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. 2004 Apr 19;32(7):2138-46.
doi: 10.1093/nar/gkh539. Print 2004.

Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex

Tim J M Welting  1 , Walther J van Venrooij, Ger J M Pruijn
Affiliations

Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex

Tim J M Welting et al. Nucleic Acids Res. .

Abstract

The eukaryotic ribonuclease for mitochondrial RNA processing (RNase MRP) is mainly located in the nucleoli and belongs to the small nucleolar ribonucleoprotein (snoRNP) particles. RNase MRP is involved in the processing of pre-rRNA and the generation of RNA primers for mitochondrial DNA replication. A closely related snoRNP, which shares protein subunits with RNase MRP and contains a structurally related RNA subunit, is the pre-tRNA processing factor RNase P. Up to now, 10 protein subunits of these complexes have been described, designated hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38 and Rpp40. To get more insight into the assembly of the human RNase MRP complex we studied protein-protein and protein-RNA interactions by means of GST pull-down experiments. A total of 19 direct protein-protein and six direct protein-RNA interactions were observed. The analysis of mutant RNase MRP RNAs showed that distinct regions are involved in the direct interaction with protein subunits. The results provide insight into the way the protein and RNA subunits assemble into a ribonucleoprotein particle. Based upon these data a new model for the architecture of the human RNase MRP complex was generated.

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Figures

Figure 1
Figure 1
Recombinant GST-fusion proteins of RNase MRP subunits. The composition of the recombinant GST-fusion protein preparations was analysed by SDS–PAGE. The asterisks (*) indicate the full-length GST-(fusion) proteins. Lane 1, GST; lane 2, GST–hRrp42p; lane 3, GST–hPop1; lane 4, GST–hPop4; lane 5, GST–hPop5; lane 6, GST–Rpp14; lane 7, GST–Rpp20; lane 8, GST–Rpp21; lane 9, GST–Rpp25; lane 10, GST–Rpp38. The faster migrating polypeptides most likely represent truncated versions of the full-length recombinant proteins. The positions of molecular mass markers are indicated on the left.
Figure 2
Figure 2
GST pull-down analysis of RNase MRP protein–protein interactions. GST-fusion proteins were incubated with 35S-labelled in vitro translated proteins and after precipitation by glutathione–Sepharose, co- precipitated radiolabelled proteins were analysed by SDS–PAGE and autoradiography. In each panel, lane 1 shows the radiolabelled input protein, lanes 2 and 3 the negative control precipitations with GST and GST–hRrp42p, respectively, and lane 4 the material precipitated by the GST-tagged RNase MRP protein. (A) Interaction between GST–hPop1 and hPop4. (B) Interaction between GST–hPop4 and hPop1. (C) Interaction between GST–hPop4 and hPop4. (D) Interaction between GST–Rpp20 and Rpp25. (E) Interaction between GST–hPop5 and Rpp25.
Figure 3
Figure 3
Summary of protein–protein interactions between the RNase MRP protein subunits detected by GST pull-down analyses. The dark-grey boxes indicate efficient interactions (more than 20% of input radiolabelled protein precipitated) that were detected with either one or the other interacting partner fused to GST; the grey boxes mark efficient interactions that were detected in only one of the reciprocal pull-downs; the light-grey boxes indicate relatively weak interactions (<20% of input protein precipitated). Because GST–Rpp30 and GST–Rpp40 were not available, these proteins were not analysed.
Figure 4
Figure 4
Direct interactions between GST-tagged RNase MRP proteins and the human RNase MRP and RNase P RNAs. The eight bacterially expressed GST-fusion proteins were incubated with a mixture of 32P-labelled, in vitro transcribed, full-length human RNase MRP RNA, RNase P RNA and hY1 RNA, a small RNA associated with human Ro RNPs which was included as a negative control. Binding of these RNAs was determined by GST pull-down followed by denaturing gel electrophoresis of the co-precipitated RNAs and autoradiography. Ten percent of the input RNA was loaded in lane 1. The GST fusion proteins are indicated above the lanes (lanes 3–10). GST alone was used as a control (lane 2). The positions of RNase P RNA (P), RNase MRP RNA (MRP) and hY1 RNA (hY1) are indicated on the left.
Figure 5
Figure 5
Schematic structure of deletion mutants of RNase MRP RNA. The RNA on the left represents the predicted secondary structure of the human RNase MRP RNA. The designations of the phylogenetically conserved helices (P1–P19) is based upon the RNase P RNA numbering described by Frank et al. (37). In the structures of the deletion mutants MRP67–267, MRP67–197, MRP67–167, MRP1–82, MRPΔ87–115 and MRPΔ132–176 only the remaining regions are shown.
Figure 6
Figure 6
GST pull-down analysis of RNase MRP protein–RNA interactions. GST-fusion proteins were incubated with radiolabelled in vitro transcribed mutants of RNase MRP RNA (see Fig. 5) and after precipitation by glutathione–Sepharose, co-precipitated RNAs were analysed by gel electrophoresis and autoradiography. In all panels, lane 1 shows the mixture of radiolabelled input RNAs and lane 2 the negative control precipitation with GST. Lane 3 contains the RNAs precipitated by GST–hPop1 (A), GST–Rpp20 (B and C) and lane 4 contains the RNAs precipitated by GST–Rpp25 (B) or GST-Rpp38 (C), respectively.
Figure 7
Figure 7
Summary of protein–RNA interactions between the RNase MRP subunits detected by GST pull-down analyses. The efficiency of binding by the GST-fusion proteins to the deletion mutants of RNase MRP RNA was determined by three independent experiments. The most efficient interactions are indicated by dark-grey boxes, intermediate interactions by grey boxes and weak interactions by light-grey boxes. In agreement with the lack of interaction with the full-length RNase MRP RNA (Fig. 4), GST–hPop5 and GST–Rpp14 did not shown detectable interactions with any of the RNase MRP RNA mutants.
Figure 8
Figure 8
Model for the human RNase MRP complex. Using the data obtained in this study and previously published UV-crosslinking data (25), a structural model for the human RNase MRP complex was generated. In this model, all detected protein–RNA interactions, except for the interaction of Rpp21, which seemed to be non-specific, are combined with all detected protein–protein interactions, except for the most weak interactions. Note that the size of the depicted subunits is not proportional to their molecular masses.
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