NSUN4 is a dual function mitochondrial protein required for both methylation of 12S rRNA and coordination of mitoribosomal assembly

Abstract

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.

Figure 1. Conditional inactivation of the Nsun4 gene in the germline.

A. Schematic representation of the targeting strategy for disruption of the Nsun4 gene. Exon II was flanked by loxP-sites. The puromycin resistance gene is flanked by Frt-recombination sites and was used for ES cell selection. Mice with an Frt-flanked puromycin gene were crossed with mice with ubiquitous expression of Flp-recombinase to remove the Puromycin resistance gene. Transgenic mice expressing cre-recombinase were used for breeding with animals with a loxP-flanked Nsun4 gene to disrupt Nsun4. B. Morphological comparison between wild type and whole-body Nsun4 knockout embryos at E∼8.5. Scale bar = 1 mm.

Figure 2. Conditional inactivation of the Nsun4 gene in heart and skeletal muscle.

A. Decreased lifespan of mice with a muscle-specific knockout of Nsun4. Survival curve for control (L/L; n = 20; open squares) and mutant mice (L/L, cre; n = 16; filled squares). B. Heart- to body-weight ratio in control (L/L; open squares) and mutant mice (L/L, cre; filled squares). Number of analyzed animals at 5 weeks L/L n = 6, L/L, cre n = 6; at 10 weeks L/L n = 7, L/L, cre n = 8; at 15 weeks L/L n = 5, L/L, cre n = 7; at 20 weeks L/L n = 12, L/L, cre n = 12. Data are represented as mean +/− SEM. *, p<0.05; **, p<0.01; ***p<0.001. Student's t test. C. BN-PAGE analysis of levels of assembled respiratory chain complexes in control (L/L) and knockout (L/L, cre) mice at different ages. D. Western immunoblotting of steady-state levels of NSUN4 in heart mitochondrial extracts from control (L/L) and knockout (L/L, cre) mice at different ages. VDAC was used as a loading control.

Figure 3. Mitochondrial translation and ribosome assembly in Nsun4 knockout hearts.

A. Pulse-labeling of mitochondrial translation products in isolated heart mitochondria from 20 weeks-old control (L/L) and knockout mice (L/L, cre). The Coomassie-stained gel is a loading control. Known mitochondrial polypeptides are indicated. B. Western blot analysis of steady-state levels of mitochondrially and nucleus-encoded OXPHOS proteins in mitochondria from control and knockout hearts at different ages. VDAC was used as a loading control. *, cross reaction. C. Analysis of mitoribosomal assembly by sucrose gradient ultracentrifugation of heart mitochondrial extracts from control (L/L) and mutant (L/L, cre) mice. Sedimentation of 28S (SSU, fraction 6), 39S (LSU, fraction 8) and 55S (assembled ribosomes, fraction 10) was determined by western blot analysis using MRPS15- and MRPL13-specific antibodies. D. Western blot analysis of steady-state levels of MRPL13 and MRPS15 in heart mitochondrial extracts from control (L/L) and knockout (L/L, cre) mice at different ages. VDAC was used as a loading control.

Figure 4. Analysis of rRNA methylation in control, Nsun4 and Mterf4 heart tissue specific knockout.

A. Relative methylation levels of 12S and 16S rRNA determined after sequencing of cDNA obtained from bisulfite treated RNA from heart mitochondria of control (L/L; n = 3) mice at age 20 weeks. Nucleotide numbers are relative to the 5′-end of the mouse mtDNA gene for tRNA^Phe. B. Relative methylation levels of 12S and 16S rRNA in NSUN4 knockout (L/L, cre; n = 3) at age 20 weeks. Analysis performed as in panel a. C. Relative methylation levels of 12S rRNA in control (N = 1) and MTERF4 knockout (N = 2) at age 15 weeks and in control (N = 1) and MTERF4 knockout (N = 2) at age 20 weeks.

Figure 5. rRNA binding by NSUN4 and MTERF4 and model of the role of the NSUN4/MTERF4 complex in regulation of ribosome assembly.

A. Location, along mtDNA, of the RNA fragments identified after CLIP analysis on HeLa cells expressing “trap” mutant of NSUN4, NSUN4^C258A-FLAG. B. Location, along mtDNA, of the RNA fragments identified after PAR-CLIP analysis on HeLa cells expressing MTERF4-FLAG. C. Model for the role of NSUN4 in mitoribosomal assembly. (1) NSUN4 methylates 12S rRNA. Red star denotes the methyl group donated by SAM. (2) The NSUN4/MTERF4 complex is incorporated into the LSU. (3) Release of the NSUN4/MTERF4 complex from LSU enables the interaction between both subunits.