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Molecular Genetics > Lecture 5 - Pre-mRNA And ncRNA Processing > Flashcards

Lecture 5 - Pre-mRNA And ncRNA Processing Flashcards

1
Q

What is on the 5’ end of every mRNA?

A

A cap

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2
Q

Why is the CTD tail important for RNA pol II?

A

scaffold for RNA processing proteins, regulates phosphorylation and is essential for life

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3
Q

The CTD tail recruits capping enzymes - how does this happen after the nucleotides are synthesised? What happens to the cap?

A

5’ triphosphate of the primary transcript is cleaved and a guanosine residue is added via 5’-5’ linkage. The guanosine cap is methylated

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4
Q

What happens if the mRNA is not capped?

A

The mRNA is degraded

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5
Q

What are the functions of the 5’ cap to mRNA?

A

Protects from degradation, promotes pre-mRNA splicing, needed for export from the nucleus and required for efficient translation in the cytoplasm

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6
Q

What produces RNA’s which do not have a cap?

A

Pol I and pol III

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7
Q

Polydadenlation and cleavage factors are also recruited to the CDT tail of pol II. What are the 2 important factors recruited here?

A

CPSF - cleavage and polyadenylation specificity factor
CstF - cleavage stimulating factor

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8
Q

When is CPSF and cstF recruited to the tail?

A

After phosphorylation of certain amino acids

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9
Q

What happens when the mRNA with the correct signal for termination goes past the CTD tail?

A

The CPSF and cstF will bind

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10
Q

What is the termination signal in the mRNA?

A

AAUAAA

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11
Q

What happens to the RNA after CPSF and cstF have bound?

A

It cleaves and recruits a polyA binding protein and a non-templates tail.

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12
Q

What does the recruitment of poly-A polymerase (PAP) and the template do to the mRNA?

A

Stabilises it

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13
Q

Is the poly-A tail encoded into the RNA?

A

No

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14
Q

You have cleaved the RNA tail and added a polyA tail on for protection but how does the ribosome know to stop translating?

A

Could be that the ribosome does not have a cap and is therefore being degraded

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15
Q

What is splicing?

A

The exons get transcribed but the introns need to be removed and spliced out

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16
Q

What do you need to splice?

A

enzymes for cutting and then join the two ends together

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17
Q

what type of process is splicing?

A

Catalytic

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18
Q

what does splicing form?

A

An intron lariat -

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19
Q

How does splicing then happen?

A

An A residue in the intron nucleophilic attacks the 5’ splice site creating a lariat structure (a loop) then the 5’ exon is joined to the 3’ exon

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20
Q

How is the 5’ and 3’ splice sites recognised?

A

Specific sequences both minor and major

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21
Q

How do proteins recognise specific sequences in RNA?

A

Through contacts between amino acids and bases or stacking interactions.
Or through a protein using a guide RNA (a ribonucleoprotein complex)

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22
Q

How does the proteins identify the sequence in splicing?

A

protein using a guide RNA (a ribonucleoprotein complex)

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23
Q

What is the names of the ribonucleoproteins involved in splicing?

A

U1, U2, U3, U4, U5 and U6.

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24
Q

The ribonucleoproteins involved in splicing are called what?

A

SnRNPs - small nuclear ribonucleic proteins

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25
Q

What do snRNPs recognise?

A

5’ splice site and branch site.

26
Q

What do the snRNPs help do?

A

They help catalyse RNA cleavage and joining reactions

27
Q

What does U1 recognise?

A

5’ splice site

28
Q

What does BBP and U2Af bind to?

A

Branch point and 3’ splice site - starts making loop

29
Q

What does U2 do?

A

Displaces BBP, binds to branch point making the A bulge

30
Q

What does U4, U5, and U6 do?

A

They join the complex and U6 displaces U1 -

31
Q

What does U4 specifically do?

A

Is displaced causing active site formation and catalysis

32
Q

What does U5 specifically do?

A

Brings 2 exons together = final reaction

33
Q

What do U6 and U2 bridge?

A

The 5’ and 3’

34
Q

Can introns self cleave in some cases?

A

Yes

35
Q

Where is nuclear free pre-mRNA splicing derived from?

A

The self cleave mechanism

36
Q

Alternative splicing - can this be constitutive?

A

Yes -

37
Q

What is alternative splicing?

A

The idea that you can make functional mature messanger RNA from a locus due to the combination of exons put together

38
Q

Can alternative splicing be used as a regulatory mechanisms.

A

yes

39
Q

Why is alternative splicing important?

A

Evolution of new proteins

40
Q

How do we define which exons should be joined together?

A

Regulatory proteins

41
Q

What is one type of regulatory proteins?

A

SR proteins

42
Q

What do SR proteins do?

A

They bind to exons and contact splicing machinery (exonic splicing enhancers - ESEs). This helps cooradinate splicing in specific areas

43
Q

What is the regulatory protein which binds to RNA but not the splicing machinery?

A

hnRNPs (heterogenous ribonucleoproteins) these are intronic/ exonic splicer silencer

44
Q

What do SR proteins do?

A

Promotes splicing

45
Q

What do hnRNPs do?

A

Inhibits splicing

46
Q

Can splicing be regulated in a cell specific way?

A

yes - if there is a repressor near a splice site if a repressor is there then no splicing happens but if a repressor is not there splicing happens.

This is the same with activators

47
Q

Can alternative splicing affect the untranslated regions as well as reading frames?

A

yes

48
Q

Why is splicing medically relevant?

A

Many point mutation from splicing cause inherited human diseases

49
Q

How does mutations impact alternative splicing?

A
  • an exon migt get skipped
  • the normal splice site is altered and another cryptic splice site is used.
  • a mutation could just cause a new splice site to be used.
50
Q

What happens when a mutation causes an exon to get skipped?

A

It will just join with the third exon

51
Q

What is a cryptic splice site?

A

Sequence that has the potential for interacting with the spliceosome

52
Q

Example of a splicing defect - deficiency’s in STAT-1. What was wrong with the splice site?

A

Mutation in the donor splice site skipping exon 3 - this makes the gene smaller and so moves through the gel quicker.

53
Q

MRNA isn’t the only product of Pol II what are others?

A

MicroRNA’s

54
Q

What are microRNA’s?

A

a class of non-coding RNA that are produced in a pol II transcripts (normally in the introns) before being moved into the cytoplasm.

55
Q

How to microRNA’s moved to the cytoplasm? First stop the nucleus

A

Form a stem loop structure which is recognised by enzymes in the micro-processor of the nucleus (DROSHA and DGCR8)

56
Q

What happens once the microRNA has been processed by DROSHA and DGCR8?

A

They are cleaved making a pre-miRNA which can go to the cytoplasm

57
Q

What happens to pri-miRNA in the cytoplasm?

A

Processed by DICER1 to make it smaller and it then binds to AGO1-4

58
Q

What happens to pri-miRNA once it is in the AGO1-4?

A

One strand is kept in the miRISC

59
Q

Where does the miRISC take the single stranded miRNA?

A

To the mRNA to regulate it

60
Q

What happens to the mRNA in the miRISC

A

The miRNA is a guide and can inhibit translation and mRNA degradation processes

61
Q

Where can microRNA’s be produced from?

A

Exons and introns

Molecular Genetics (20 decks)

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