Week 9 - RNA Splicing Flashcards
E complex
Formation of commitment complex in which U1 is Base paired with 5’ splice site
A complex
U2 addition to base pair with branch site in the presence of ATP
B1 complex
Joining of U4/6 and U5 tri-snRNP
B2 complex
U1 and U4 release Formation of catalytic centre in which U6 base pairs with 5’ splice site; U6 also Base pairs with U2; U2 remains Base paired with branch site; U5 interacts with both exons through its loop
C1 complex
The first step of transesterification 5’splice sites are cleaved, lariat formed
C2 complex
The second step of transesterification, 3’ splice sites cleaved, exons ligated
RNA can … with other nucleic acid molecules
Hydrogen bonds
What is pre-MRNA?
The nuclear transcript that is processed by modification and splicing to give an mRNA
What is RNA splicing?
The process of excising introns from RNA and connecting the exons into a continuous mRNA
How is RNA modified?
In the nucleus by additions to the 5’ and 3’ ends and by splicing to remove the introns
What is the first modification made to eukaryotic mRNA ?
5’ 7-methylguanosine cap
How is the 5’ 7-methylguanosine cap formed?
By adding a guanine through the 3’ phosphodiester bond
What is the function of 5’ 7 methylguanosine cap?
To protect the mRNA from degradation by endonuclease s
What recognises the 5’ 7- methylguanosine cap?
Cap binding protein heterodimer (CBP20/80) to facilitate export from the nucleus
What does the cap structure influence?
mRNA stability, splicing, export and translation
When does the capping process take place?
During transcription
Name the components of the capping enzyme?
RNA triphosphatase and guanyltransferase
How does capping occur?
- The Gamma phosphate is removed from a nucleotide on the emerging pre-mRNA 2. Guanyltransferase will add guanine to the beta phosphate 3. Nitrogen in position 7 is methylated
Why is RNA pol II involved in the production of mRNA?
It has a large carving terminal domain that provides a docking site upon activation
How is CTD on RNA pol II activated to induce transcript capping?
Ser-5 is phosphorylated by TFIIH
What are the splice sites?
Sequences immediately surrounding the exon-intron boundaries?
The 5’ splice site at the end of intron in U2 splice sites, contain what?
The consensus sequence GU
The 3’ splice site at 3’ end of the intron in U2 splice site contains what?
The consensus sequence AG
The GU-AG splice site is present in what % of human introns?
98
In GU-AG splice sites, what is the branching nucleotide ?
A
In U12 splice sites, what is at the 5’end of the intron?
The consensus sequence AU
In U2 splice what is the at the 3’ end of the intron?
The consensus sequence AC
The AU-AC forms what % of human introns?
0.1%
True or false: GU-AG splice sites have a pyramiding rich rich tract?
True
What does splicing require?
The 5’ and 3’ splice site and a branch site upstream of the 3’ splice site
When is lariat formed
When the intron is cleaves at the 5’ splice site and the 5’ end is joined to a 2’ position at the A at branch site in intron
How is lariat formed?
By two transesterification reaction
How is the intron released as a lariat?
It is cleaves at 3’ splice site and left and right exons are ligated together
Describe the transesterification reaction needed to release the lariat?
2’-hydroxyl on branching nucleotide initiates nucleophilic attack on 5’ splice site 3’-hydroxyl generated at the end of 3’ end of the exon during the first transesterification initiation a nucleophilic attack on the phosphodiester bond at the 3’- splice site
What are small cytoplasmic RNA ?
RNAs that are present in the cytoplasm involved in the modification of rRNA
What are small nuclear RNAs?
One of many small RNA specifies confided to the nucleus that are important in the recognition of the boundaries and creation of the splicing machinery
What are small nucleolar RNA?
A small nuclear RNA that is localised to the nucleolus and is involved in the processing of tRNA
Name the 5 snRNPs involved in splicing of a U2 splice site?
U1, U2, U5, U4 and U6
What is the size of spliceosome?
12 MDa
What do all snRNPs except U6 Contain?
