Gene Structure

Question 1

What is alternative splicing?

Answer 1

Alternative splicing generates different proteins from one gene
Genes contain multiple exons
Changing which exons are found in the final RNA changes the function of the protein

Question 1

What is the mechanism of splicing?

Answer 1

Doesn’t require ATP
Can splice over large distances (10s of kilobases)
Pre-mRNA contains introns and exons
OH group from branch point adenine attacks phosphyl bridge between last base of exon 1 and first base of intron
Lariat structure formation
OH from last base of exon 1 attacks bridge between exon 2 and lariat
Formation of spliced mRNA and lariat structure
Look at image

Question 2

What are the spliceosome and spliceosome co-factors responsible for?

Answer 2

Bringing splice sites into close proximity
Using the correct splice site
Exon skipping
Avoiding cryptic splice sites

Question 3

What is a cryptic splice site?

Answer 3

Selection of alternative splice sites: tissue and developmental-stage specific
Splice site selection must be tightly regulated
Many genetic diseases can be caused by point mutations that activate cryptic splice sites or delete splice sites
Cryptic splice sites can be useful when pre-existing and damaging when introduced by a mutation
Changes frame of protein so rest of protein sequence is missense
Missing important part of coding sequence so protein looses its function

Question 4

What is the structure of the spliceosome?

Answer 4

Made of 5 small nuclear RNAs (snRNAs)
snRNAs associate with proteins (at least 50) to form 4 small nuclear ribonucleic particles snRNPs
snRNPs: U1, U2, U4, U5, U6 (4 and 6 combine)
Spliceosome is made of snRNPs + accessory proteins
These attach to pre-mRNA transcript and form complexes
Specific complexes are formed in a sequential manner

Question 5

What are the steps of assembly and action of the spliceosome?

Answer 5

1) U1 and U2 assemble onto pre-mRNA in a co-transcriptional manner
U1 binds to splice donor site
U2 binds to start of 3’ exon (acceptor site)
2) The U1 and U2 snRNPs interact with each other to form the pre-spliceosome (complex A)
Brings ends of introns together
3) The preassembled tri-snRNP U4-U6-U5 is recruited to form complex B (tri-catalytic complex)
4) Complex B undergoes a series of rearrangements to form a catalytically active complex B
U1 and U4 are eliminated from the complex –> now made of U5, U6, U2
5) Complex B carries out first catalytic step of splicing, generating complex C which contains free exon 1 and the intron-exon2 intermediate
6) Complex C undergoes rearrangements and then carries out second catalytic step
Results in post-spliceosome complex that contains lariat intron and spliced exons
7) Release of spliced mRNA and lariat
RNA helicase unwinds RNA of lariat so RNA and U2, U5, U6 can be recycled

Question 6

DSCAM gene as an example of transcript variation

Answer 6

Example DSCAM (down syndrome cell adhesion molecule) gene
Expressed in developing neurons
Protects neural projections in dendrites from forming connections with themselves
If protein on a dendrite is the same then it will avoid it
Some exons are always included and other exons are randomly selected
24 exons permits 38,016 protein variants

Question 7

How is alternative splicing a method of regulation? Transcriptional control

Answer 7

Insertion/deletion of specific domains
Ex. It can regulate antibody and neuropeptide production

Question 8

How is alternative splicing a method of regulation? mRNA production

Answer 8

Splicing can affect amount of mRNA created
A premature stop codon in an exon that is not the final exon
If this exon is included it leads to nonsense-mediated RNA degradation
Unspliced intron leads to unprocessed RNA which is not transported into the cytoplasm or if it is transported then truncated due to premature stop codon in the intron

Exons are retained or skipped
Introns are excised or retained
5’ and 3’ splice site positions are moved (cryptic splice sites) to make exons longer or shorter

Question 9

What are the effects of alternative splicing?

Answer 9

Rate of translation of mRNA
mRNA degradation and susceptibility
Insertion/deletion of amino acids
Insertion/deletion of functional domains
Polypeptide truncation due to premature stop codon
Protein properties and functions can change due to splicing:
Make a smaller/larger protein
Soluble or membrane bound
Subcellular location changes
Affinity changes for substrate

Question 10

What are the 3 groups of alternatively spliced transcripts

Answer 10

5’ transcript ends differ from one another
3’ ends differ from one another
Middle portions differ

Question 11

5’ ends differ

Answer 11

5’ ends differ due to different transcription start sites
Example skipping the starting exon
Example in mouse alpha amylase gene:
In the liver transcription is initiated further down so the starting exons are skipped
Leads to salivary gland and liver having different affinities in tissues

