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RDT > Pattys Lectures L18-22 > Flashcards

Pattys Lectures L18-22 Flashcards

1
Q

Is there proof-reading in transcription in Eukaryotes?

A

No

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2
Q

How many RNA pols are there in Euks Transcription?

A

3

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3
Q

What is the structure of RNAPs

A

2 large subunits and multiple (10-14) smaller subunits

Similar to proks

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4
Q

What is the CTD heptad repeat that distinguishes RNAPII from the other RNAPs?

A

YSPTSPS

young stinky prostitute tickles sams penile spasms

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5
Q

How many copies of the CTD are there in humans?

A

52
Can be a substrate for kinases
flexible scaffold dock for proteins
Proteins come on and off the scaffold via phosphorylation

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6
Q

How many NTs must RNAPII transcribe for it to escape?

A

5
4NTs= Escape commitment
>5NTs=Clash, Escape (8-9NTs)

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7
Q

After how many NTs does promoter-proximal pausing occur?
TFIIH phosphorylates CTD Ser5/Cap addition
NELF (negative elongation factor) and DSIF inhibit elongation

A

+20 to +40

Rate limiting step (check-point)
5’ CAP ADDITION

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8
Q

CTDK-1/P-TEFb

NB/ RNAPII is very processive

A

phosphorylates inhibitory proteins NELF/DSIF and ser2
NELF phosphorylation by P-TEFb releases elongation inhibition
DSIF phosphorylation by P-TEFb recruits elongation factors (elongin/ELL)
CTD phosphorylation by P-TEFb on Ser2 recruits factors for 3’ end formation

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9
Q

Pre-mRNA 5’ Cap addition

7-methyl guanosine m7G

A

Addition of m7G to 5’ end of pre-mRNA is known as capping
Capping is a co-transcriptional process
Capping enzyme complex is bound to CTD of RNAPII

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10
Q

What is the importance of the 5’ CAP?

A

Promotes mRNA stability by preventing 5’ end degradation
Promotes intron excision (mRNA splicing)
Acts as a tag to promote mRNA export from nucleus
Stimulates mRNA translation in the cytoplasm

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11
Q

3 step process: pre-mRNA 5’ Capping enzyme

A

bifunctional enzyme

1) NT phosphohydrolase
2) RNA guanylyl transferase

Guanine N7 methyl transferase
Nucleoside-2-O-methyl transferase

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12
Q

In Eukaryotes is there a transcriptional termination signal?

A

NO

3’ ends of mRNAs are formed by CLEAVAGE immediately followed by poly-adenylation

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13
Q

What is the importance of a polyA tail?

Poly(A) polymerase does not require a template

A

Promotes mRNA stability-protects from 3’ exoribonucleases
Promotes mRNA export
Affects translational EFFICIENCY (the longer the poly (A) tail the more efficient translation is)

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14
Q

How does termination occur in transcription? Eukaryotes

A

After the mRNA has been made the RNAPII continues beyond 3’ end cleavage and poly(A) tail addition. 3’ end cleavage allows entry of 5’-3’ exoribonucleases which act like a “torpedo” to degrade nascent RNA.
These exoribonucleases will eventually catch up with the RNAPII and will cause the complex to dissociate.

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15
Q

Do both Euks and Proks undergo splicing?

A

NOOOOOO
Only Euks
Pros are dependent on the genome they have

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16
Q

What are the main NTs in introns and exons?

A

introns (and promoters) -A/T rich

exons-G/C rich

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17
Q

Splice donor site and acceptor site?

Within intron

A

Donor-GU (100% invarient)
Acceptor-AG (99.9% conserved)
Branch point = unpaired A
Pyrimidine rich region adjacent to the A site will assist with the splicing reaction IN HIGHER EUKS with large introns

18
Q

Why is U5 snRNP important in lariat formation?

A

Once you have made the lariat you put the mRNA in a dangerous position to be degraded by exonuclease
U5 snRNP plays an important role and bridges exons that are going to be joined and keeps them in place in close proximity

19
Q

What is the debranching enzyme used for?