Conserved sequence that binds to Sm proteins that are recognised by antibodies ?
What is a splicing factor?
A protein component of the spliceosome that is not part of one of the snRNPs
How does the complex assemble on pre-MRNA?
Sequentially
How are Antibodies (anti-SM) generated?
autoimmune diseases
What are U4 and U6 round together as?
A Di-snRNP
Name a non-snRNP protein that is needed for spliceosome pathways?
Splicing factor 1 (Branch point binding protein)
What does splicing factor 1 bind to?
The branch point in the intron and when U1 interacts to the complementary sequence with the 5’end
What does U2AF bind to?
The polypyrimidine tract and AG at 3’ splice site
How does U1 snRNP initiate splicing?
Bind to 5’ splice site by means a of an RNA-RNA pairing reaction
What does the commitment complex ( E complex) Contain?
U1 snRNP and the U1AF protein
What is the role of splicing regulatory proteins?
Initiation the formation of commitment complex
How can the pairing of splices sites be accomplished?
By intron definition or exon definition
What do splicing regulatory proteins contain?
Large amounts of serine and Lysine to recognise sequences within exons
What does the interaction of SR proteins and U1 do?
Bring the 5’ intron close to the branching site in the intron
Describe how pre-MRNA is committed to the splicing pathway?
U1 and SR proteins define 5’ splice site U2AF bind to pyramidine tract and 3’ splice site Branch point binding protein recognises the branch site SR proteins bridge the complexes at the 5’ and 3’ splice site to form commitment complex
List the order of complexes in the spliceosome assembly pathway?i
E complex, A complex, B1 complex, B2 complex, C1 complex and C2 complex
What causes the commitment complex to progress to A complex?
In the presence of ATP, U2 will base pair with branch site
How is A complex converted to B1 complex?
The recruitment of U5 and U4/U6 snRNPs which will interact with the pyramidine tract
How is B2 complex formed ?
U1 snRNP is released to allow U6 snRNA to interact with 5’ splice site
What occurs to form the C complex from B2 complex?
U4 is released which allows U6 to interact with 5’ end of the intron. U5 and U6 will create enzymatic site which will alllow transesterification to occur
What happens with the C1 complex?
The lariat is formed with 5’ splice site cleaves by transesterification
What happens with C2 complex?
The second step of transesterification 3’ splice site is cleaves and the exon is ligated
What do Exons Contain which are binding sites for SR proteins?
Exotic splicing enhancer
What do exonic splicing enhancers do?
When bound, SR proteins recruit U1 snRNP to the downstream splice site and U2AF to the pyramidine tract and AG dinucleotide of the upstream 3’-splice site
What does U2AF recruit ?
snRNP to the Branch point sequence in cross-exon recognition
What can mutations in splice sites or ESE lead to?
Skipping of entire exons or intron rentention
What does BRCA1 mutation destroy?
An acceptor site leading to a 59 bp insertion
What do mutation in splice site induce?
exon skipping
What snRNP do alternative spliceosome use?
U11 and U12, in addition to different U4 and U6
Where are Autocatalytic introns found?
Organelles and bacteria: Group I and II introns
Which intron excise themselves from RNA by an autocatalytic splicing event ?
Group II introns
How does splicing begin and how does it continue
Begins as cotranscriptional process and continues as posttranscriptipnal process
What machineries are physically and functionally integrated?
Transcription and splicing
What is splicing connected to ?
mRNA export and stability control
What does alternative splicing contribute to?
Structural and functional diversity of gene products
Alternative splicing of the CaMKII delta gene
3 different alternative exons target the kinase to different cellular compartments
Delta A
Target to membrane
Delta B
Target to nucleus
Delta C
Localise in the cytoplasm
What is Alternative splicing associated with ?
Weak splice sites
Give two examples of binding proteins that can promote or suppress splice site selection in context dependent fashion?
Nova and Fox families
What are the 3 steps of transcription termination?
- Cleavage at poly(A) site 2. Addition of poly(A) at new 3’end 3. Transcription termination downstream from the cleavage site
What is the function of polyadenylation ?
