RNA binding properties of conserved protein subunits of human RNase P

Abstract

Human nuclear RNase P is required for transcription and processing of tRNA. This catalytic RNP has an H1 RNA moiety associated with ten distinct protein subunits. Five (Rpp20, Rpp21, Rpp25, Rpp29 and Pop5) out of eight of these protein subunits, prepared in refolded recombinant forms, bind to H1 RNA in vitro. Rpp20 and Rpp25 bind jointly to H1 RNA, even though each protein can interact independently with this transcript. Nuclease footprinting analysis reveals that Rpp20 and Rpp25 recognize overlapping regions in the P2 and P3 domains of H1 RNA. Rpp21 and Rpp29, which are sufficient for reconstitution of the endonucleolytic activity, bind to separate regions in the catalytic domain of H1 RNA. Common themes and discrepancies in the RNA-protein interactions between human nuclear RNase P and its related yeast and archaeal counterparts provide a rationale for the assembly of the fully active form of this enzyme.

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

“…A human homolog of Pop4 (Rpp29) appeared to interact directly with the RNA component of human RNase P (Jiang et al 2001;Reiner et al 2011). Yeast three-hybrid studies and pull-down experiments suggested interactions between Pop4 and S. cerevisiae RNase P as well (Chu et al 1997;Houser-Scott et al 2002).…”

“…Although we did not observe crosslinks between Pop4 and the P1 stem (it should be noted that a large part of the P1 stem was not accessible to the analysis that involved primer extension), the proximity of the identified Pop4-binding site and the P1 stem supports the existence of Pop4-P1 stem interactions. The footprinting results obtained for the in vitro-transcribed human RNase P RNA in a complex with the human Pop4 homolog (Reiner et al 2011) also position Pop4 so that it can bridge the two RNA domains, although the location of the protected area differs from that of the crosslinks observed in the yeast holoenzyme.…”

“…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.…”

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

“…Crystals of SeMet Δ76 PRORP1 and WT Δ76 PRORP1 in the presence of Sr and Ca were obtained by adding 0.02 M SrCl 2 or CaCl 2 into the crystallization solution described above. Crystals of Δ76 PRORP1 in the presence of Mn were obtained through soaking with a solution containing 0.05 M MnSO 4 . Diffraction data were collected on beamline GM/CA-CAT 23-ID-D at the Advanced Photon Source, Argonne National Laboratory (Argonne, IL).…”

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

“…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.…”