A

Loop/lariat is insensitive to exonucleases so need debranching enzyme to open up the lariat
It is now exposed and subjected to nucleases so we are able to reuse the NTs

20
Q

What type of disorders arise form cis or trans acting mutations?

A

Cis-loss of function/frame-shifts
Trans-Retinitis pigmentosa
Spinal muscular atrophy

21
Q

What proteins bind to ESEs?
Alternative splicing
exonic splicing enhancer

A 
SR proteins
serine/arginine
These bind to ESEs and recruits U2AF to upstream intron (recruits U2 snRNP)
Recruits U1 snRNP to downstream intron
SR proteins also promote mRNA export
22
Q

How is drosphila sex determined?

A

By a ration
X:A = # X chromosomes:sets of autosomes (A)
FEMALE (XX)=2:2 MALE (XY)=1:2
No dominant gene in drosophila whereas in humans dominant gene is on the Y chromosome

23
Q

What protein promotes female development in drosophila?

This protein has a premature stop codon in exon 3

A

Sxl (sex-lethal) - transacting factor
There are two promoters-early and late
Sxl early protein promotes alternative splicing of sxl late pre-mRNA
Sxl regulates its OWN pre-mRNA splicing
Sxl early is a SPLICING REPRESSOR -prevents U2AF from binding within intron2
Exon 3 out of 8 is skipped out in females by using sex early protein
Sxl early then produces functional Sxl LATE PROTEIN

24
Q

What other protein other than Sxl is needed for female development?

A

Tra
Gene only has 4 exons
Premature stop codon exists in exon2

25
Q

What protein encodes 2 different function proteins with different splice variants and is the third pathway in the drosophila developmental pathway?

A

Dsx (Doublesex gene) Tra2 SR protein
Exon skipping in males of exon 4 out of 6 exons
Exon 4 has ESE site and Tra made previously in females is recruited to Tra2 SR protein present on Exon 4 ESE. This includes exon 4
Tra recruits U2AF to intron 3
Exon 4 uncovers a new poly(A) site

Males exon1,2,3,5,6
Females exon 1,2,3,4Poly(A)tail

26
Q

What is the cause of myotonic dystrophy?

A 
Sequesters trans-acting splicing factors
CUG repeats in the 3'UTR (non-coding seq)
n=50-4000
n=38-49 premutation
n=5-37 normal
  1. Expanded triplet repeat in 3’ UTR forms HAIRPIN
  2. Hairpin sequesters Muscleblind-like I splicing factor, which effects alternative splicing
  3. Affects splicing of pre-mRNAs requiring these factors
27
Q

Muscle blind like I splicing factor causes what diseases?

A 
Insulin receptor:Insulin resistance
Chloride channel:Myotonia
Cardiac troponin T:Cardiac abnormalities
Ryanodine receptor:Muscle weakness
NMDA receptor 1:CNS effects

Myotonic dystrophy

Muscle blind is involved in a lot of alternative splicing factors

28
Q

B-RAF kinase
B-RAF V600E

What is it inhibited by?

A

Leads to inhibitor resistance
BRAF involved in MAPK cascade
RAS-BRAF-MEK-ERK-CCND1-cell proliferation
p61 BRAF V600E alt splicing isoform lacks exons 4-8 and activates MEK- is oncogenic and becomes constitutively active
RAS binding domain is deleted

BRAF V600E is inhibited by vemurafenib
Resistance to vemurafenib is associated with 61kDa variant of BRAF V600E arising from alternative splicing

29
Q

RNA editing:mRNA variation not caused by alternative-splicing

A

Cytidine deaminase (occurs in the NUCLEUS)
Changes NH2 to O
In a deamination reaction it creates URACIL

EXAMPLE:ApoB gene makes 2 different variants:
Large-liver-fatty acid metabolism
Small-intestine-dietry lipids, chylomicron proteins
introduced immature stop codon CAA->UAA

ADARs Adenosine deaminases acting on dsRNA
Substrate is ds pre-mRNA (DOUBLE STRANDED)
A changed to I (inosine)
I is recognised as a G (CAG=CIG=CGG)

