A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.
PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.
Schwer
B
B
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Guthrie
C
C
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001483:Base Sequence; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020365:RNA Helicases; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000378:metabolism*
D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; C027900:RNA-dependent ATPase; D020365:RNA Helicases