MBB 267 Week 10: Mitchel 7 Flashcards
Explain the mechanism of pre-mRNA splicing?
The chemical reaction mechanism of pre-mRNA splicing involves two transesterification reactions and is dependent upon recognition of the pre-mRNA sequences at the 5’ splice site, the 3’ splice site and the branchpoint. There is no requirement for ATP hydrolysis. In the first step, an ester linkage is formed between the 2’ hydroxyl group of the branchpoint adenosine and the phosphate group at the 3’ end of the 5’ exon, while the phosphoester linkage at the 3’ end of the 5’ exon is lost. The first step generates a free 5’ exon and a 3’ exon-lariat intermediate where the intron contains a looped sequence centred on the branchpoint adenosine. In the second step, an ester linkage is formed between the hydroxyl group at the 3’ end of the 5’ exon and the phosphoester at the 5’ end of the 3’ exon. The second step joins the two exons and releases the intron lariat
What happens to the released intron lariat?
subsequently degraded by a specific debranching enzyme and exoribonucleases
What is the spliceosome complex made out of?
The spliceosome is a dynamic complex, consisting of; five Ribonuceloprotein (RNP) complexes, the Prp19 complex (19 complex; NTC) and other proteins.
What are RNP complexes?
-and what are their structures?
A ribonucleoprotein (RNP) is a complex of ribonucleic acid and RNA-binding protein
-The spliceosome is a dynamic complex of small nuclear RNP complexes ("snurps") called U1, U2, U4, U5 and U6 snurps and protein complexes that is assembled and disassembled on the pre-mRNA during each splicing event. Individual snurps contain a single snRNA and between 6-10 proteins. Some proteins are unique, others are common. Sm proteins are found in pol II snRNAs (U1,2,4 &5) while U6 snRNP contains Lsm (like Sm) proteins.What do Sm and Lsm proteins do?
The Sm and Lsm proteins form ring-shaped structures containing seven distinct but structurally similar proteins. The Sm and Lsm complexes are wrapped around the snRNA polynucleotide chain
How often does spliceosome assemble on pre-mRNAs?
The snRNP complexes assemble and disassemble on the pre-mRNA during each splicing event, the assembly and disassembly steps during this cycle being driven by various spliceosome- associated proteins.
Explain the formation of the spliceosome cycle
Assembly is initiated by the binding of U1 snRNP to the 5’ splice site, generating the E complex. The U2 snRNP then binds to the branchpoint, giving the A complex. A U4/U6 snurp complex is formed that has extensive base-pairing between the snRNAs. This complex then associates with U5 snRNP to give the U4/U6.U5 tri-snurp, which is recruited to the spliceosome to give complex B. The spliceosome undergoes a major rearrangement to generate the activated B complex, wherein U6 and U4 are dissociated and U4 is lost from the complex. The U1 snRNP is also lost from the complex at this time. The Prp19 complex is recruited upon U4 release and remains associated through the first and second step of splicing. The first step of splicing occurs after formation of the active B complex, giving the C complex. Another structural rearrangement is required to generate the C2 complex before the second step of splicing can proceed. The spliced product is then released and the U2, U5 and U6 snRNPs and the NTC are disassembled
Where does base pairing occur between snRNAs & pre-mRNA?
The pre-mRNA splicing reaction is driven by base-pairing interactions between the snRNAs and the pre-mRNA. The 3’ end of the U1 snRNA base-pairs with the 5’ splice site (exon on 5’ side), while U2 snRNA selects the branchpoint through base-pairing with the conserved UACUAAC (“TACTAAC”) sequence across the branchpoint such that the branchpoint adenosine is kinked out of the duplex. In the catalytically active spliceosome, U6 snRNA base-pairs with nucleotides close to the 5’ (5’ end of intron) splice site and is extensively base-paired with U2 snRNA. The U1/pre-mRNA interaction has been lost but replaced by U5 snRNA base-pairs at the 3’ end of the 5’ exon. Some base-pair interactions such as the U5/5’ exon interaction are very short and involve only a few base- pairs. These interactions are stabilized by the Sm proteins, three of which have basic unstructured C-terminal tails that interact with RNA.
What catalyses the assembly and disassembly of the spliceosome complex?
The cycle of assembly and disassembly steps and structural rearrangements within the spliceosome cycle are driven by RNA helicases.
Why are there so many checkpoints when assembling spliceosome complexes?
-What are those checkpoints?
Checkpoints are in place at various points during the spliceosome cycle to ensure the fidelity of pre-mRNA splicing; in the absence of the appropriate RNA helicase activity, the complex is disassembled and the pre-mRNA degraded.
-RNA helicases often function as chaperones to facilitate structural rearrangements within RNP complexes. The base-pairing interaction between U4 and U6 is driven by Prp24. Prp5 and Sub2 are required for complex A formation, Brr2 drives release of U4 from U6 snRNA, while Prp28 is required for complex B formation. Prp2 is required for activation of the B complex, while Prp16 is required to prime the spliceosome for the 2nd step
What does Prp16 do?
-What do Prp16 mutants with a decreased ATP hydrolysis rates have an affect on?
Prp16 facilitates the transition between the first and second step of splicing. Prp16 is required for a structural transition that aligns the 5’ exon and 3’ exon before the second step of splicing.
- prp16 mutants with decreased ATP hydrolysis rates improve splicing of suboptimal substrates.
- If the exons are not correctly aligned after ATP hydrolysis, the pre-mRNA is normally released and degraded. A slowed ATP hydrolysis rate enables further time for exon alignment and productive splicing
How is splicing analysed in vitro?
Employ 32P- labelled RNA substrates and nuclear extracts and run them through PAGE. The unspliced RNA will decrease in concentration as the incubation time increases because the unspliced RNA is slowly being spliced and therefore used up. In the other hand, the fully spliced RNA begins to be seen after a while of incubation, as we start with no spliced mRNAs at the beginning. The 5’ exon and the 3’ exon+lariat loop are seen at the same time as they are both products of the first step of splicing. The intron lariat is seen last, because it is one of the last steps of splicing (same time as spliced mRNA).
