Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

Abstract

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

FIGURE 1.

Glycerol gradient centrifugation of NuMRP and MtMRP. Purified NuMRP or MtMRP containing 50–80 ng of the MRP RNA were centrifuged in parallel on a 15%–30% glycerol density-gradient, and fractioned as described in the Material and Methods. RNA isolated from the gradient fractions and the MRP RNA was detected by Northern analysis. (A) MtMRP. (B) NuMRP.

FIGURE 2.

Protein analysis of NuMRP and MtMRP. (A) Protein samples were separated on a 7.5%–17.5% SDS-PAGE gel and visualized using MALDI compatible silver staining. Protein components were excised, digested by trypsin, and analyzed by MALDI-TOF mass spectrometry (see Materials and Methods). M, molecular-weight markers. (Lane 1) 10 μL of TAP-tag purified NuMRP prior to glycerol gradient. All 10 NuMRP protein components were confirmed in this preparation (not shown). (Lane 2) Precipitated proteins from MtMRP containing 1000 picograms (pg) of the MRP RNA. (Lane 3) Precipitated proteins from NuMRP containing 1000 pg of the MRP RNA. Five of the 10 proteins successfully identified by mass spectrometry of this sample are indicated. (B) Proteins precipitated from both MtMRP and NuMRP containing 1000 pg of the MRP RNA were separated on a 12.5% SDS-PAGE gel, transferred to nylon membrane, and Pop4 was detected using anti-TAP antibody. Both preparations were made from the same yeast strain expressing a Pop4:TAP fusion as its only source of Pop4 protein. Part of the protein is specifically cleaved during the NuMRP purification giving a smaller protein, but leaving the antibody epitope. The arrow indicates the size of the full-length uncleaved protein in the MtMRP preparation.

FIGURE 3.

In vitro cleavage assays of RNase MRP on three different substrates. RNA substrates 3′-end labeled with ³²P were incubated with either NuMRP or MtMRP for 30 min at 37°C. The cleavage reactions were analyzed on a 6% polyacrylamide, 7 M urea gel as described in Materials and Methods. (A) Mitochondrial Ori5 substrate. (B) rRNA A3 substrate. (C) CLB2 mRNA substrate, (D) MtMRP and NuMRP were mixed together on the mitochondrial substrate at varying ratios.

FIGURE 4.

Enzyme titration and time course of RNase MRP cleavage by NuMRP and MtMRP. (A) The ³²P-labeled mitochondrial substrate was incubated with increasing amounts of MtMRP or NuMRP for 30 min. Enzyme levels were quantitated by the MRP RNA levels, which are indicated. (B) The ³²P-labeled mitochondrial substrate was incubated with the MtMRP or NuMRP for increasing amounts of time. The cleavage reactions were analyzed on a 6% polyacrylamide, 7 M urea gel as described in Materials and Methods.

FIGURE 5.

Effect of ionic strength on NuMRP and MtMRP activity. The ³²P-labeled mitochondrial ORI5 substrate was incubated with nuMRP and mtMRP for 30 min in buffers with different concentrations of K⁺ or Mg²⁺. Except for the indicated ion, standard reaction conditions were used. The cleavage reactions were analyzed on a 6% polyacrylamide, 7 M urea gel as described in Materials and Methods.

FIGURE 6.

Effect of lithium on RNase MRP catalytic activities. (A) The ³²P-labeled substrate was incubated for 30 min with NuMRP or MtMRP under standard reaction conditions with increasing concentrations of lithium and either the ORI5 or A3 substrates. (B) Effect of lithium or potassium on cleavage of the A3 substrate by NuMRP. The cleavage activity without adding lithium or potassium is set as 100%.