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

Abstract

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

“…However, we previously showed that coexpression of several insoluble RNase P/MRP proteins resulted in the formation of soluble and structurally homogeneous complexes (Perederina et al 2007(Perederina et al , 2010b. Yeast two-hybrid studies (Houser-Scott et al 2002), pull-down experiments (Welting et al 2004;Aspinall et al 2007), as well as our data (Khanova et al 2012; E Khanova, AS Krasilnikov, unpubl. ) indicated interactions between Pop1 and another RNase P/MRP protein component, Pop4, prompting us to consider coexpression of Pop1 and Pop4 as a possible solution to the insolubility of Pop1.…”

“…The size of Pop1, its presence exclusively in the eukaryotic RNases P/MRP Marvin and Esakova and Krasilnikov 2010;Jarrous and Gopalan 2010), the effects of Pop1 mutations on the holoenzymes' assembly (Lygerou et al 1994;Chamberlain et al 1998;Xiao et al 2006), and the apparent spread of the RNase P/MRP RNA regions involved (directly or not) in interactions with Pop1 (Ziehler et al 2001;Houser-Scott et al 2002;Hipp et al 2012;Khanova et al 2012), are all consistent with a potential central role played by Pop1 in the stabilization of the global structure of the RNA components of eukaryotic RNases P/MRP. However, insolubility of individually expressed Pop1 (Ziehler et al 2001;Xiao et al 2006) has previously hindered direct studies of this protein and its interactions with other RNase P/MRP components.…”

“…While the specificity of RNase MRP differs from that of eukaryotic RNase P, the two enzymes share most of their protein components (Chamberlain et al 1998;Walker et al 2010), as well as many RNA features (Fig. 1A,B, 2), and demonstrate similarities in their structural organizations (Esakova et al 2008;Khanova et al 2012;Hipp et al 2012).…”

“…It was suggested that this RNA domain serves as a protein-binding hub in eukaryotic RNase P and RNase MRP (Ziehler et al 2001;Perederina and Krasilnikov 2010;Perederina et al 2010b) and thus may be indirectly involved in the stabilization of the tertiary structure of RNA (Perederina and Krasilnikov 2010;Perederina et al 2010b). In RNase P, the Pop6/Pop7 heterodimer was shown to mediate a tertiary interaction between the P3 RNA domain and P15 RNA stem (Esakova et al 2008;Khanova et al 2012). However, this interaction appears to be too localized to fulfill the role of the structural scaffold that is played by tertiary RNA-RNA interactions in bacteria.…”

“…UV-crosslinking analysis of RNase P/MRP holoenzymes (Khanova et al 2012) shows that interactions of Pop1 with RNA components involve direct interactions with the CR-IV/mCR-IV regions (Fig. 1A,B).…”