Heterodimerization regulates RNase MRP/RNase P association, localization, and expression of Rpp20 and Rpp25

Abstract

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function.

Footprinting analysis demonstrates extensive similarity between eukaryotic RNase P and RNase MRP holoenzymes

“…In S. cerevisiae RNases MRP and P RNAs, the P3 helix was shown to directly interact with the protein components Pop6 and Pop7 (Perederina et al 2007); similar results were obtained for the human RNase MRP (Welting et al 2007). Pop6 and Pop7 were shown to form a heterodimer that binds P3, protecting a segment of the lower strand of the internal loop of P3 and part of the adjacent helical stem (nucleotides 30-38, RNase MRP numbering) (Perederina et al 2007).…”

Crystal Structure and Functional Analysis of an Archaeal Chromatin Protein Alba from the Hyperthermophilic ArchaeonPyrococcus horikoshiiOT3

“…40,41) Furthermore, the heterodimerization of Rpp20 and Rpp25 regulates their RNA-binding activity, subcellular localization, and expression. 19) A recent study showed that human nuclear RNase P is required for transcription of tRNA and other small noncoding RNA genes by pol III, and Rpp20 and Rpp25 are expected to play an essential role in this function as chromatin-binding proteins. 20) In the present study, we found that PhoAlba in the hyperthermophilic archaeon P. horikoshii was capable of binding to PhopRNA and pre-tRNA Tyr , but that it had little effect on the pre-tRNA processing activity of reconstituted R-5P or R-4P composed of PhopRNA and five or four proteins respectively.…”

“…It was reported recently that Rpp20 and Rpp25, belonging to the Alba protein family, bind to each other, and that heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, though their catalytic contribution to human RNase P remains unclear. 19) Furthermore, a recent study indicates that human nuclear RNase P is required for transcription of tRNA and other small noncoding RNA genes by pol III, and Rpp20 and Rpp25 are expected to play an essential role in this function as chromatinbinding proteins. 20) We have found via a reconstitution experiment that RNase P RNA (PhopRNA) and four proteins, PhoPop5, PhoRpp21, PhoRpp29, and PhoRpp30, are essential to the RNase P activity of the Pyrococcus horikoshii OT3.…”

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

“…Protein components Pop6 and Pop7 (as well as their human homologs) were shown to form a heterodimer that binds directly to the in vitro-transcribed P3 RNA subdomain of both RNase P and RNase MRP (Pluk et al 1999;Welting et al 2004Welting et al , 2007Perederina et al 2007;Hands-Taylor et al 2010), and the crystal structure of a complex of the Pop6/Pop7 heterodimer with the P3 RNA subdomain has been reported (Perederina et al 2010b). Our results obtained for the active holoenzymes ( 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.…”