Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Abstract

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP

“…The formation of a complex between Pop5 and Rpp1 is consistent with the available results of two-hybrid studies and pull-down experiments performed for yeast RNases MRP/P (HouserScott et al 2002;Aspinall et al 2007) as well as with the results obtained for archaeal homologs of Pop5 and Rpp1 (Tsai et al 2006;Pulukkunat and Gopalan 2008;Honda et al 2010).…”

“…1A,B) in a complex with protein components Pop6 and Pop7 (Perederina and Krasilnikov 2010;Perederina et al 2010a,b). Two-and three-hybrid studies, UV-cross-linking studies, as well as pull-down assays have been performed on yeast and human RNases MRP/P (Pluk et al 1999;Houser-Scott et al 2002;Welting et al 2004;Aspinall et al 2007;Reiner et al 2011), but how most of the multiple protein components interact with the catalytic RNA moiety and what are the roles of the proteins, remain unclear.…”

“…One of the major challenges in studying eukaryotic RNases MRP/P is that most of the proteins from Saccharomyces cerevisiae RNases MRP/P are not soluble or tend to aggregate when individually expressed in Escherichia coli (Aspinall et al 2007, and references therein), requiring the presence of other components for their proper folding. A coexpression approach to studying ''difficult'' eukaryotic RNase MRP/P proteins has been previously applied to the structural characterization of protein components Pop6 and Pop7 (Perederina et al 2007(Perederina et al , 2010a.…”

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

“…The results of three-hybrid studies and pull-down experiments (Chu et al 1997;Houser-Scott et al 2002;Welting et al 2004;Aspinall et al 2007) suggest that Pop4 is involved in interactions with the RNA component of RNase MRP. Our crosslinking data indicate that Pop4 binds to the ymP5 stem of the RNA components of RNase MRP ( Fig.…”

“…RNA-protein interactions in eukaryotic RNases P/MRP have been studied using pull-down, three-hybrid, UV-crosslinking, and footprinting assays, mostly in the context of partially assembled complexes (Pluk et al 1999;Jiang et al 2001;Houser-Scott et al 2002;Welting et al 2004Welting et al , 2007Aspinall et al 2007;Perederina et al 2007Perederina et al , 2011Esakova et al 2008;Reiner et al 2011); however, the structural organizations of eukaryotic enzymes of the RNase P/MRP family-in particular, the locations of the binding sites of individual proteins on RNA-remain unclear.…”