What is Splicing Mechanism? Write down the mechanism of splicing mechanism.
In Eukaryotes, the RNA transcribed from DNA almost invariably undergoes RNA splicing to yield mature RNA sequences (rRNA, mRNA, tRNA). It involves removal of sequences mainly corresponding to introns in split genes.
These excision of introns & joining together of all the exons of a gene in a proper sequences to yield the mature mRNA is called Splicing.
G1 introns: The first one is called group 1 introns which is found in some nuclear, mitochondrial & chloroplast genes coding rRNA. G1 Splicing reaction requires a Guanine nucleotide or nucleotide cofactor. But this cofactor is not used as a source of energy. The 3'-OH grp of guanocine is used as a nucleophile in the 1st step of splicing
pathway. The guanocine 3' OH firms a normal 3',5'-phosphodiester bones with 5' end of the introns. The 3' OH of the exon i.e. displaced in this stop acts as a nucleophile in a similar reaction at the 3' end of the introns. The results is precise excision of the introns & lygation of the exons. Many of this introns are self splicing, no proteins or enzyme are involved.
 |
| Fig: Formation of primary transcript & splicing of introns to from mature mRNA.
|
Group II introns: G2 introns are generally found in the primary transcript of mitochondrial & chloroplast mRNA. In G2 introns the pattern is similar except for the nucleophile in the first step. Instead of an external co-factor the nucleophile is the 2' OH- group of an adenylates residue within the intron. An unusual bronchiole lariate structure is formed as an intermediate lariate structure is formed as an intermediate. The mechanism involved in self Splicing & no proteins & enzymes are required. Here, the RNA acts as an enzyme itself i.e. auto catalytic in nature.
Group 3. The third group of introns are found in nuclear mRNA primary transcript. They are not self splicing. Splicing requires the action of splice RNA protein complexes. Containing a class of eukaryotic RNA is called small nuclear RNAs (snRNA). 5 snRNA -U1, U2, U3, U4 & U5 are involved in splicing reaction.
These are complex with the protein to form small nuclear ribosomal protein snRNPs of an reffered as SNURPs. IQ snRNA has a ss sequence complementary to consensus sequence at 5' splice site of introns.
fig:
It pairs directly with 5' terminal 11 nucleotide of this complementary splice site of introns. Addition of U2,U4,U5 & U6 SnRNPs lead to formation of a complex called spliceosome with this spliceosome actual mechanism of splicing occurs.
In 1st step U1 SnRNP is paired with 5’ splice site & involves in interaction between one of its protein ( U1-70K) & the protein ASF/SF2 (A splicing factor). The U2A ( U2 Auxilary factor) binds to a pyrimidine tract down stream of a branch site UACUAAC box. The binding of U2 SnRNA requires ATP hydrolysis.
B1 complex is formed when A treamer containing U5 & U4/U6 SnRNP bind together. This complex is (which is found as a single paratide) B1 complex then transformed into B2 complex when U1 is released . The calalytic reactia is triggered by the release of U4 & thus this spliceosome is able to proceed which released as hydrolysis of ATP. When U4 dissociate the attachmen t region of U6 become free to take up another structure. The 1st part of its pair with U2 & the 2nd part forms an intramoleucular hairpin. The 5’ splice site is actually now close to U6 srquence immediately on 5’ site of stretch bound to U2. This sequence in U6 SnRNA contracts the conserve GU at the 5’ splice site.
Base pairing between U2 & U6 creates as structure that ressemble the active centre of the group 2 introns. U5 shifts from axon to introns & a C1 complex is formed. The U5 SnRNA is then immediate adjacent to the first base positions in both axons. Transesterification is possibly catelysed by U6/U2 components . U5 binds to the axon at 3’ splice site . The 5’ site is cleaved & a lariat structure is formed by base pairing od U2 SnRNA to the branch site displaces & activates the Adenine to form such structure.
The second splicing reaction follows rapidly binding of U5 SnRNA to the 3’ splice site is needed for this reaction. There is no region of complementary available is single stranded form but the protein components the SnRNA are involved. U2,U5&U6 remain bound to lariate 3’ site is cleaves & axons are ligated then the 3’ consensus sequence nearest to the 3’ site of the branch becomes the target for the 2nd transesterification reaction.
Finally, spliced RNA is released . The lariat structure is debranched .
Group 4: The fourth class of introns (group iv) is found in certain tRNAs is distinguished from group 1 & group 2 introns in that its splicing requires ATP. In this case a splicing endonuclease cleaves the phosphodiester bonds at bond ends of introns & the two axons are joined . The joining reaction is similar to DNA ligase mechanism.
figL
In 1st step U1 SnRNP is paired with 5’ splice site & involves in interaction between one of its protein (
U1-70K) & the protein ASF/SF2 (A splicing factor). The U2A ( U2 Auxilary factor) binds to a pyrimidine tract down stream of a branch site UACUAAC box. The binding of U2 SnRNA requires ATP hydrolysis. B1 complex is formed when A treamer containing U5 & U4/U6 SnRNP bind together. This complex is
(which is found as a single paratide) B1 complex then transformed into B2 complex when U1 is released . The calalytic reactia is triggered by the release of U4 & thus this spliceosome is able to proceed which released as hydrolysis of ATP. When U4 dissociate the attachmen t region of U6 become free to take up another structure. The 1st part of its pair with U2 & the 2nd part forms an intra moleucular hairpin. The 5’ splice site is actually now close to U6 sequence immediately on 5’ site of stretch bound to U2. This sequence in U6 SnRNA contracts the conserve GU at the 5’ splice site.
U1-70K) & the protein ASF/SF2 (A splicing factor). The U2A ( U2 Auxilary factor) binds to a pyrimidine tract down stream of a branch site UACUAAC box. The binding of U2 SnRNA requires ATP hydrolysis. B1 complex is formed when A treamer containing U5 & U4/U6 SnRNP bind together. This complex is
(which is found as a single paratide) B1 complex then transformed into B2 complex when U1 is released . The calalytic reactia is triggered by the release of U4 & thus this spliceosome is able to proceed which released as hydrolysis of ATP. When U4 dissociate the attachmen t region of U6 become free to take up another structure. The 1st part of its pair with U2 & the 2nd part forms an intra moleucular hairpin. The 5’ splice site is actually now close to U6 sequence immediately on 5’ site of stretch bound to U2. This sequence in U6 SnRNA contracts the conserve GU at the 5’ splice site.
Base pairing between U2 & U6 creates as structure that ressemble the active centre of the group 2 introns. U5 shifts from axon to introns & a C1 complex is formed. The U5 SnRNA is then immediate adjacent to the first base positions in both axons. Transesterification is possibly catalysed by U6/U2 components . U5 binds to the axon at 3’ splice site . The 5’ site is cleaved & a lariat structure is formed by base pairing od U2 SnRNA to the branch site displaces & activates the Adenine to form such structure.
The second splicing rxn follows rapidly binding of U5 SnRNA to the 3’ splice site is needed for this reaction. There is no region of complementary available is single stranded form but the protein components the SnRNA are involved. U2,U5&U6 remain bound to lariate 3’ site is cleaves & axons are
ligated then the 3’ consensus sequence nearest to the 3’ site of the branch becomes the target for the 2nd transesterification reaction.
Finally, spliced RNA is released . The lariat structure is debranched .