The MRPP1/MRPP2 complex is a tRNA-maturation platform in human mitochondria

Abstract

Mitochondrial polycistronic transcripts are extensively processed to give rise to functional mRNAs, rRNAs and tRNAs; starting with the release of tRNA elements through 5'-processing by RNase P (MRPP1/2/3-complex) and 3'-processing by RNase Z (ELAC2). Here, we show using in vitro experiments that MRPP1/2 is not only a component of the mitochondrial RNase P but that it retains the tRNA product from the 5'-processing step and significantly enhances the efficiency of ELAC2-catalyzed 3'-processing for 17 of the 22 tRNAs encoded in the human mitochondrial genome. Furthermore, MRPP1/2 retains the tRNA product after ELAC2 processing and presents the nascent tRNA to the mitochondrial CCA-adding enzyme. Thus, in addition to being an essential component of the RNase P reaction, MRPP1/2 serves as a processing platform for several down-stream tRNA maturation steps in human mitochondria. These findings are of fundamental importance for our molecular understanding of disease-related mutations in MRPP1/2, ELAC2 and mitochondrial tRNA genes.

Figure 1.

Role of MRPP1/2 in RNase P function on tRNA^Tyr. (A) Schematic representation of the light-strand (LS) tRNA cluster Tyr-Cys-Asn-Ala and its complementary heavy-strand (HS). (B) Secondary structure of human pre-tRNA^Tyr(38,17) (Y(38,17)) having 38 nt as 5′-leader and 17 nt as 3′-trailer. The discriminator base is boxed. (C) RNase P dilution series on 200 nM tRNA^Tyr(38,17). Top: MRPP3 dilution (from 800 to 3.1 nM) and MRPP1/2 constant (800 nM). Bottom: MRPP1/2 dilution (from 800 to 3.1 nM) and MRPP3 constant (50 nM). The molar ratio of tRNA to protein is given in brackets. The reactions were incubated for 1 h. (D–F) Gel filtration chromatograms of MRPP1/2:pre-tRNA^Tyr(5,6) prior and after MRPP3 treatment as well as of MRPP3 alone (D). Peaks of interest are numbered and the peaks tRNA and protein content are visualized by urea PAGE (E) and SDS-PAGE (F), respectively.

Figure 2.

Role of MRPP1/2 in RNase P function on tRNA^His. (A) Schematic representation of the heavy-strand (HS) tRNA cluster His-Ser(AGY)-Leu(CUN). The 5′- and 3′-region of tRNA^His (in black) are highlighted in red and blue, respectively. (B) Secondary structure of pre-tRNA^His(25,tRNA^Ser(AGY)) (H(25,S)) having 25 nt as 5′-leader (in red) and the complete tRNA^Ser(AGY) as 3′-trailer (in blue). The discriminator bases are boxed. (C) RNase P dilution series on 200 nM tRNA^His(25,S). Top: MRPP3 dilution (from 800 to 3.1 nM) and MRPP1/2 constant (800 nM). Bottom: MRPP1/2 dilution (from 800 to 3.1 nM) and MRPP3 constant (50 nM). The molar ratio of tRNA to protein is shown in parentheses. The reactions were incubated for 1 h. (D–F) Gel filtration chromatograms of MRPP1/2:pre-tRNA^His(25,S) prior and after MRPP3 treatment (D). Peaks of interest are numbered, and tRNA and protein content of the peaks are visualized by urea PAGE (E) and SDS-PAGE (F), respectively.

Figure 3.

MRPP1/2 supports ELAC2 based 3′-tail removal. (A and B) ELAC2 activity in the absence and presence of 800 nM MRPP1/2 at different salt concentrations (15–200 mM KCl) on pre-tRNA^Tyr(0,17) (Y(0,17)) (A) and pre-tRNA^His(0,S) (H(0,S)) (B). Mis-cleavage on Y(0,17) is indicated by asterisks. The reaction mixes of H(0,S) were incubated for 1 h.

Figure 4.

Role of MRPP1/2 in RNase Z function on tRNA^Tyr and tRNA^His. (A) RNase Z dilution experiments on pre-tRNA^Tyr(0,17) (Y(0,17)). Top: ELAC2 dilution series (800–3.1 nM) in the presence of 800 nM MRPP1/2. Middle: MRPP1/2 dilution series (800–3.1 nM) in the presence of 50 nM ELAC2. Bottom: ELAC2 dilution series (800–3.1 nM) in the absence of MRPP1/2. The reactions were incubated for 1 h. The molar ratio of tRNA to protein is shown in parentheses. (B–D) Gel filtration chromatograms of MRPP1/2:pre-tRNA^Tyr(5,6) after treatment with MRPP3 and ELAC2, as well as of MRPP3 and ELAC2 alone. Peaks of interest are numbered and tRNA and protein content of the peaks is visualized by urea PAGE (C) and SDS-PAGE (D), respectively. For comparison, also pre-tRNA^Tyr(5,6) and pre-tRNA^Tyr(0,6) from Figure 1E are shown in (C). (E) RNase Z dilution experiments on pre-tRNA^His(0,S) (H(0,S)). Reaction condition are as given in (A). (F–H) Gel filtration chromatograms of MRPP1/2:pre-tRNA^His(0,S) prior to and after treatment with ELAC2. Peaks of interest are numbered, and tRNA and protein content of the peaks is visualized by urea PAGE (G) and SDS-PAGE (H), respectively.

Figure 5.

Mitochondrial tRNA processing order and CCA-adding enzyme modify MRPP1/2 bound tRNA. (A and B) Processing order of RNase P and RNase Z on pre-tRNA^Tyr (38,17) (Y (38,17)) (A) and pre-tRNA^His(25,S) (H(25,S)) (B). For activity measurements, 800 nM MRPP1/2, 50 nM MRPP3 and 50 nM ELAC2, or combinations, were used. (C and D) Activity of CCA-adding enzyme (CCA-E, 50 nM) on pre-tRNA^Tyr(0,0) (Y(0,0)) (C) and pre-tRNA^His(0,0) (H(0,0)) (D) in the absence and presence of 800 nM MRPP1/2. Hyper-modified tRNA is indicated by asterisks. (E–G) Gel filtration chromatograms of MRPP1/2:pre-tRNA^Tyr(0,0) after incubation with CCA-adding enzyme and rNTPs (E). Peaks of interested are numbered, and tRNA and protein content of the peaks is visualized by urea PAGE (F) and SDS-PAGE (G), respectively.