Question 12

3’ ends differ

Answer 12

5’ ends the same, 3’ ends differ
When different poly A sites are used for transcription termination
Example in immunoglobin chains of antibodies:
Alternative 1: polyA site is after exon 4
Alternative 2: polyA site can after exon 6 so have 2 extra exons which code for a transmembrane anchor

Question 13

Centre differs

Answer 13

5’ ends the same, 3’ ends the same but middle differs
Can’t be explained by differential promotor use or cleavage
Example: troponin T gene in skeletal muscle
64 different ways found in different muscle types
Tissue specific splicing factors act on the pre-mRNA to decide which exons are included
Used to screen for people thought to have heart attacks as it expresses a specific muscle type of troponin T gene

Question 14

Alternative splicing vs exon shuffling

Answer 14

Alternative splicing and exon shuffling are different mechanisms for creating diversity
Alternative splicing acts on RNA to create diversity during lifespan of an organism
Exon shuffling created diversity on evolutionary scale over many generations

Question 15

What is Exon shuffling? How does it occur?

Answer 15

Exon shuffling: exons jump around in the genome so exons from different genes are rearranged or combined to give new gene structure
Many eukaryotic proteins are mosaics of motifs
How it occurs:
Illegitimate non-homologous recombination during meiosis - slightly misaligned
LINES (long interspersed nuclear elements): An exon nearby the LINE element can be transcribed and included in the RNA, then moved to a different location
DNA transposons (similar to LINES): can collect exons and move them into genes

Question 16

What are tissue specific splicing factors?

Answer 16

Tissue specific splicing factors: proteins that recognise cis-acting factors within the RNA transcript
Bind to pre-mRNA before it is spliced and it decides which exons are included
Promote or inhibit splice sites in different cases
Factor binds 1st intron and promotes splicing of exon 1 to exon 2
Factor binds 1st intron and inhibits splicing of exon 1 to exon 2, allowing splicing of exons 1 and 3

Question 17

Sex lethal (Sxl) gene

Answer 17

Autoregulates its own splicing
If sxl is present: inhibits inclusion of exon 3 which contains a stop codon = production of sxl protein
In males: No expression of early promotor so never get sxl protein
In females: Early promotor causes burst of sxl so stop codon is never included so get sxl protein
Sxl determines sex in somatic cells

Question 18

Transformer (Tra) gene

Answer 18

Tra splicing is regulated by sxl
In males: no sxl –> tra with early stop codon –> short protein with no function
In females: sxl binds at proximal splice site in intron 1 –> prevents U2AF binding –> binds cryptic splice site in exon 2 (distal splice site) –> allows splicing out of stop codon –> tra protein is expressed
Sxl promotes exclusion of an exon

Question 19

Doublesex (Dsx) gene

Answer 19

Dsx is responsible for sex determination
Tra is a splicing factor and regulates dsx (doublesex) gene
Dsx encodes a transcriptional repressor that determines development
Tra promotes inclusion of an exon
Females: Tra is recruited to exon splicing enhancer (ESE) by tra 2 –> tra binds to exon 4 –> recruits U2AF –> inclusion of exon 4 –> exon 4 contains stop site –> transcription termination
Dsx mRNA ends with exon 4 –> shorter protein isoform with different properties
Males: exon 4 is not included
2 isoforms of dsx protein –> regulates genes in different ways to get more female or male characteristics

Question 20

Fruitless (fru) gene

Answer 20

Fru is responsible for sex determination
Tra also regulates splicing of fru (fruitless) gene
Encodes a transcriptional regulator that determines development
In females: tra promotes splicing from the end of exon 2 –> inclusion of stop codon –> transcription termination no functional protein (truncated protein) and no male isoform is produced
In males: tra is absent –> get splicing from start of exon 2 (before stop codon) –> excludes stop codon –> functional protein
Tra forms a male specific isoform of fruitless which is important for male specific behaviour

Question 21

Explain the summary of the sex determination hierarchy in drosophila

Answer 21

Splicing factors can act positively (ex. Tra) to promote the use of a splice site
Or act negatively (ex. Sxl) to inhibit the use of a splice site
In females: 2 copies of X –> Sxl expression –> regulates it’s own splicing –> regulates Tra –> functional tra –> splices dsx and fru differently to get female characteristics
Sxl also regulates MSL-2 which is important for dosage compensation
In males: no Sxl expression –> no Tra –> male versions of dsx and fru to give male characteristics

Question 22

How does fruitless control mating behaviour?

Answer 22

Fruitless controls male mating behaviour
Male orientates itself at 45 degree to female, taps abdomen of female, sings, licks and attempts copulation

Question 23

How is alternative splicing used for response to signals with SLO gene?

