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CMB2001 - Control of Eukaryotic Gene Expression > L7 - Post-transcriptional control of gene expression > Flashcards

L7 - Post-transcriptional control of gene expression Flashcards

1
Q

What is the central dogma of gene expression?

A

We’ve evolved a system which comes from an RNA world, where the RNA was the genetic information, to a protein world, where proteins are catalytic, ending up in the DNA world, where the genetic information is the DNA, and the RNA does the functional roles between DNA & proteins

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2
Q

Transcription & translation in prokaryotes

A

Single membrane round the outside

As the RNA is being produced, ribosomes are being attached to translate it as its being transcribed

Transcription and translation are coupled (translation is co-transcriptional)

Translation is occurring very rapidly after the gene is being turned on, so translation will happen after transcription, but most happens at the same time

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3
Q

Transcription & translation in eukaryotes

A

Have an inner membrane compartment – nucleus, where the genetic material is stored

DNA is in the nucleus where it is transcribed to mRNA

Transcription and translation happen in different places due to compartmentalisation

Therefore, there is a lag between transcription and translation, they are not coupled

Gives us a level of control that’s not available in bacteria

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4
Q

Regulation of eukaryotic gene expression

A

Regulation is possible at each step

Even if transcription levels are constant, the amount & type of mRNA can vary dramatically – changes can happen in translation

Genetic diseases are caused by defects in these events

Viruses alter or use them to their advantage

Need to get RNA thorugh nuclear pore which is a difficult event

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5
Q

Why is it difficult to get RNA through the nuclear pore?

A

mRNA is quite a charge molecule and the pore is a hydrophobic place so difficult to get through the membrane

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6
Q

What is the coding sequence in mRNA?

A

The section between AUG & TER

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7
Q

What are the eukaryotic specific elements in mRNA?

A

5’ cap & polyA tail

They are not encoded in the genome – are added after transcription

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8
Q

What is unfinished mRNA?

A

Precursore mRNA / pre-mRNA

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9
Q

What does mRNA have to go through to become finished?

A

Capping
Splicing
Polyadenylation
Editing

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10
Q

What events are coupled to transcription via the RNA pol II C terminal domain?

A

Capping
Splicing
Polyadenylation

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11
Q

CAPPING

What is the cap made of?

A

Cap made of a G and a methyl group at position 7

5’ m7G cap

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12
Q

CAPPING

How does capping work?

A

Capping – 2 step event:

  1. GpppN structure
  2. Methylation

RNA initially contains triphosphate at 5’ end
Cap is added backwards – 5’ onto 5’

Methylation alters chemical behaviour of base – mRNA is chemically very different to a normal gene

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13
Q

CAPPING

Functions of the 5’ m7G cap

A

Protects mRNA from degradation by 5’-3’ nucleases

Facilitates splicing

Facilitates export from the nucleus

Critical for translation of most messages

Functions mediated through protein binding, serves as a binding site to allow proteins to bind to dictate what happens to the RNA, the cap itself doesn’t do this it is just a binding site that is recognised

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14
Q

CAPPING

What does the 5’ m7G cap act as a binding site for?

A

CBP80/CBP20 in nucleus (processing/export)

eIF4 complex in cytoplasm (translation)

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15
Q

Structure of eukaryotic genes

A

Have promoter, poly A, termination signal, but in a most genes there is no discernible open reading frame – it isn’t continuous

Instead we have group of exons which is split up across the gene – the coding sequence is separated by introns

To produce an mRNA this all has to be joined together to produce a coding sequence so we can translate

Pre-mRNA contains both introns and exons and is transcribed all the way through

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16
Q

SPLICING

What is splicing?

A

Splicing cuts out of all of the introns and joins all of the exons to produce one mRNA with a proper open reading frame

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17
Q

SPLICING

What are the conserved sequences in introns?

A
  • 5’ splice site
  • 3’ splice site
  • Branch site

Sequences define limits of exon & intron

Sequences recruit the splicing machinery required to remove the intron and join the exons

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18
Q

SPLICING

What happens if the sequence is cut wring by 1 nucleotide?

A

If cuts wrong by one nucleotide then you have a frame shift and then the incorrect mRNA is produced so don’t produce the protein you want

19
Q

What does Y mean in an mRNA sequence?

A

Pyrimidine

C or U

20
Q

What does R mean in an mRNA sequence?

A

Purine

A or G

21
Q

What does N mean in an mRNA sequence?

A

Any nucleotide

22
Q

SPLICING

What are the 2 trans-esterification reactions?

A

Step 1
• Cut at 5’ splice site
• Creation of bond between 5’ end of intron & branch site

Step 2
• Cut at 3’ splice site to release intron lariat
• Ligation of 2 exons

The lariat structure is then degraded, the cell turns it over

23
Q

SPLICING

What is the spliceosome?

