Lecture 5 - Pre-mRNA And ncRNA Processing

Question 1

What is on the 5’ end of every mRNA?

Answer 1

A cap

Question 2

Why is the CTD tail important for RNA pol II?

Answer 2

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

Question 3

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

Answer 3

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

Question 4

What happens if the mRNA is not capped?

Answer 4

The mRNA is degraded

Question 5

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

Answer 5

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

Question 6

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

Answer 6

Pol I and pol III

Question 7

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

Answer 7

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

Question 8

When is CPSF and cstF recruited to the tail?

Answer 8

After phosphorylation of certain amino acids

Question 9

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

Answer 9

The CPSF and cstF will bind

Question 10

What is the termination signal in the mRNA?

Answer 10

AAUAAA

Question 11

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

Answer 11

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

Question 12

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

Answer 12

Stabilises it

Question 13

Is the poly-A tail encoded into the RNA?

Answer 13

No

Question 14

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

Answer 14

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

Question 15

What is splicing?

Answer 15

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

Question 16

What do you need to splice?

Answer 16

enzymes for cutting and then join the two ends together

Question 17

what type of process is splicing?

Answer 17

Catalytic

Question 18

what does splicing form?

Answer 18

An intron lariat -

Question 19

How does splicing then happen?

Answer 19

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

Question 20

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

Answer 20

Specific sequences both minor and major

Question 21

How do proteins recognise specific sequences in RNA?

Answer 21

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

Question 22

How does the proteins identify the sequence in splicing?

Answer 22

protein using a guide RNA (a ribonucleoprotein complex)

Question 23

What is the names of the ribonucleoproteins involved in splicing?

Answer 23

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

Question 24

The ribonucleoproteins involved in splicing are called what?

Answer 24

SnRNPs - small nuclear ribonucleic proteins

Question 25

What do snRNPs recognise?

Answer 25

5’ splice site and branch site.

Question 26

What do the snRNPs help do?

Answer 26

They help catalyse RNA cleavage and joining reactions

Question 27

What does U1 recognise?

Answer 27

5’ splice site

Question 28

What does BBP and U2Af bind to?

Answer 28

Branch point and 3’ splice site - starts making loop

Question 29

What does U2 do?

Answer 29

Displaces BBP, binds to branch point making the A bulge

Question 30

What does U4, U5, and U6 do?

Answer 30

They join the complex and U6 displaces U1 -

Question 31

What does U4 specifically do?

Answer 31

Is displaced causing active site formation and catalysis

Question 32

What does U5 specifically do?

Answer 32

Brings 2 exons together = final reaction

Question 33

What do U6 and U2 bridge?

Answer 33

The 5’ and 3’

Question 34

Can introns self cleave in some cases?

Answer 34

Yes

Question 35

Where is nuclear free pre-mRNA splicing derived from?

Answer 35

The self cleave mechanism

Question 36

Alternative splicing - can this be constitutive?

Answer 36

Yes -

Question 37

What is alternative splicing?

Answer 37

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

Question 38

Can alternative splicing be used as a regulatory mechanisms.

Answer 38

yes

Question 39

Why is alternative splicing important?

Answer 39

Evolution of new proteins

Question 40

How do we define which exons should be joined together?

Answer 40

Regulatory proteins

Question 41

What is one type of regulatory proteins?

Answer 41

SR proteins

Question 42

What do SR proteins do?

Answer 42

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

Question 43

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

Answer 43

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

Question 44

What do SR proteins do?

Answer 44

Promotes splicing

Question 45

What do hnRNPs do?

Answer 45

Inhibits splicing

Question 46

Can splicing be regulated in a cell specific way?

Answer 46

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

Question 47

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

Answer 47

yes

Question 48

Why is splicing medically relevant?

Answer 48

Many point mutation from splicing cause inherited human diseases

Question 49

How does mutations impact alternative splicing?

Answer 49

- 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.

Question 50

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

Answer 50

It will just join with the third exon

Question 51

What is a cryptic splice site?

Answer 51

Sequence that has the potential for interacting with the spliceosome

Question 52

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

Answer 52

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

Question 53

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

Answer 53

MicroRNA’s

Question 54

What are microRNA’s?

Answer 54

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

Question 55

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

Answer 55

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

Question 56

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

Answer 56

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

Question 57

What happens to pri-miRNA in the cytoplasm?

Answer 57

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

Question 58

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

Answer 58

One strand is kept in the miRISC

Question 59

Where does the miRISC take the single stranded miRNA?

Answer 59

To the mRNA to regulate it

Question 60

What happens to the mRNA in the miRISC

Answer 60

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

Question 61

Where can microRNA’s be produced from?

Answer 61

Exons and introns