Answer 23

SLO gene encodes potassium channel which is important for action potential in neurons
STREX domain –> SLO is reacts quicker to allow K+ to pass through channel
During action potential calcium is high –> binds to camkinase proteins –> regulates factor that binds to CAR region in pre-mRNA –> exclusion of STREX domain –> potassium channel is less sensitive and slows down action potential

Question 24

What are the regulating factors?

Answer 24

ESE - exonic splicing enhancer ISE - intronic splicing enhancer ESS - exonic splicing silencer ISS - intronic splicing silencer SR proteins - Stimulate splicing hnRNPs - hinder splicing, bind to exon silencing elements

Question 25

What are SR proteins?

Answer 25

Serine arginine repeat regions within splicing factors Bind 5' splice site and promote binding of U1 snRNP to promote splicing Can bind within exonic splice enhancers (ESEs) within downstream exon to promote U2AF binding and promote splicing

Question 26

What happens if RNA polymerase is elongating at a slower or faster rate?

Answer 26

Splicing occurs as transcription is still going on Rate of elongation can affect splicing pattern Slow: more likely to include exons with weak acceptor sites Fast: more likely to skip exons with weak acceptor site (as protein won't bind tightly)

Question 27

What controls regulation of splicing?

Answer 27

RNA sequences - elements within RNA that can recruit proteins Constitutive or tissue specific trans-acting factors (example Tra-2 is always present) Splice site strength: ability to bind to factors (U1 / snRNP) and presence/absence of ESEs

Question 28

Origins of introns early and late theory

Answer 28

Intron early theory: Introns originated in prokaryotes and due to evolution lost them to have more compact genomes Introns were kept in more complex organisms No evidence for anything that resembles introns in bacteria Intron late theory: Introns only evolved in eukaryotes Prokaryotes and archaea never had introns or spliceosome machinery

Question 29

Origins of introns theory with LECA

Answer 29

Last eukaryotic common ancestor (LECA) Prokaryotes invaded archaea like cell, co-opted to generate energy for the cell, became mitochondria of eukaryotes Bacteria contained retroelements (group II introns / self splicing RNAs) which invaded the archaea genome Happened after endosymbiosis Created precursors of introns / origin of spliceosomal introns in pieces

Question 30

How can introns be a burden to the host?

Answer 30

Spliceosome complex is huge and forms a large part of the genome So need to transcribe more RNA which requires energy and time (60 nt / s) Vulnerability as errors in splicing causes mutated proteins ex. Need recognition of cis-regulatory sequences

Question 31

Roles of introns

Answer 31

Sequence dependent functions ex. intron contains non coding RNA like microRNA Length dependent functions ex. Large introns take a long time to transcribe Splicing dependent functions ex. interaction between splicing machinery and RNA polymerase

Question 32

Life phases of an intron

Answer 32

Genomic intron Transcribed intron Intron being spliced Excised intron EJC-harbouring transcript (marks where exons have been spliced together)

Question 33

What is a genomic intron?

Answer 33

Still in the DNA Location of gene's cis-regulatory elements Contain transcription initiation sites (modulate main promotor action) Enhancers, silencers, TF binding sites Often found in most 5' introns 40% of TF binding sites are within introns

Question 34

Alternative transcription initiation due to genomic intron - AFP

Answer 34

Alpha-fetoprotein (AFP) plasma protein made in the liver and yolk sack in the foetus regulates osmotic pressure Tissue specific expression Alternative transcription initiation due to genomic introns Use of upstream TSS of exon 1 or TSS in the first intron

Question 35

Alternative transcription termination due to genomic intron

Answer 35

Intron sequences regulate polyadenylation and cleavage Different transcription termination depending on which polyA site is used (Intron needs to be harbouring the site) Example Flt-1 gene Soluble form is more abundant than the membrane bound form Membrane bound form has a later polyA site so includes exon 14 (longer protein) Soluble form has an earlier polyA site so excludes exon 14 (shorter protein)

Question 36

Nested genes of genomic introns

Answer 36

Introns can encode nested genes Same orientation or reverse strand 800 in drosophila May have their own promotor and different expression profile Non-coding (ex. microRNA) and protein-coding genes

Question 37

How does length of intron affect timing of when protein is made?

