Banerjee D, McDaniel PM, Rymond BC

The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-mRNA and rRNA maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 proteins are implicated as co-factors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary ...

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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 Y2H interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (aa 102-149) does not impede rRNA processing but impacts snoRNA biogenesis resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by 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.

Date: Mar. 25, 2015

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