Lecture 10 Flashcards
Process of capping (4 steps)
- phosphatase removes gamma phosphate from 5’ end of nascent RNA.
- Guanyl transmethyl transferase adds a guanosine monophosphate
- guanine 7-methyl transferase adds a CH3 to the guanosine at position N7
- 2’-O-Methyl transferase adds a CH3 to the 1st ribose in mRNA chain (only in some mRNA)
Function of 5’capping (3)
- regulates export of mRNA out of nucleus by Cap Binding Complex
- Required for efficient translation of mRNA into protein (CBC is replaced by translation initiation factor)
- Prevents 5’degradation, increasing half life of mRNA
Process of polyadenylation
- CPSF (Cleavage & polyadenylation specificiy factor) recognize and binds to poly-A signal
- CstF (Cleavage stimulating factorF) binds to GU sequence
- Cleavage factor binds to CA sequence
- All the three factors recruit Poly A polymerase.
- Cleavage factor cleave then PAP polymerize A nt.
What is the sequence of poly A signal?
AAUAAA
Function of 3’ poly A tail (4)
- PABP binds to poly A protecting mRNA thus increasing half life of mRNA
- Stimulate initiation of translation as PABP interact with translation initiation factor.
- Facilitates transcription termination
- Aids in export of mRNA from nucleus.
What are the three conserved sequences in pre-mRNA direct splicing?
- Splice donor site (GU)
- Splice acceptor site (AG)
- Branch point sequence (A)
Steps of splicing (5)
- U1 snRNP binds to 5’ end of intron (splice donor site)
- U2 snRNP binds to branch point
- U4/6 and U5 bind to U1 and U2, forming a loop (lariat)
- U4 snRNP is released activating spliceosome
- Cleavage and ligation occurs at the same time resulting into mature RNA and intron in lariat shape
What is spliceosome made up of?
small nuclear ribonucleoproteins (snRNP) such as U1,2,4,5,6
What is snRNP made up of ?
Short-nuclear RNAs and proteins
How can alternative splicing carried out?
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.
What is Limb Girdle muscular dystrophy?
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.
Relationship between Beta thalassemia with mutation at splice site.
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.
What is cryptic splice site?
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
2 types of RNA editing
Adenosine to Inosine
Cytidine to Uridine
What enzyme carries out A-I RNA editing?
Adenosine deaminase acting upon RNA enzyme (ADARs)
Mechanism of A-I editing
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.
Where is ADAR1 found?
Liver, required for normal erythrocyte development
Where is ADAR2 found
Brain, for nomal brain development
What happens if RNA editing (A-I) is not carried out in glutamate receptor mRNA in brain?
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
Which enzyme does C-U editing need?
Cytidine deaminase
What happens if C-U editing is not done in apolipoprotei B mRNA?
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.
Difference between adenosine deaminase and cytidine deaminase
Other than differenth editing, adenosine needs stem-loop structure to detect editing region, but cytidine doesn’t.
Relationship of systemic lupus erythematosis to splicing.
Patients has auto-antibodies that react with U1 RNA component of spliceosome, which prevents normal splicing.