Answer 37

Length of intron affects the timing of when the protein is made RNA polymerase II has an elongation rate of 50kb / min Intron transcription may take hours Time delay between gene activation and translation of protein Must splice and export from nucleus before translation

Question 38

HES7 gene transcribed introns

Answer 38

HES7 gene (mus muscula) in mice Transcription factor Forms a negative feedback loop Controls timing of somite segmentation during embryonic development Somites form vertebrae, bones, cartilage Made in sequence as organism is growing Oscillations in HES7 expression Peak in oscillation forms somites so somites are formed at regular intervals

Question 39

Timing of expression and feedback loops in transcribed introns

Answer 39

Timing of oscillations affected by transcription Longer mRNA = transcription and translation takes longer = longer oscillations Negative feedback loop Delays due to time for transcription, splicing, translation HES7 is a transcription factor and represses its own transcription As protein levels increase, transcription decreases Once protein levels are low, transcription begins again Leads to oscillations in protein production Unstable protein required Introns are important for producing the correct period of oscillation mRNA stability, regulatory elements for feedback loops, length of mRNA Knockout HES7 mouse embryo, then reintroduced gene but without the introns --> oscillations occurring very frequently

Question 40

How are introns spliced? What do they affect?

Answer 40

Splicing occurs co-transcriptionally Linked via RNAPII C-terminal domain Splicing can affect initiation, elongation, termination

Question 41

How do introns affect initiation of splicing?

Answer 41

U1 of spliceosome binds to 5' end of intron in the pre-mRNA U1 promotes binding of pre-initiation complex TFIIH and TFIID Intron at the start of the gene enhances transcription

Question 42

How do introns affect elongation of splicing?

Answer 42

RNA polymerase doesn't always elongate, sometimes falls off Machinery to make sure RNA polymerase stays on U1 promotes TAT-SFI which binds to RNApol machinery and enhances elongation

Question 43

How do introns affect termination of splicing?

Answer 43

Endonucleolytic cleavage and polyA tail addition If a potential termination (polyA) site close, CPSF protein combines U2 binds to 3' end so if U2 is close to CPSF, CPSF is enhanced which increases the probability that transcription will end U1 binds to 5' end so if U1 is close, CPSF is inhibited which prevents termination Prevents early termination in an intron ex. a cryptic termination site

Question 44

What are excised introns?

Answer 44

When an intron is excised it forms a lariat structure and undergoes debranching and degradation Embedded RNA genes may be expressed in a removed intron Can contain non-protein coding RNAs (ncRNAs), such as microRNAs (miRNAs) and small nuclear RNAs (snoRNAs)

Question 45

Excised introns - mirtrons

Answer 45

Intron is excised (mirtron) by splicing to form pre-miR Pre-miR is exported from the nucleus and cut up by Dicer to form microRNA duplex Unwinded and loaded onto RISC which regulates expression of RNA Either leads to mRNA degradation or affects translation

Question 46

Excised introns - snoRNAs

Answer 46

60-150 nucleotides long Fundamental to RNA modifications in archaea and eukaryotes Modify RNAs (tRNAs, rRNA, snRNA) Ex. Methylated before they move into the nucleolus Released after splicing

Question 47

What are EJC harbouring transcripts?

Answer 47

Exon junction complex Binds 25 nucleotides upstream of exon-exon junction (where the intron was) on mRNA transcript Acts as a marker of where the intron was 4 core proteins (MAGO, YI4, eIF4AIII, MLN51) Present from splicing until translation

Question 48

Roles of EJC

Answer 48

Nuclear transport Translation activation mRNA localisation Nonsense mediated decay (NMD)

Question 49

EJC nuclear transport role

Answer 49

Mature mRNAs bind to mRNA specific transport factors Shuttles through nuclear pore complexes Transport rates are 10x higher for spliced transcripts with EJC which increases the expression of the transcript Spliceosome or EJC recruits ALY/REF export factor which allows it to export transcript to the cytoplasm more efficiently

Question 50

EJC translation activation role

Answer 50

Presence of EJC on the mature mRNA enhances translation EJC core component MLN51 interacts with eIF3 which is important for translation initiation

Question 51

EJC Cytoplasmic localisation role

Answer 51

Subcellular regions targeted within cytoplasm Localisation permitted by shuttling proteins Oskar mRNA needs EJC for localisation important for anterior posterior polarization of embryo

Question 52

EJC Nonsense mediated decay (NMD) role

Answer 52

Prevent expression of proteins/degrade proteins with a premature stop codon as this would truncate the protein To prevent dominant negative/gain of function proteins Normal stop codon: Ribosome kicks off EJC from mRNA Premature stop codon: splicing dependent, if EJC is more than 50nt downstream of a termination codon it is termed as premature Ribosome pauses at stop codon Proteins bound to ribosome and EJC can interact with each other UPF1 and UPF2 interact causing phosphorylation which tags the RNA for degradation Only occurs when stop codon is upstream of the EJC

Question 53

What are overlapping genes?

