The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102-149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.KEYWORDS Saccharomyces cerevisiae; spliceosome; rRNA processing; DExD/H-box protein; pre-mRNA splicing N UMEROUS genome-wide interaction and gene expression studies identify ribosome biogenesis as a central integrative feature of Saccharomyces cerevisiae metabolism (see, e.g ., Mnaimneh et al. 2004;Davierwala et al. 2005;Gavin et al. 2006;Collins et al. 2007;Hu et al. 2007;Magtanong et al. 2011). In rapidly dividing organisms such as this budding yeast, ribosomal RNAs (rRNAs) make up the lion's share of cellular nucleic acid by mass, while ribosomal protein transcripts can account for more than half the transcribed messenger RNA (mRNA). Because ribosome biogenesis is so energetically costly, eukaryotes have evolved multiple means to regulate rRNA and ribosomal protein production in response to changes in cellular demand and surveillance systems to remove aberrant ribosomal protein complexes formed during assembly or after environmental insult (Jorgensen et al. 2002;Fingerman et al. 2003; FromontRacine et al. 2003;Jorgensen et al. 2004;Marion et al. 2004;Henras et al. 2008;Kressler et al. 2010;Lafontaine 2010). The coordination of pre-mRNA processing with ribosome biogenesis is especially relevant in the intron-poor environment of the yeast genome, where the highly expressed ribosomal protein transcr...