Increase stability of MRNA
What is signal sequence for cleavage to generate a 3’end of MRNA ?
AAUAAA
What is needed for polyadenylation?
Specificity factor, an endonuclease and a poly(A) polymerase
What does CstF bind to?
GU rich sequence
What does CTD bind to?
Both CPSF and CSTF
What does Ssu74 and PC4 interact with?
CPSF and CstF
What machinery is needed for polyadenylation only?
PABII
What does PAB II do?
Interact with CPSF, stimulate rate of poly(A) addition and controls poly(A) tail length
What is needed for both cleavage and polyadenylation?
CPSF, PAP and symplekin
What does CPSF bind to?
PolyA signal AAUAAA
What does PAP do and what is it recruited by?
Adds the poly(A) tail and is recruited by CPSF
What does symplekin do?
Help to assemble or stabilise the CstF complex and holds the entire cleavage/polyadenylation machinery together
How many A residues does the specificity factor and poly(A) polymerase add to the 3’ end?
~200
What does the poly(A) control?
mRNA stability and influences translation
What plays a role in Xenopus embryonic development?
Cytoplasmic polyadenylation
What is the function of Xrn2?
Digests downstream RNA until it reaches RNA pol II
How is transcription terminated in Polymerase I?
2 discrete termination sites are recognised by DNA-binding protein and cleavage mediated by the endonuclease Rnt1
How is transcription terminated in Polymerase III?
Defined terminator sequence in the DNA molecule (oligo dT) Signals release of RNA polymerase
What is expression of histone mRNA ?
Replication dependent
When is the expression of histone mRNA regulated?
cell cycle
True or false: histone mRNA are polyadenylated
False
How are the 3’ end of histone mRNA generated?
Cleavage reaction that depends on a conserved hairpin structure in the mRNA
What is needed for 3’end of histone mRNA to be formed?
Steric loop binding protein (SLBP) Bind to a stem-loop structure and the U7 snRNA to pair with adjacent single-stranded region
What enzyme catalysed the 3’ end formation of histone mRNA ?
CPSF73
What is REF1, how is it recruited and what is its function?
RNA export factor 1 Recruited by splicing factor UAP56 Targets mRNA to nuclear pore
What is exon junction complex and what is its function?
A protein complex that assembles exon-exon junctions during splicing and assists in RNA transport, localisation and degradation
After the mRNA has been exported, what factors remain and what factors dissociate ?
mRNA export factors dissociate and factors involved in nonsense mediated decay remain bound
mRNA is exported from the nucleus to the cytoplasm via what structure?
Nuclear pore
What is non-sense mediated mRNA decay?
A pathway that degrades an mRNA that has a nonsense mutation prior to last exon
Describe what happens if there is a premature stop codon in the mRNA ?
The ribosome moves along mRNA, translating it into a protein but it will remove exon junction complexes along the way. Due to the presence of premature stop codon, the ribosome is realised and the EJc remain intact. This junction can’t be removed and so it recruits UPF which activates the capping enzyme, resulting in the 5’ being cleaves and mRNA being degraded
What does tRNA splicing involve?
Successive cleave and ligation reactions
How does RNA polymerase III terminate transcription?
poly(U)4 sequence embedded in a GC-rich sequence
What is needed to create the alternative confirmation of the tRNA arm?
All tRNA introns include a sequence that is complementary to the tRNA anticodon and this creates alternative confirmation
How are tRNA introns removed?
An endonuclease cleaves Both end of the intron in the pre-tRNA
What does tRNA intron release generate?
Two half-tRNA with unusual ends that contain 5’ OH and 2’-3’ cyclic phosphate and a linear transformation
How is the normal tRNA structure formed after splicing?
Polynucleotide kinase phosphorylates the 5’-OH end. Cyclic phosphate group is opened by phosphodiesterase in order to generate a 2’-phosphate terminus and 3’OH Group. The exon ends are joined by RNA ligase and the 2’-phosphate is remixed by a phosphatase