Answer 53

Adjacent genes located on either DNA strand sharing one or more nucleotides in coding sequence Found in mitochondria, microbes, eukaryotes In humans 10% of genes are overlapping Complete/internal/embedded/nested overlaps: Small gene within a larger gene Partial/terminal overlaps: Involving only small 5' or 3' overlap of coding sequences

Question 54

Same strand overlapping (unidirectional)

Answer 54

3' end of one gene overlapping with 5' end of another gene Genes may be regulated by a common promotor (so expressed at same time) Very common in bacteria

Question 55

Different stand overlapping

Answer 55

Convergent: 3' ends overlap Divergent: 5' ends overlap. Bidirectional promotors can be active to drive expression at the same time

Question 56

Examples of types of overlapping genes

Answer 56

Genes sharing same locus on same strand but coding for different proteins (Genes have same TSS and same first exon but rest of exons and termination sites are different) Genes sharing same promotor region (Are not physically overlapping) Nested gene (Gene is overlapping or falling within a single intron) Embedded gene (Exons of smaller overlapping gene are falling within introns but not same intron) Genes on opposite strands with overlapping locus but no overlap within the exonic region Tail to tail overlap in the exonic region (Example only 3' UTRs are overlapping) Head to tail overlap involving 5'-UTRs and coding sequences Draw image in notes

Question 57

What is gene phase?

Answer 57

Have to consider relative phase that the overlapping genes exist in relative to another Overlaps can be in the same reading frame or shifted 1-2 base pairs One gene is the reference gene and create base comparisons from that If overlapping genes result in same reading frames = in phase

Question 58

What are in phase overlaps?

Answer 58

In phase overlaps common in bacteria and viruses Result in the same reading frame 2 categories: involving different initiation and different termination of translation

Question 59

In phase overlaps initiation

Answer 59

Alternative translation start site New internal promotor formation (different transcription starts) Genes share terminator Different N-terminal domains Same C-terminal domains Ex. Bind same substrate with C-terminal but catalyse different reactions using N-terminal domains

Question 60

In phase overlaps termination

Answer 60

Same initiator codon Termination occurs at distinct codons Ex CS3 genes in E. coli form 5 polypeptides that form hair like appendages in bacteria

Question 61

Example of in phase initiation thermos flavus

Answer 61

Thermus flavus aspartokinase has different translation start sites askA: alpha subunit to give 405aa protein askB: starts at 3' end of askA, beta subunit to give 161aa protein Dependent on shine Dalgarno sequence to help initiate protein synthesis

Question 62

Out of phase overlaps in bacteria

Answer 62

Genes overlapping in ways that don't result in identical reading frames Can be on same strand or different strands Same strand: If base/open reading frame shifted one along = phase 1 If base/open reading frame shifted two along = phase 2 If both are in phase 0 then they are considered in phase Short overlaps are often in phase 2, large overlaps in phase 1 Due to genetic code probabilities - greater probability of stop codons so harder to get larger overlaps in phase 2 Different strand: Phase 0 is the backwards reading frame (opposite strand) No bias between phase 1 and phase 2 overlaps Genes overlapping in phase 2 will produce the same amino acid sequence

Question 63

Out of phase overlaps in eukaryotes Arf (P14) and Ink4a (P16)

Answer 63

Tumour suppressor made of two genes: Arf (P14) and Ink4a (P16) 2 TSS/first exons (1alpha and 1beta) that are transcribed from different promotors One for Arf at 5' end and one for Ink4a further down the gene Both splice to exon 2 but reading frames are different Arf gene will stop at end of exon 2 but Ink4a will continue to exon 3 This is an out of phase overlap between the two genes

Question 64

Partial overlap of genes

Answer 64

Small overlaps on 5' or 3' end Common for prokaryotes with functionally dependent genes Terminator site of 1 gene overlaps with initiator of another

Question 65

Example of partial overlap of genes with Tryp operon

Answer 65

5 genes involved in tryptophan synthesis Some have very short overlaps Overlap by one nucleotide between the stop codon of trpD and start codon of trpE Same with trpB and trpA (Shine Dalgarno sequence with trpB) Translation coupling is dependent on this ratio Allows protein to be synthesized in equimolar ratios Proximity of trpB stop codon to trpA start codon influences trpA translation By changing overlap between genes don't get equimolar production of proteins Degree of overlap can affect translation rates

Question 66

What is translational recoding

Answer 66

Ribosomes can be directed to: Use alternative start sites Bypass or recode termination codons Or site specific programmed shift of reading frame (PSRF) - can change frames while translating

Question 67

What is a ribosomal frame shift?

