Splicing Flashcards
What is primary transcript for 60s ribosome? (eukaryotes)
5.8S and 28S rRNA
What is primary transcript for 40s ribosome? (eukaryotes)
18S rRNA
Describe major steps in making tRNA through splicing
- Remove leader sequence at 5’ (RNase P)
- Remove trailor sequence at 3’ (RNase D)
- Base modification (e.g. pseudouridine)
- Add CCA arm to 3’ end
- generate anticodon region (endonuclease + ligase)
- Tyr added to CCA arm
what is responsible for anticodon region production in tRNA made?
endonuclease + ligase
Chemicals needed to attach CCA arm to 3’ of tRNA
2 CTP + 1 ATP
what helps add CCA arm to tRNA?
tRNA nucleotidyl transferase
How does capping occur in eukaryotic pre-mRNA?
i) hydrolysis of triphosphate (one phosphate grp released)
ii) oxygen on beta phosphate at 5’ end of transcript attack alpha phosphate on GTP
–> form 5’ to 5’ triphosphate linkage
What enzymes are involved in capping of eukaryotic pre-mRNA?
guanylyl transferase
S-adenosyl methionine (methylation)
Product from capping of eukaryotic pre-mRNA
5’ to 5’ triphosphate linkage
–> PPi + P
Function of guanyly transferase in capping of pre-mRNA
cataylze formation of 5’ to 5’ triphosphate linkage
What enzyme helps with methylation on pre-mRNA?
S-adenosyl methionine (SAM)
Why is PolyA tail needed?
increase stability of mRNA
where does transcription stop?
after transciption pass consensus sequence of AAUAAA –> poly A addition site
What helps with formation of poly A tail?
Poly A polymerase
What is the function of CPSF?
recognize poly A site and G/U (3’ of poly A site)
What is the function of cleavage factors (CF)?
cut transcript 3’ from poly A site after CPSF binds
What happens before poly A tail formed?
i) 7 methyl G cap formed
ii) CPSF recruited and recognize poly A site and G/U
iii) CF binds to transcript and cleave transcript 3’ of poly A site
iv) CFs dissociate –> PAP come in to form poly A tail
What is RNA editing?
changes one or more nucleotides in RNA transcript by deamination
(A to I or C to U)
Base modifications that can occur during RNA editing?
A to I
C to U
Example of RNA editing
without editing
–> Apo b 100 formed with CAA
with RNA editing
–> stop codon UAA formed
–> truncated Apo B-48 formed
Difference between truncated and normal version of ApoB
Truncated version lack LDL receptor binding region
–> only has lipoprotein assembly part
function of U5 in splicing
i) bind to 5’ splice site, aligning splice sites
ii) after lariat intermediate formed, align 3’OH of exon 1 to 5’ end of exon 2 for 2nd transesterification rxn
What happens when A is deaminated to I?
I will pair with C instead
(different from A pairing with U)
What happens after C is deaminated to U?
U pair with A instead
(different from C pairing with G)
How does RNA editing increase protein diversity?
i) Alter amino acid coding possibilities
ii) introduce premature stop codon (e.g. in Apo-B)
iii) change splice site in transcript
Features of intron splicing on transcript
i) on 5’ splice site, have invariant GU
ii) on 3’ splice site, have invariant AG
iii) on branch site, have invariant A
Major chemical reaction during splicing
transesterification
Which snRNA(s) align splice sites for splicing to occur?
U5
Function of U4 in splicing
inhibit the activity of U6 when binding to it
Function of U1 in splicing
recognize 5’ splice site
Function of U2 in splicing
recognize the branch site
Where are the phosphodiester bond located in branch site after splicing?
Three phosphodiester bonds
–> 2’ connect to 5’ splice site, 3’, 5’ (from previous base)
Function of U6, U2
bind tgt to catalyze transesterification rxn
Function of calcitonin
regulates Ca2+, phosphorous metabolism
Which snRNA(s) recognize branch site?
U2
Which snRNA(s) recognize 5’ splice site?
