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, 18 (4), 720-8

Structural Organizations of Yeast RNase P and RNase MRP Holoenzymes as Revealed by UV-crosslinking Studies of RNA-protein Interactions

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Structural Organizations of Yeast RNase P and RNase MRP Holoenzymes as Revealed by UV-crosslinking Studies of RNA-protein Interactions

Elena Khanova et al. RNA.

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.

Figures

FIGURE 1.
FIGURE 1.
Typical results of the primer extension analysis of RNase P/MRP RNA crosslinked to specific proteins. (A) RNase MRP RNA crosslinked to Pop1; (B) RNase P RNA crosslinked to Pop7; (C) RNase MRP RNA crosslinked to Pop7; (D) RNase P RNA crosslinked to Pop4. (Lanes 1,2,12,13,23–26,37,47,48) Sequencing markers; (lanes 3,14,27,38) primer extension reactions for deproteinated RNAs (controls); (lanes 11,22,35,46) primer extension reactions for UV-treated (1280 mJ/cm2) deproteinated RNAs (controls); (lanes 4–10,15–21,28–34,39–45) primer extension reactions for UV-crosslinked RNAs, the UV dose increasing from 0 to 640 mJ/cm2. The arrows and boxed regions indicate the locations of the RNA–protein crosslinks.
FIGURE 2.
FIGURE 2.
Secondary structure diagrams of the RNA components of (A) yeast RNase MRP; (B) yeast RNase P; and (C) bacterial (Thermotoga maritima) RNase P. (Arrows) The identified sites of RNA–protein crosslinking in eukaryotic RNases P/MRP. (Gray lines) Tertiary interactions in the bacterial RNase P. The diagrams are based on Esakova and Krasilnikov (2010).
FIGURE 3.
FIGURE 3.
UV-induced RNA–protein crosslinks mapped onto the outline of the RNA component of RNase P. The outline is based on the crystal structure of bacterial RNase P (Reiter et al. 2010). (Gray) RNase P RNA; (light gray) tRNA product; only the RNA elements that are universally conserved from bacteria to eukaryotes are shown. (Solid spheres) The locations of the identified crosslinking sites for individual proteins. (Red) The location of the Pop1 crosslink; (green) the location of the Pop4 crosslink; (brown) the location of the Pop5 crosslink (carried over from RNase MRP); (magenta) the locations of the Pop7 crosslinks. Corresponding approximate locations of proteins are shown as semitransparent in matching colors. The structures of Pop6 and Pop7 are modeled according to Perederina et al. (2010b); Pop5 and Rpp1 are modeled according to Perederina et al. (2011). RNA structural elements are marked according to the nomenclature used in Figure 2B.
FIGURE 4.
FIGURE 4.
Isolation of His-tagged proteins from RNase P/MRP holoenzymes. (Lanes 1,2) Protein Pop1 (yeast strain EK-Pop1NHis); (lanes 3,4) protein Pop3 (yeast strain EK-Pop3); (lanes 5,6) protein Pop4 (yeast strain EK-Pop4); (lanes 7,8) protein Pop5 (yeast strain EK-Pop5); (lanes 9,10) protein Pop6 (yeast strain EK-Pop6); (lanes 11,12) protein Pop7 (yeast strain EK-Pop7); (lanes 13,14) protein Pop8 (yeast strain EK-Pop8); (lanes 15,16) protein Rmp1 (yeast strain EK-Rmp1); (lanes 17,18) protein Rpp1 (yeast strain EK-Rpp1); (lanes 19,20) protein Snm1 (yeast strain EK-Snm1); (lanes 21,22) protein Rpr2 (yeast strain EK-Rpr2). Odd lanes are isolated mixtures of RNase P/MRP holoenzymes; even lanes are pull-downs of tagged proteins (marked by black stars). Silver-stained SDS-polyacrylamide gels.

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