Communication between U1 and U2 snRNPs is critical during pre-spliceosome assembly; yet, direct connections have not been observed. To investigate this assembly step, we focused on Prp5, an RNA-dependent ATPase of the DExD/H family. We identified homologs of Saccharomyces cerevisiae Prp5 in humans (hPrp5) and Schizosaccharomyces pombe (SpPrp5), and investigated their interactions and function. Depletion and reconstitution of SpPrp5 from extracts demonstrate that ATP binding and hydrolysis by Prp5 are required for pre-spliceosome complex A formation. hPrp5 and SpPrp5 are each physically associated with both U1 and U2 snRNPs; Prp5 contains distinct U1- and U2-interacting domains that are required for pre-spliceosome assembly; and, we observe a Prp5-associated U1/U2 complex in S. pombe. Together, these data are consistent with Prp5 being a bridge between U1 and U2 snRNPs at the time of pre-spliceosome formation.