?
A
7
Q
Generally speaking, what are the components that make up the Splicesome?
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8
Q
Provide a detailed step by step description of how the splicesome complex
A
- U1 and U2 assemble onto pre-mRNA in a co-transcriptional manner (as transcription is taking place) U1 binds to donor site and U2 binds to acceptor site.
- The U1 and U2 snRNPs interact with each other to form the pre-spliceosome (complex A).
- The preassembled tri-snRNP U4–U6•U5 is recruited to form complex B
- Pre catalytic spliceosome (Complex B) undergoes a series of rearrangements to form a catalytically active complex B (complex B*) - involves ejection of U1 and U4 –> Complex B* is ready to perform first catalytic step.
- Complex B* then carries out the first catalytic step of splicing, generating complex C, which contains free exon 1 and the intron–exon 2 lariat intermediate
- Complex C undergoes additional rearrangements and then carries out the second catalytic step, resulting in a post-spliceosomal complex that contains the lariat intron and spliced exons
- Release of spliced mRNA and lariat uses helicase to help unwind intron lariat so that the snRNPs can be reused.
9
Q
Before the 1980s, what was the school of thought about the relationship between DNA and RNA?
A
Until the 1980’s: genes thought to have only 1 mRNA
But then they realized that the DNA didn’t always match the RNA produced because 1° transcripts can have 2 or more splicing pathways –> they are related but different mRNAs
10
Q
Is the number of genes a good reflection of protein diversity?
A
Yeast 0.1% of genes undergo splicing whereas in humans its 95% - so number of genes in an organism is not a good reflection of protein diversity
11
Q
What are the different sources of variation created by splicing?
A
12
Q
Outline how splicing is used for regulatory purposes?
A
Adds another layer of regultion on top of the regulation that occurs transcriptional/on the DNA level
13
Q
List the different effects that Splicing can have on mRNAs and proteins?
A
14
Q
What are some specific effects that Splicing has on protein structure and function?
A
15
Q
Does splicing play an important role in tissue differentiation and development?
A
16
Q
What are the three main products of alternative splicing?
A
17
Q
Outline what is meant by 5’ ends differ - 3 basic products of splicing
A
18
Q
Example of alternative splicing - 5’ transcript with ends that differ?
A
19
Q
Outline what is meant by 3’ ends differ - 3 basic products of splicing.
Include example
A
20
Q
Outline what is meant by centre differs - 3 basic products of splicing
A
Centre differs –> same 5’ and 3’ end but different center - can be explained by different tissue specific splicing factors acting on the pre mRNA
Example: Troponin T gene (skeletal muscle) - 64 different isoforms (found in different muscle types)
21
Q
How does a cell know whether to include/skip exons?
A
Tissue specific splicing factors
22
Q
How is the sex of drosophila determined?
A
Background info: Female - 2 sex chromosome / Male - 1 sex chromosome
Gene hierarchy determines sex in fruit flies - Basically each gene product (e.g. RNA/protein) controls splicing of the next gene in hierarchy - like a cascade
23
Q
Outline the first step in Drosophila sex determination - SXL autoregulation.
A
Starts with autoregulation of splicing in Sxl (Sex lethal gene)
- Males and females expressed differential quantities of SXL protein due to different ratio of sex chromosomes
You may ask how it starts off - different promoter is used in early stages of development to kick start process in females – excludes exon 3
- Net result: Allows females to have a higher Sxl expression
- Sxl protein in females binds to the Sxl pre-mRNA changing the splicing pattern - removes exon 3 to create more functional protein
How? - Sxl binds to pre-mRNA blocking the use of the exon 3 acceptor site
What happens in males?
- Opposite occurs in males - no Sxl expression –> so exon 3 is kept in the mRNA transcript - exon 3 includes a stop codon creating a truncated protein.
24
Q
Outline the second step in Drosophila sex determination - SXL regulation of tra?
A
25
Outline the third step (A) in Drosophila sex determination - tra regulation of dsx?
26
Outline the third step (B) in Drosophila sex determination - tra regulation of fruitless?
The Fru gene has 4 promoter regions, but the transcripts produced by P1 are only susceptible to Tra binding.
1. **In males**, where tra is not present, splicing yields a transcript with a male specific N-terminus (FruM).
2. **In females** binding of tra to the P1 transcript results in the exclusion of this male specific N-terminus (FruCOM).
Consequence?
As the Fru proteins belong to the BTB zinc protein family, we can suggest that the splice variants have different impacts on transcription regulation, resulting in the sex differentiation of CNS cells (Yamamoto & Koganezawa, 2013).
27
Outline the overall hierarchy invovled in the sex determination of Drosophila.
28
What are the different RNA recognition motifs and what are the trans factors that bind to them?
29
What do the regulation factors SR proteins and hnRNPs do?
Both of these bind to ‘RNA recognition Motifs’ and manipulate splicing
30
Summarize the different factors that influence the splicing outcome.
31
Can the rate of transcription impact the splicing outcome?
32
How do we identify the different splice variants/ study splicing?
How we studying splicing?
In the Past – microarray
Present - Next gen sequencing
Basic Idea - sequence all the mRNA in the cell then examining which exons are being included/excluded
33
Summary of splicing?
