RNA helicase II/Gu (RH-II/Gu) is a nucleolar DEAD-box protein that unwinds double-stranded RNA and introduces secondary structure to a single-stranded RNA. We recently identified its paralogue, RH-II/Gu(beta), in contrast to the original RH-II/Gu(alpha). Their similar intron-exon structures on chromosome 10 suggest gene duplication. To determine functional differences, their expression, localization, and enzymatic activities were compared. RH-II/Gu(alpha) is expressed two- to threefold more than RH-II/Gu(beta) in most tissues. Both proteins localize to nucleoli, suggesting roles in ribosomal RNA production, but RH-II/Gu(beta) also localizes to nuclear speckles containing splicing factor SC35, suggesting possible involvement in pre-mRNA splicing. The C-terminus responsible for nuclear speckle localization of RH-II/Gu(beta) contains an arginine-serine-rich domain present in some RNA splicing proteins. In vitro assays show weaker ATPase and RNA helicase activities of RH-II/Gu(beta). RH-II/Gu(alpha) unwinds RNA substrate with a 21- or 34-nt duplex and 5' overhangs, but RH-II/Gu(beta) unwinds only the shorter duplex. Although RH-II/Gu(beta) has no RNA folding activity, it catalyzes formation of an RNA complex with unidentified structure, which is not observed when assayed with a mixture of the two enzymes. Instead, the presence of RH-II/Gu(beta) stimulates RH-II/Gu(alpha) unwinding activity. Our data suggest distinct and complex regulation of expression of the two paralogues with nonredundant gene products. (c) 2002 Elsevier Science (USA).