A

Enzymatic complex that catalyses the removal of introns

Requires ATP

Large complex containing >200 proteins that assembles onto each intron

24
Q

SPLICING

What proteins make up the spliceosome?

A

RNA-binding proteins
ATPases
GTPases

Also contain several snRNPs

25
Q

SPLICING

What are snRNPs?

A

Small nuclear ribonucleo-protein particles

They are small RNAs that form a simple structure, each contain a conserved sequence – the red box

They have different structures but are very conserved between eukaryotes

Also have a protein binding site – the Sm binding site (consensus sequence)

That sequence binds the same set of proteins – Sm proteins

7 membranes all having similar fold and they sit together to form a donut like ring, the RNA goes through the hole in the centre, so the RNA binding surface is in the centre

A very important structure as the elements are essential for splicing

26
Q

SPLICING

What is anti-Sm & lupus?

A

Anti-Sm antibodies react against the Sm proteins.

Anti-Sm antibodies are very rare unless you have the autoimmune disease systemic lupus erythematosus (SLE)

~20% of patients with SLE have anti-Sm antibodies

27
Q

SPLICING

What is the splicing mechanism?

A
  • U1 recognises 5 prime splice site
  • U2 recognises the branch point
  • U4,5,6 recognise the structure of U1 and U2 and the rest of the intron
  • U1 and U4 then leave as not needed so are recycled, U4 is chaperone for U6
  • Still no catalysis done
  • Complex of U2, 5 and 6 on the intron mediate the processing
  • Catalysis is split between U2 and U6, they are the catalytic components, U5 functions to hold the two exons together
  • Step 1 produces lariat 2 – exons separate
  • Step 2 exon ligation
  • Coming in step wise on to the mRNA as its being produced and recognising the conversed sequence elements by base pairing
28
Q

SPLICING

What is alternative splicing?

A

Process that enables a messenger RNA (mRNA) to direct synthesis of different protein variants (isoforms) that may have different cellular functions or properties

It occurs by rearranging the pattern of intron and exon elements that are joined by splicing to alter the mRNA coding sequence

But can lose different parts of a genome by doing alternative splicing

29
Q

SPLICING

Mutations in the 5’ splice site

A

If we have a mutation in the 5’ splice site, then it could mean the exon isn’t recognise and it is skipped, so we lose an element of the RNA we may need

30
Q

SPLICING

Diseases that arise from mutations in the 5’ splice site

A

Spinal muscular atrophy

Retinitis Pigmentosa

Myotonic dystrophy

31
Q

What is spinal muscular atrophy?

A

Result in the loss of motor neurons and progressive muscle wasting

Most common genetic cause of infant mortality, any kid wont reach adult hood

32
Q

What is Retinitis Pigmentosa?

A

Reduced visual capabilities and blindness, which develops with age, by middle age blind

33
Q

What is myotonic dystrophy?

A

A muscle wasting disease

34
Q

What is polyadenylation?

A

The addition of a poly(A) tail to a messenger RNA

35
Q

POLYADENYLATION

What is the starting sequence you need for polyadenylation?

A

AAUAAA

The polyadenylation sequence

36
Q

POLYADENYLATION

What does the polyadenylation sequence do?

A

This sequence triggers endonuclease cleavage of the RNA

RNA is cut after this sequence, then addition of A’s by PolyA polymerase where it is cleaved

37
Q

POLYADENYLATION

What is the process linked to?

A

Transcription termination

Once cleaved the AAUAAA then the mRNA is free to go be exported and translated

38
Q

POLYADENYLATION

Structure of the polyA site

A

Conserved AAUAAA 10-35 nucleotides upstream of the poly(A) site.

G/U or U rich tract just downstream of poly(A) site

U-rich upstream element “USE”

39
Q

POLYADENYLATION

What proteins are required for polyadenylation?

A

CPSF

CstF

PAP

40
Q

POLYADENYLATION

What is CPSF?

A

Cleavage & polyadenylation specificity factor (CPSF)

Binds AAUAAA

41
Q

POLYADENYLATION

What is CstF?

A

Cleavage stimulatory factor (CstF)

Binds G/U

42
Q

POLYADENYLATION

What is PAP?

A

PolyA polymerase

43
Q

POLYADENYLATION

How do CPSF, CstF & PAP work?

A

Factors come in, like the ‘pac-man’ like nuclease

Come in and cut at the poly A site

CstF binds G/U sequence, get cleavage to produce 3’ end of mRNA

polyA pol (PAP) comes in and adds the A, not templated by the genome

44
Q

POLYADENYLATION

Functional significance of the polyA tail

A

Enhances export of RNA

Stabilises the 3’ end of the mRNA

Enhances translation of mRNA

CMB2001 - Control of Eukaryotic Gene Expression (10 decks)

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