Exploring the roles of ATP and hPrp5 in the 1(st) ATP-dependent step of spliceosome assembly

Abstract

Conserved sequences within pre-mRNA are crucial for correct splicing of the message. One of these is the branch site, specifically the branch nucleotide (A), which participates in the transesterification reactions. The branch nucleotide is identified by U2 snRNP in the early, ATP-dependent Complex A, and correct assembly of this complex is important for progression through the splicing cycle. Formation of this U2 snRNP-containing complex is the first ATP-dependent step of spliceosome assembly, and this ATP requirement is not well understood. To study this ATP-dependent event, we identified and characterized a set of minimal intron-derived substrates that recapitulate the ATP requirement for U2 snRNP engagement, and another set that is relieved of the ATP requirement. We found that the ATP requirement during Complex A formation is associated with sequence 5' of the branch site, and that the ATP is most likely required for an incoming factor, not for activity directly related to the RNA (i.e., secondary structure rearrangement).;Prp5, a DEAD-box putative RNA helicase previously identified only in the yeast S. cerevisiae, is a good candidate for the factor that requires ATP in mediating complex formation. We have identified the human homologue of ScPrp5, which we call hPrp5, and have found it associated with U2 and U1 snRNP. We also found a different ATP-dependent activity likely to represent a subsequent rearrangement of Complex A protein components. Thus, we have separated part of the complex assembly pathway of the spliceosome into separate activities. These activities represent rearrangements required for U2 snRNP addition, and for progression along the splicing pathway.