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Abstract:
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Pre -mRNA splicing by the spliceosome requires the precise and regulated efforts of the five snRNAs (U1 , U2 , U4 , U5 , and U6 ) and numerous associated proteins . Following assembly and activation of the spliceosome , two consecutive reactions result in intron removal and exon ligation from pre -mRNA substrates . It has been established that several members of the DExH /D -box family of helicases act transiently on the spliceosome prior to the chemical steps to authorize the successive reactions by hydrolyzing ATP and consequently inducing structural rearrangements . While it has been suggested that these changes produced in the structure of the spliceosome result in optimal positioning of the reactive species , the mechanisms and products of these reorganizations remain uncharacterized .
The work presented here describes the genetic strategy for accumulating and purifying spliceosomes arrested in vivo , during the catalytic steps of the splicing cycle . Using these complexes , we have defined the components required to proceed through the first and second steps of splicing , in addition to the factors required for the release of the spliced message . Analysis of these functional , synchronized particles has also allowed us to define a function for Prp2p in initiating the first step of pre -mRNA splicing . Our data suggest that Prp2p may act in an ATP -independent manner to remodel the spliceosome prior to using its ATPase function to displace the SF3 complex . We propose that the SF3 complex , in addition to its role in identification of the branchpoint , also acts to sequester the reactive 2’OH of the branchpoint adenosine to prevent premature reactivity .
Following the two catalytic steps of the splicing cycle , the spliceosome must disassemble and recycle its snRNPs for further rounds of splicing . The essential U6 snRNP component Prp24p , mediates one of the early assembly events - the annealing between the U4 and U6 snRNAs . We have discovered that although Prp24p is essential for viability , its function (s ) can be bypassed by overexpressing the U6 snRNA . Additionally , biochemical characterizations of various forms of the U4 /U6 snRNP provide evidence that Prp24p must be released before other components of the U4 /U6 snRNP are permitted to interact and facilitate tri -snRNP formation . |