Answer 67

When ribosome pauses on mRNA and moves forwards or backwards 1 nucleotide before continuing Changes the reading frame Get more than one protein per mRNA Depends on the mRNA regulatory sequence and structure All mRNA structure must be unfolded (mRNA forms secondary structure which must be unfolded for translation) This can affect codon/anti-codon binding and leads to uncoupling Formation of energetic barriers is important for PRF Slippage can happen at any stage of translation PRF can diversivy the proteome

Question 68

Ribosome structure

Answer 68

A site=aminoacyl tRNA site that allows entry of new tRNA attached to an amino acid P site=peptidyl tRNA site that holds the growing polypeptide chain E site=exit of used tRNA and protein chain out of ribosome Small subunit=binds incoming tRNA Large subunit=facilitates protein synthesis

Question 69

-1 programmed ribosomal frame shifting mRNA requirements

Answer 69

Very common in prokaryotes mRNA requires a slippery sequence, a spacer sequence and a downstream stimulatory sequence Slippery sequence: where the shift takes place 7 nucleotides X XXY YYZ (original reading frame) --> XXX YYY Z (shifted reading frame) XXX: 3 identical nucleotides YYY: AAA/UUU Z: any nucleotide but not often G Spacer sequence: 12 nucleotides or less Downstream stimulatory sequence: Pseudoknots, kissing stem loop Energetic barrier for ribosome to overcome One of the secondary structures that the ribosome has to unfold before it translates Aids positioning over slippery site Causes ribosome to pause over the slippery site

Question 70

-1 programmed ribosomal frame shifting when does slippage occur?

Answer 70

Slippage may occur during distinct points of translation elongation cycle During accommodation of the A-site tRNA Or during EF-G catalysed translocation (EF-G catalyses translocation of tRNA and mRNA down the ribosome) Just after peptidyl transfer

Question 71

-1 programmed ribosomal frame shifting mechanism

Answer 71

Elongating ribosome encounters frameshifting signal (pseudoknot) and pauses Ribosome slips back one base Reading frame from G GGU UUA --> GGG UUU A Ribosome unwinds the pseudoknot and continues translating in new -1 frame

Question 72

+1 PRF example

Answer 72

OAZ1 in yeast Mammalian equivalent ornithine decarboxylase antizyme (OAZ) OAZ is involved in ubiquitin independent degradation of ornithine decarboxylase (ODC) ODC produces polyamines Usually OAZ has stop codon but +1 PRF shift adds an extra sequence to the end that targets ODC for degradation Autoregulation/negative feedback loop: ODC increases polyamine levels --> polyamines stabilize pseudoknot in OAZ mRNA--> causes the +1PRF on OAZ mRNA --> frameshift allows production of functional OAZ protein --> OAZ promotes degradation of ODC --> less polyamines are produced

Question 73

-1 PRF example

Answer 73

HIV virus Gag is produced as a 55kDa precursor protein that forms the virus particle (capsid) -1 PRF leads to formation of 160kDa GagPol polyprotein extended ORF which encodes for a protease, reverse transcriptase and integrase This only occurs 5% of the time If amount of frameshift is disrupted, can impede the growth of the virus Higher % (so makes more GagPol) it is less efficient for the cell Drug developed to stabilize stem-loop structure and increase amount of PRF to make viruses less efficient

Question 74

Why do we have overlapping genes and PRF?

Answer 74

Allows for genome compression PRF provides another method to increase the diversity of the proteome (can create many different proteins from the same stretch of DNA/RNA) Allows control of stoichiometry: PRF and shared promotors allow proteins to be expressed at stable levels relative to each other - allows for coordinated control

Question 75

Advantages of overlapping genes in viruses

Answer 75

Small genome size so gene compression is important Have limited space in the capsid so need to compress genome Smaller genome and smaller capsid allows viruses to replicate faster

Question 76

Disadvantage of overlapping genes

Answer 76

In evolution overlapping genes can cause evolutionary constraint Want to make a mutation that allows adaptation to the environment --> changing base pairs in one gene can affect the coding sequence of the overlapping gene

Question 77

Eukaryotes overlapping genes

Answer 77

Have larger genomes so have more types of overlapping genes Contain introns so overlapping genes may be located in introns More abundant different strand overlaps Lower proportion of divergent different strand overlaps At 5' end there is TF binding site and promotors, enhancers and overlapping which may lead to constraint

Question 78

Prokaryotes overlapping genes

Answer 78

Features exons primarily so exon overlapping is common Unidirectional overlapping is the most common - operons could be a driving force PRF may be more prevalent due to genome size restrictions

Question 79

Gene regulation by antisense transcription

Answer 79

Antisense transcription can impact gene expression at 3 different stages: transcription initiation, during transcription and post transcription Antisense is the non-coding strand

Question 80

Antisense transcription affecting initiation

Answer 80

ANRIL on antisense strand recruits polycomb complex to induce histone modification or DNA methylation--> represses transcription initiation

Question 81

Antisense transcription affecting during transcription

Answer 81

What would happen when two overlapping antisense genes are transcribed at the same time? RNA polymerases would collide

Question 82

Antisense transcription affecting post transcription

Answer 82

BACE1 antisense strand makes RNA that binds to base RNA which protects RNA from being degraded by an miRNA

Question 83

Sense-antisense pairs as self-regulatory circuits

Answer 83

Fine tuning: antisense expression slightly modulates expression of the sense gene Bistable switch: strong mutal repression Can go quickly from off state to on state of a gene

Question 84

What is RNA editing?

