Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA

Abstract

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.

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

“…However, we did not identify crosslinks between Pop6, the other member of the Pop6/Pop7 heterodimer, and RNA, even though interactions between Pop6 and the P3 RNA (albeit less extensive than those for Pop7) were observed in the crystal structure (Perederina et al 2010b) and were consistent with in vitro reconstitution experiments (Hands-Taylor et al 2010). Apparently, this discrepancy reflects the fact that the presence of an RNA-protein contact does not guarantee that a UV-induced crosslink will form.…”

“…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.…”

Of proteins and RNA: The RNase P/MRP family

“…In addition to Pop6 and Pop7, the P3 RNA subdomain is expected to bind to another protein component, Pop1 (Ziehler et al 2001), and, possibly, others. The P3 subdomain in human RNases P/MRP is likely to have a similar structural organization (Hands-Taylor et al 2010;Perederina et al 2010b).…”

The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins

“…The ITC experiments were performed in 20 mM Tris, 200 mM KCl, 0.2 mM EDTA, 1 mM DTT (pH 7.25) at 298 K using a Microcal ITC-200 microcalorimeter as previously described (Hands-Taylor et al 2010). In each titration, 20 injections of 2 mL each of a RNA solution, at a concentration of 200 mM, were added into a 20 mM protein solution, using a computer-controlled 40-mL microsyringe.…”