U1
Which snRNA(s) catalyze transesterification rxn?
U2,6
How much major snRNP are present per nucleus?
100,000
mutation in thalassemia
A to G mutation at branch site
Effect of mutation in thalassemia
introduce new 5’ splice site
–> may form abnormal transcript
–> lower amount of beta globulin produced –> Hb decrease
Example of tissue specific splicing
In thyroid, only intron spliced
–> form calcitonin that regulates Ca2+, phosphorous metabolism
In brain
–> exon 4 spliced
–> form CGRP that is related to calcitonin
Function of CGRP
related to vasolidation
–> calcitonin gene related protein
final step of forming a mature tRNA
anticodon region adds Tyr to CCA arm
How does group 1 self-splicing introns work?
i) Group 1 introns align with upstream exon with its internal guide sequence (IGS)
ii) G cofactor introduced and carry out nucleophilic attac to phosphate at 5’ splice site
iii) G incorporated in 5’ of intron, then 3’OH of upstream exon attack phosphate in5’ of 2nd exon
iv) introns released
Similarities between self splicing RNA and spliceosome
i) OH attack 5’ splice site
ii) 3’OH of 1st exon to 3’ splice site
What carries out first nucleophilic attack in group 2 introns?
2’OH within group II intron
Difference between group 1 and 2 self-splicing introns
i) Group I has G cofactor incoroporated for 1st attack, while nucleophilic site of group 2 introns is included within introns
ii) 3’OH used for 1st attack in Group I, 2’OH used for 1st attack in Group II
Where are group 1 introns usually found?
eukaryotic genome
Where are group 2 introns usually found?
mitochondrial genomes or lower eukaryotes
What carries out first nucleophilic attack in group 1 introns?
3’OH in introduced G cofactor
What happens to spliced group I introns after being removed?
i) 3’OH attacks G –> circularize
ii) the loop then opens up
iii) four nt removed –> circularizes
iv) L19 RNA formed in linear form (catalytically active)
Where does G cofactor bind in self-splicing grp 1 intron?
major groove of DNA
How does G cofactor bind to self-splicing grp 1 intron?
triple base pair
G cofactor uses 2 H as donor with N7 and O6 in G
Order of proteins recruited to CTD within pre-mRNA
i) capping enzymes
ii) splicing factors
iii) polyadenylation factors
How to test for G cofactor binding in group 1 intron?
site-directed mutagenesis
i) Change G in intron to A
–> no activity
ii) change C in intron to U
–> no activity
iii) use 2AP (aminopurine) to replace G cofactor
–> recovered
What is catalytic efficiency stand for?
V (velocity)/Km (affinity)
–> higher the better
Function of L19
promote nucleotidyl transfer rxns
Example of action by L19
i) C5OH pair with G rich region in L19
ii) 3’OH in intron attack C in C5 (1st transesterification)
iii) C4OH released
iv) another C5OH joins in, which its 3’OH attack G on 3’ of intron
v) C6OH made
Product from L19 reaction of one round
C4OH, C6OH
(If C5OH is added in both steps)
How to test for Mg2+ role in group 1 intron?
i) base either connected by O or S to G cofactor
ii) test against Mg2+ or Mn2+
iii) gel electrophoresis
(only two bands seen in Mg2+ with O linkage)
What does Mn2+ bind to in experiment?
oxygen and sulphur
What system is responsible for most of the RNA splicing in eukaryotes?
spliceosome
How does Mg2+ help stabilize structure of group I intron?
mg2+ with phosphate
Mg2+ with water with base
Mg2+/H2O/2’OH
From experiment on splicing of 26S rRNA, What are the findings?
i) Mg2+ help with structure of introns
ii) IVS isn’t a protein as bands are seen in presence of protease
Examples of viral RNA
i) hammerhand ribozyme
ii) hairpin ribozyme
iii)nhepatitis delta virus ribozyme
Who’s awarded nobel prize for discovery of ribozymes?
Thomas Cech, Sid Altman