Answer 84

RNA editing are mechanisms that change the sequence of RNA transcripts encoded by genes in a wide range of organisms Only found in eukaryotes

Question 85

RNA editing vs RNA splicing

Answer 85

Overall similarities: mRNAs, tRNAs, rRNAs are substrates for RNA editing and splicing Alternative splicing and editing generate protein diversity Splicing and editing are developmentally regulated Overall differences: Splicing removes RNA sequences encoded by a gene Editing adds/changes the information encoded by a gene Splicing is often a RNA catalysed reaction (snRNPs), while editing is always protein catalysed

Question 86

RNA editing in trypanosoma parasite

Answer 86

Has a kinetoplast (extended mitochondria) Editing involves insertion or deletion of uridines Two thirds of mitochondrial genes are edited Information for editing is encoded by gRNAs transcribed from minicircles RNA editing process can be developmentally regulated to alter the proteome Editing occurs post-transcriptionally

Question 87

The kinetoplast

Answer 87

Kinetoplast: network of circular DNA inside the mitochondria that contains many copies of the mitochondrial genome It has around 4,000,000 bp DNA forms looping structure of maxicircles and minicircles 10,000 minicircles (1kb in length) and 50 maxicircles (20kb) Maxi circles encode components of the mitochondrial oxidative phosphorylation machinery

Question 88

Discovery of RNA editing in COXII

Answer 88

Cytochrome oxidase II (COXII) gene is very well conserved in eukaryotes Found premature stop codon in a very well conserved gene Sequenced RNA Found insertion of 4 uridines in RNA that removed the premature stop codon Cytochrome oxidase III (COXIII) gene Found a lot of uridine insertions (heavily edited) Editing occurs post-transcriptionally

Question 89

What is the function of insertion or deletion of uridines / RNA editing?

Answer 89

Form start codons Correct frameshift mutations Create complete ORFs Remove premature stop codons Form appropriate stop codons

Question 90

Minicircles

Answer 90

Minicircles encode guide RNAs (not like ones from CRISPR) About 1kb in size Thousands of copies in kDNA (10,000) Heterogenous sequences

Question 91

Maxicircles

Answer 91

Encodes mRNA and rRNA genes About 22Kb size Tens of copies in kDNA (50)

Question 92

How do minicircles and maxicircles edit RNA?

Answer 92

Mitochondrial genes and minicircles are transcribed as polycistronic RNAs Guide RNAs produced from minicircles bind to mRNAs produced from maxicircles and guide RNA editing Mature edited mRNA is translated All information to remove or insert genes comes from circles

Question 93

Mechanism of Trypanosome (T. brucei) RNA editing

Answer 93

Annealing of guide RNA that has base pair complementarity to target mRNA Insertions: template guides TUTase to insert uridine into mRNA, ligation and translation Deletions: exonuclease cuts mRNA and removes uridine, ligation and translation Editing occurs post-transcriptionally

Question 93

Structure of gRNA

Answer 93

Triphosphate at 5' end Polyuridine at 3' end added post-transcriptionally Anchor sequence - base pairs to target pre-mRNA Guiding sequence - directs insertion or deletion of uridine into mRNA Editing occurs post transcriptionally and can lead to either insertions or deletions These guide RNAs should not be confused with ones used in CRISPR!

Question 94

Editosome

Answer 94

20S editosome U-insertion and U-deletion domains RNA binding and zinc-finger proteins to help bind mRNA Enzymes used for cutting or adding in uridines - TUTase, exonuclease, ligase

Question 95

How does alternative RNA editing generate protein diversity in trypanosomes?

Answer 95

Trypanosome takes part in RNA editing to rapidly alter mitochondrial function and change its metabolism for when it is in different stages of its life cycle Editing causes shift from trypanosome quiescent to proliferative mode for more mitochondrial activity to give correct proteins Example COXIII gene is heavily edited to make COXIII protein but it can also be alternatively edited to make AEP-1 by changing N-terminus

Question 96

What is AEP-1?

