To identify splicing factors in proximity of the 5′ splice site (5′SS), we followed a crosslinking profile of site-specifically modified, photoreactive RNA substrates. Upon U4/U5/U6 snRNP addition, the 5′SS RNA crosslinks in an ATP-dependent manner to U6 snRNA, an unidentified protein p27, and the 100-kDa U5 snRNP protein, a human ortholog of an ATPase/RNA helicase yPrp28p. The 5′SS:hPrp28p crosslink maps to the highly conserved TAT motif in proximity of the ATP-binding site in hPrp28p. We propose that hPrp28p acts as a helicase to unwind the 5′SS:U1 snRNA duplex, and at the same time as a 5′SS translocase, which, upon NTP-dependent conformational change, positions the 5′SS for pairing with U6 snRNA within the spliceosome. This repositioning of the 5′SS takes place regardless of whether the 5′SS is originally duplexed with U1 snRNA.