Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

Olga Esakova,Anna Perederina,Igor Berezin,Andrey S Krasilnikov

doi:10.1093/nar/gkt432

Abstract

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

Highlights

RNase MRP is a catalytic ribonucleoprotein complex that is closely related to RNase P [a universal RNA-based enzyme responsible for the maturation of the 50-end of tRNA and involved in some other activities [1]]
Interactions between the RNA component of the RNase MRP holoenzyme and its substrate To identify regions of the RNA component of RNase MRP that were involved in interactions with substrates, we isolated the active RNase MRP holoenzyme from S. cerevisiae, subjected it to UV irradiation in the presence of a photoreactive RNase MRP substrate, and we located the sites of the UV-induced RNA–RNA crosslinks that appeared in the presence of the photoreactive substrate
The design of the substrate used in cross-linking experiments was based on the results of in vitro selection of RNase MRP substrates [49], which demonstrated that a typical RNase MRP substrate has a CUC/CAC/CGC/ UUC/AUC triad in the positions +2 to +4 relative to the cleavage site, no guanines in the positions+1 and À1 and a U-rich stretch located 50 to the cleavage site

Summary

Introduction

RNase MRP is a catalytic ribonucleoprotein complex that is closely related to RNase P [a universal RNA-based enzyme responsible for the maturation of the 50-end of tRNA and involved in some other activities [1]]. RNase MRP is involved in the maturation of the 5.8S rRNA, cleaving the precursor molecule at a specific site (A3) within the internal transcribed spacer 1 [11,12,13,14]. RNase MRP may participate in additional, earlier steps of rRNA maturation [15], but the exact nature of this activity has not been determined. RNase MRP was shown to be involved in the regulation of the cell cycle in yeast by participating in the cleavage of specific mRNAs [16,17,18], in the processing of U2 snRNA, as well as in the metabolism of a number of other RNAs [18,19,20]. Defects in the activity of RNase MRP result in a variety of pleiotropic diseases in humans [21,22,23]

Methods

Results

Conclusion