Answer 96

AEP-1 binds in the kinetoplast Important for integrity of kinetoplast DNA network Affects fitness of trypanosomes

Question 97

RNA editing in slimemold

Answer 97

RNA editing in Physarum/slimemold Additions of GU and CU C to U changes (deamination) Extremely accurate Occurs co-transcriptionally (unlike trypanosomes occurs post-transcriptionally)

Question 98

RNA editing events in plant mitochondria and plastids

Answer 98

Mitochondria: C to U transitions occur, U to C transitions are rare, mRNAs, rRNAs, tRNAs are edited, high frequency (2%), post transcriptional Plastids: C to U transitions occur, U to C transitions are rare, mRNAs are edited, low frequency (0.04%), post transcriptional Editing tends to be higher in mitochondria in plants

Question 99

RNA editing mechanism in plants

Answer 99

Protein binds to specific sequence that brings in transaminase to alter RNA NO guide RNA that has base pair complementarity

Question 100

Mammalian mRNA editing

Answer 100

C to U and A to I changes occur

Question 101

Apolipoprotein B mRNA editing (C to U editing)

Answer 101

Apolipoprotein has C to U editing that produces 2 proteins: one specific to liver and one specific to intestine RNA editing of C to U produces premature stop codon which truncates the protein and leads to protein specific to intestine Need formation of hairpin loop and 5' efficiency element to recruit ACF that performs editing

Question 102

Adenosine deaminases (ADARs)

Answer 102

Adenosine deaminases that act on RNA (ADARs) are a class of RNA editing enzymes In vertebrates there are 3 different types of ADARs proteins Contain 1-3 double stranded RNA binding motifs ADARs found in a wide range of organisms from yeast to mammals Converts adenosines to inosines (A to I) mRNAs, tRNAs, viral RNAs and non-coding RNAs can be substrates Alters specific codons to change amino acids or changes stop codons

Question 103

ADAR structure and mechanism

Answer 103

ADAR is a single protein (not a complex) Sequence in an intron adjacent to an exon that allows recruiting of ADAR protein

Question 104

Editing of serotonin receptor in mammals

Answer 104

Higher editing = reduced efficiency of serotonin receptor Over and under editing of this mRNA are associated with genetic diseases (Prader-Willi syndrome) and with depression

Question 105

Editing of AMPA glutamate receptor in mammals

Answer 105

Glutamate receptor must be edited from glutamine to arginine Lack of editing of GluA2 Q to R results in a large influx of Ca2+ into neurons and causes death Low levels of GluA2 Q to R has been observed in patients with major depressive disorder and schizophrenia

Question 106

RNA editing and development ADAR1

Answer 106

ADAR1 mutants are embryonic lethal Phenotypes include impaired haematopoiesis and defects in liver formation Embryonic stem cells have high editing levels ADAR1 levels impact efficiencies of cellular reprogramming Exact roles and contributions to these developmental defects are not yet clear

Question 107

RNA editing and cancer

Answer 107

ADAR1 down regulation can reduce proliferation of chronic leukaemia in mice ADAR2 down regulation inhibits cellular proliferation in different types of brain tumours ADAR silencing in breast cancer leads to less cell proliferation and more apoptosis

Question 108

Higher levels of RNA editing in the nervous system

Answer 108

Editing is important in the nervous system/brain ADAR mutants (knock-outs) lead to brain related phenotypes (behavioural phenotypes) 3 ADAR proteins in mammals ADAR2 mutants die from seizures ADAR3 can't do RNA editing but can block RNA editing by ADAR2, it is only expressed in the brain Alu repeats in the genome are heavily edited Alu harbouring genes are also enriched for neuronal genes

Question 109

RNA editing as a driving force of brain evolution

Answer 109

Theory More editing occurs in the brain The bigger the brain, the more RNA editing is occurring More RNA editing = more developed brain and nervous system Intensity of RNA editing is higher in humans than in mice Alu repeats suggests increase in editing from monkeys to humans

Question 110

Why does the artery have a lot more editing compared to other tissues

Answer 110

Because it has to adapt to a wider range of conditions

Question 111

ADAR1, ADAR2, ADAR3 functions

Answer 111

ADAR1 edits repetitive sites ADAR2 edits nonrepetitive sites ADAR3 acts as an inhibitor of editing

Question 112

How much RNA editing occurs in squids compared to mammals?

Answer 112

Mammals have around 200 reported recoding sites (RNA editing that changes the protein sequence) More than 50,000 recoding sites in the squid nervous system

Question 113

Why are there high levels of editing in squid and flies?

Answer 113

Squid and flies have higher editing than humans Cold blooded organisms use RNA editing to rapidly respond to changes in the environment (ex. temperature) When temperature decreases, editing increases in a potassium channel in drosophila Theory: A to G change (G is like I) results in replacement of large R group for a small one which reduces activation energy so enzyme can work at a lower temperature