1. Alternative splicing allows for different proteins to be produced from the same genetic information
2. Splicing depends on several factors - RNA sequences, ESE’s, ISE’s, splicing factor presence and concentration
3. Several categories - 5’ end different, 3’ end different and middle different
4. Massive variation between different tissue/cell types
5. Splicing also responds to signalling - e.g. level of calcium in the cell
34
Are introns found in all eukaryotic genomes?
Introns: found in “all” eukaryotic genomes
Except, a nucleomorph in the cryptophyte of Hemiselmis andersenii
35
What are the Intron Early and Late theory?
36
What are the different life phases of an intron?
Other types of characterisation
1. ‘Sequence-dependent functions’
2. ‘Length-dependent functions’
3. ‘Splicing-dependent functions’
37
How can introns be a burden to the host?
1. Spliceosome complex is huge!
2. Energy & time ( e.g. RNAP II 60 nt s-1) cost
3. Vulnerability e.g. need recognition of cis-regulatory sequences - can be mutated --\> influence final product
38
What is meant by a genomic intron (Life Phase - 1) What is their role?
39
What is meant by a Transcribed intron (Life Phase - 2) What is their role?
Transcribed intron - Intron that has been transcribed, not spliced out yet, present in the RNA transcript
Role?
RNA polymerase takes different amounts of time to transcribe genes, **Splicing of introns** takes time and translation of the protein as well --\> e.g. Some cases it may take hours - meaning that the time gap between gene activation and when protein is produced is large.
This time difference allows for transcription, splicing (mRNA creation) and translation to be staggered - oscillates at specific frequencies
Why is this useful? --\> allows cells and tissues to develop in a specific order/sequentially
Example - HE27 gene
40
What is meant by spliced introns (Life Phase - 3) What is their role?
Spliced Introns - Introns that are being spliced from the RNA transcript
We need to remember that transcription and splicing often occur simultaneously, thus...
This allows the presence of specific introns to have an effect on the different stages of transcription...
1. Intiation
2. Elongation
3. Termination
41
How do spliced introns influence intiation?
42
How do spliced introns influence elongation?
43
How do spliced introns influence termination?
44
What happens to excised introns? (Life phase - 4)
45
Outline how MiRNAs embedded in introns (Mitron) are processed.
46
What are snoRNAs?
snoRNAs --\> released after splicing - Fundamental to RNA modifications in archaea & eukaryotes
Modify RNAs (rRNA, snRNA, tRNAs) --\> Basically, introns released and combined with protein to form the snoRNA assembly --\> used to modify (e.g. methylate) rRNA (ribosomal RNA)
47
What is the Exon junction complex (EJC)? What are its roles?
EJC harbouring transcripts - ‘Exon Junction Complex’ --\> known as intron ‘ghost’
It consists of a Protein Complex that binds ~25 nts upstream of exon-exon junction on mRNA transcript - 4 core proteins (MAGO, YI4, eIF4AIII, MLN51) --\> Present from splicing until translation.
Basically, where there was an exon-intron boundary you have an Exon junction complex remains bound to mRNA until its translated
48
Example of EJCs being used to localize mRNA to a subcellular region?
49
Why would cells have a mechanism in place to degrade mRNAs that have a premature stop codon?
50
How does the EJC work perform non-sense mediated decay?
mRNA Marking model --\> If the EJC is greater than 50 nucleotides downstream (to the right) of a termination codon = premature - basically looking at location of EJCs relative to termination codon
**Normal Process:**
1. mRNA with EJC
2. Ribosome starts translating the mRNA when doing so it kicks off the EJCs.
**Non-sense mediated decay process:**
1. mRNA with EJC + premature stop codon
2. Ribosome moves along and briefly stops at the stop codon (always happens)
3. Gives time for interaction between ribosome and EJC --\> many proteins facilitate this interaction (SURF + UPF1 – bound to ribosome / UPF2 + UPF3 – bound to EJC).
4. Once in close proximity you get interactions between UPF1 and UPF2 - signals the presence of a premature stop codon.
5. Then results in the phosphorylation of UPF1 - eventually leads to RNA degradation.
51
Summary of the different roles of the EJC? What are they?
52
Overlapping gene definition?
53
What are the two main different types of gene overlap?
54
Is the genomic distribution of overlapping gene species specific?
Yes!
The genomic distribution of these gene pairs is species specific
e.g. Convergent gene pairs are prevalent in Drosophila but rarer in human and mouse - could be due to evolutionary constraints.
55
What are the different terminlogies used to describe a gene within a gene?
56
Outline each type of overlapping gene in the image.
57
When talking about overlapping genes, why is it important to consider the relative phase?
it is important to consider the relative phase that the overlapping genes exist in relative to one another - ultimately decide the frame which defines the codons.
One gene is considered the ‘reference gene’ – base comparisons from that.
Basically used to determine the relative phase between the two genes.
58
What are the two categorize of in-phase overlap?
Basically, if the genes are in phase --\> then they must either share a different intiation site or termination site --\> otherwise the same transcript/protein would be produced
59
When talking about overlapping genes, what is meant about out of phase genes? What are the two different types? Where are they found?