30
Q

What are the consequences of adenosine demonises acting on dsRNA?
ds regions caused by hairpins formed by ss pre-mRNA/mRNA

A

affect splice acceptor sites
affect start/stop codons
modify ion channel and neurotransmitter function
defence against viruses (change of seq will change immune system,-protection of viruses)

31
Q

mRNA export into cytoplasm

A

Through nucleopores since mRNA is too large to diffuse from nucleus to cytoplasm
mRNA goes head first (5’ CAP)

32
Q

mRNA export by heterodimeric transport receptor

A

Nxf1/TAP and Nxt1 heterodimeric subunits of mRNA export receptor complex
SR proteins are dephosphorylated after splicing-permits binding of exporter complex

REF (RNA Export Factor) protein coordinates mRNA splicing and export
Marks EJC (exon-exon junction complex) at completion of splicing
Bridges mature mRNA and Nxf1/TAP exporter
REF protein is also recognised by the transporter complex

33
Q

In mRNA export how is the CAP protected in the cytoplasm?

A

CAP gets protected by binding a translational complex called Euk initiation factor 4E
EIF4E

In the cytoplasm mRNA undergoes an exchange of proteins
PABPII to PABPI

34
Q

Protecting mRNAs from degradation

A 
5' M7G CAP
3' Poly(A) addition
PABP bound to poly(A) tail (mRNA circularisation in cytoplasm)
Protects against 3'-5' deadenylases
NUCLEUS-PABPII
CYTOPLASM-PABPI
35
Q

Cytoplasmic mRNA decay

The degradation reactions will take place in specific compartments in the cell (both nuclear and cytoplasmic compartments are involved)

A

Poly(A) tail has protective function
Poly(A) Nuclease (PAN) will deadenylate the mRNA (remove the tail) - PABPI comes off
De-capping protein DCP1 removes the CAP

Allows exoribonucleases to chew up the rest of the mRNA
5’-3’ exo = XRN1 or GW/P-body
3’-5’ exo = Exosome

36
Q

What do PREs and AREs do?

A

PRE-pyrimidine rich element
inhibit deadenylation
ARE-AU rich element
enhance deadenylation

37
Q

Name 2 examples of mRNA diseases?

A

C-fos (proto-oncogene) normally short lived t1/2=1hr
2AREs de-stabilise mRNA
Deletion of ARE stabilises mRNA
Upregulation of mRNA-malignant skin cancer

Beta-globin normally long lived t1/2=20-30hr
Reduction causes thalassemia
Disrupt PRE in beta-globin
Reduce mRNA stability

38
Q

Cytoplasmic polyadenylation involves what proteins?

A

Cis-acting CPE (cytoplasmic polyadenylation element) in the 3’ UTR affects polyadenylation

Affected transcripts are usually products of spermatogenesis and oogenesis in which poly(A) tails are removed after nuclear export
These mRNAs are stored in a translationally dormant state

Cytoplasmic poly(A)+ addition makes stored mRNA competent for translation-process is faster than de novo transcription

39
Q

What are the 3 types of mRNA quality control?

A
  • Non stop mediated decay
  • Nonsense mediated decay (NMD)
  • No-go decay

Depends on the type of error in the mRNA
Processes usually involved in the pioneering round of translation (FIRST round)

40
Q

Why are histones an exception?

A

Get their poly(A) tail added in the cytoplasm whereas most mRNA get their tail added in the nucleus

41
Q

Gene expression and mRNA localisation - what mRNAs are recruited to the - and + end of the motors?
Dosophila

A
  • end dynein motor, BICOID driven to anterior end
    + end kinesin, OSKAR driven to posterior end

Actin, needed at leading end of fibroblast and it is localised to mRNA at that edge (leading) so you can make a protein where it is needed

Cis acting sorting signals (zipcodes) (usually in 3’ UTR) recruits trans-acting factors, which promote transport of the mRNAs along MTs or actin to the place they are needed

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