Lecture 10

Question 1

Process of capping (4 steps)

Answer 1

1. phosphatase removes gamma phosphate from 5’ end of nascent RNA.
2. Guanyl transmethyl transferase adds a guanosine monophosphate
3. guanine 7-methyl transferase adds a CH3 to the guanosine at position N7
4. 2’-O-Methyl transferase adds a CH3 to the 1st ribose in mRNA chain (only in some mRNA)

Question 2

Function of 5’capping (3)

Answer 2

1. regulates export of mRNA out of nucleus by Cap Binding Complex
2. Required for efficient translation of mRNA into protein (CBC is replaced by translation initiation factor)
3. Prevents 5’degradation, increasing half life of mRNA

Question 3

Process of polyadenylation

Answer 3

1. CPSF (Cleavage & polyadenylation specificiy factor) recognize and binds to poly-A signal
2. CstF (Cleavage stimulating factorF) binds to GU sequence
3. Cleavage factor binds to CA sequence
4. All the three factors recruit Poly A polymerase.
5. Cleavage factor cleave then PAP polymerize A nt.

Question 4

What is the sequence of poly A signal?

Answer 4

AAUAAA

Question 5

Function of 3’ poly A tail (4)

Answer 5

1. PABP binds to poly A protecting mRNA thus increasing half life of mRNA
2. Stimulate initiation of translation as PABP interact with translation initiation factor.
3. Facilitates transcription termination
4. Aids in export of mRNA from nucleus.

Question 6

What are the three conserved sequences in pre-mRNA direct splicing?

Answer 6

1. Splice donor site (GU)
2. Splice acceptor site (AG)
3. Branch point sequence (A)

Question 7

Steps of splicing (5)

Answer 7

1. U1 snRNP binds to 5’ end of intron (splice donor site)
2. U2 snRNP binds to branch point
3. U4/6 and U5 bind to U1 and U2, forming a loop (lariat)
4. U4 snRNP is released activating spliceosome
5. Cleavage and ligation occurs at the same time resulting into mature RNA and intron in lariat shape

Question 8

What is spliceosome made up of?

Answer 8

small nuclear ribonucleoproteins (snRNP) such as U1,2,4,5,6

Question 9

What is snRNP made up of ?

Answer 9

Short-nuclear RNAs and proteins

Question 10

How can alternative splicing carried out?

Answer 10

Repressor or activator binds to splicing site, then regulate the alternative splicing. For negative regulation, repressor is used to prevent splicing. For positive regulation, normally splicing site is too weak so no splicing occurs, but activator binds to the site, then facilitates splicing.

Question 11

What is Limb Girdle muscular dystrophy?

Answer 11

Mutation in the calpain 3 gene that generates a new splice site stronger than existing one within exon16 which results in short mRNA producing defective protein.

Question 12

Relationship between Beta thalassemia with mutation at splice site.

Answer 12

A mutation might occur in normal acceptor which skips exon resulting in shorter mRNA.
Or cryptic splice site might activated due to mutation in normal acceptor site.

Question 13

What is cryptic splice site?

Answer 13

Sequence upstream which is a weak splice site, but if there is mutation in normal strong splice site, this cryptic site can be used, leading abnormal length of mRNA

Question 14

2 types of RNA editing

Answer 14

Adenosine to Inosine

Cytidine to Uridine

Question 15

What enzyme carries out A-I RNA editing?

Answer 15

Adenosine deaminase acting upon RNA enzyme (ADARs)

Question 16

Mechanism of A-I editing

Answer 16

Before splicing, complimentary RNA sequence in exon and intron within the same RNA form a dsRNA structure, where ADAR enzyme binds and modifies adenosine to Inosine.After editing, intron is spliced out to become mature mRNA.

Question 17

Where is ADAR1 found?

Answer 17

Liver, required for normal erythrocyte development

Question 18

Where is ADAR2 found

Answer 18

Brain, for nomal brain development

Question 19

What happens if RNA editing (A-I) is not carried out in glutamate receptor mRNA in brain?

Answer 19

Glutamine instead of arginine is produced, which leads tk conductance of both Na and Ca, instead of only Na. This leads to seizures, continuous muscle contraction

Question 20

Which enzyme does C-U editing need?

Answer 20

Cytidine deaminase

Question 21

What happens if C-U editing is not done in apolipoprotei B mRNA?

Answer 21

After splicing, when editing is done, it creates stop codon, leading to shorter protein, targeting intestine. But if editing is not done, there is no stop codon, and longer protein is produced which targets liver.

Question 22

Difference between adenosine deaminase and cytidine deaminase

Answer 22

Other than differenth editing, adenosine needs stem-loop structure to detect editing region, but cytidine doesn’t.

Question 23

Relationship of systemic lupus erythematosis to splicing.

Answer 23

Patients has auto-antibodies that react with U1 RNA component of spliceosome, which prevents normal splicing.