60
Example of an out of phase overlap - Ink4a/Arf locus
61
Example of a Partial initiator or Terminal Overlap?
62
What is translational recoding?
63
What is a ribosomal frameshift?
64
What is required for -1 programmed frameshift which is commonly found in Prokaryotes?
65
Outline what happens -1 programmed frameshift occurs.
66
When can slippage (ribosomal frameshift) occur during translation?
67
Outline the example of a +1 Programmed Frame Shift example - Saccharomyces cerevisiae
68
Outline the negative feedback loop between ODC and OAZ which uses a +1 ribosomal frameshift.
69
Outline the example of the HIV virus that uses -1 frameshift to create two different proteins.
Useful - allows for simple solution for the correct proportion of protein to be produced - considering the small size of a viral genome.
70
Outline how the Stem-Loop in the HIV -1 ribosomal frameshift could be targetted using a drug.
71
Why do overlapping genes and PRF exist? What main functions do they play?
72
Advantages and disadvantages of overlapping genes?
73
Difference in overlapping genes between prokaryotes and eukaryotes?
74
Apart from overlapping genes, what else might they be overlapping that may play an important role in gene regulation?
75
Provide an example of how antisense transcritpion can influence the transcription intiation.
76
Provide an example of how antisense transcritpion can influence the regulate transcription during transcriptional elongation.
77
Provide an example of how antisense transcritpion can regulate gene expression post-transcriptionally.
78
Why is using Sense–antisense pairs useful for gene expression regulation?
79
What is RNA editing?
RNA editing describes a number of diverse mechanisms that change the sequence of the RNA transcripts (post-transcriptionally) encoded by genes in a wide range of organisms
Important - ONLY found in Eukaryotes
80
Does RNA editing resemble RNA splicing? (Similarities & Differences)
**Similarities**
1. mRNAs, tRNAs and rRNAs can all be substrates for RNA editing and splicing
2. Alternative splicing and editing generate protein diversity
3. Splicing and editing are developmentally regulated --\> temporally + different cell types
**Differences:**
1. Splicing removes large RNA sequences encoded by a gene
2. Editing adds/changes the information encoded by a gene (changes are smaller)
3. Splicing is often an RNA catalysed reaction, whereas editing is always protein catalysed
81
Outline the discovery of RNA editing in Trypanosoma brucei
82
How many genes in the Kinetoplast in Trypanosoma discovered to have RNA editing?
83
Functions of inserting and deleting uridine bases during RNA editing?
84
Where is the information coming from to perform RNA editing on this scale with such reproducibility - how does the Trypanosoma cells know what to edit?
Trypanosoma brucei
It turns out that maxicircles code for mitochondrial genes whereas the minicircles code for small guide RNAs (gRNA) gRNA bind to the maxicircle gene mRNAs and alter the sequence
85
What is the general structure of the gRNA for RNA editing?
86
Outline the process of RNA editing in the trypanosome
87
What is the complex that is responsible for RNA editing?
88
Why is RNA editing important for the trypanosome life cycle?
89
Apart from regulating mitochondrial activity in trypanosomes, what else does RNA editing allow?
90
Another example of a organism that performs RNA editing?
91
Summarize the characteristics of RNA editing in plant mitochondria and plastids
C - U transition
U - C transition
Type of RNA edited
Frequency
When - Temporal?
92
General mechanism of RNA editing in Plant Mito/Plastids?
Why transaminase?
C (amine) and U (Carbonyl) --\> Interchange
93
Is Mammalian mRNA editing possible? What forms of base changes occur?
94
What is an example of RNA editing in Mammals?
95
How does RNA editing compare to ribosomal frameshifitng?
Both are similar as they yield the same result using different routes
Frame shifting we shift the reading frame to introduce/remove stop codon/change reading frame
RNA editing manually edits in or der to introduce/remove stop codon/change the reading frame
96
Outline how Apolipoprotein B mRNA editing takes place
97
What is the most common type of RNA editing in mammals? How enzyme is responsible for this conversion?
98
Even though ADAR utilizes no co-factors, what is requires to allow RNA binding?
99
Outline the Role of ADAR in serotinin receptor editing
100
Outline the role of ADAR in glutamate receptor editing in mammals.
101
Is ADAR editing important in mammal development?
YUP
102
Can RNA editing influence the expression or function of oncogenes or tumour suppressors?
Oncogenes - An oncogene is a gene that has the potential to cause cancer
Tumour suppressors - Genes that help regulate cell growth - prevent cancer formation
103
Summary of the different roles of ADAR RNA editing in mammals?
104
Is there a high degree of RNA editing in the brain?
105
Can RNA editing be a driving force behind evolution?
106
How does the editing of repetitive and non-repetitive DNA between different tissues?
107
Different role of ADAR 1, 2 and 3?
108
Is RNA editing as a tool for acclimatization for cold blooded animals?
109
What are the sex chromosomes for male and female Drosophila flies?
Female
W-/W- --\> 2 X chromosomes
Male
W-/Y --\> 1 X chromosome & 1 Y chromosome
