New mechanistic insights into prespliceosome formation—roles of DEAD-box proteins Prp5 and Sub2

The spliceosome is a highly dynamic structure that undergoes continuous structural alterations through the sequential association and dissociation of small nuclear RNAs and protein factors during precursor mRNA splicing. These structural changes are driven by eight DExD/H-box RNA helicases that act at distinct stages of the splicing cycle. Among them, Prp5 and Sub2 are involved in prespliceosome formation, with Prp5 implicated in displacing the U2 snRNP component Cus2, and Sub2 in facilitating the release of the Msl5-Mud2 heterodimer. However, the precise mechanisms underlying the functions of these two proteins remain unclear. Here, we show that Sub2 is not essential for splicing in vitro, but it can enhance splicing independently of ATP. Strikingly, prespliceosome formation can proceed without ATP in the absence of either Sub2 or Cus2. These findings reveal a coordinated interplay among Prp5, Sub2, Cus2 Mud2, and Msl5 during prespliceosome formation.