Human NOP2/NSUN1 regulates ribosome biogenesis through non-catalytic complex formation with box C/D snoRNPs.

5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5'ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.

Relaxed selection on male mitochondrial genes in DUI bivalves eases the need for mitonuclear coevolution.

Mitonuclear coevolution is an important prerequisite for efficient energy production in eukaryotes. However, many bivalve taxa experience doubly uniparental inheritance (DUI) and have sex-specific mitochondrial (mt) genomes, providing a challenge for mitonuclear coevolution. We examined possible mechanisms to reconcile mitonuclear coevolution with DUI. No nuclear-encoded, sex-specific OXPHOS paralogs were found in the DUI clam Ruditapes philippinarum, refuting OXPHOS paralogy as a solution in this species. It is also unlikely that mt changes causing disruption of nuclear interactions are strongly selected against because sex-specific mt-residues or those under positive selection in M mt genes were not depleted for contacting nuclear-encoded residues. However, M genomes showed consistently higher dN /dS ratios compared to putatively ancestral F genomes in all mt OXPHOS genes and across all DUI species. Further analyses indicated that this was consistently due to relaxed, not positive selection on M vs. F mt OXPHOS genes. Similarly, selection was relaxed on the F genome of DUI species compared to species with strict maternal inheritance. Coupled with recent physiological and molecular evolution studies, we suggest that relaxed selection on M mt function limits the need to maintain mitonuclear interactions in M genomes compared to F genomes. We discuss our findings with regard to OXPHOS function and the origin of DUI.

TRMT1L-catalyzed m2 2G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.

tRNA modifications are critical for several aspects of their functions, including decoding, folding, and stability. Using a multifaceted approach encompassing eCLIP-seq and Nanopore tRNA-seq, we show that the human tRNA methyltransferase TRMT1L interacts with component of the Rix1 ribosome biogenesis complex and binds to the 28S rRNA, as well as to a subset of tRNAs. Mechanistically, we demonstrate that TRMT1L is responsible for catalyzing m2 2G solely at position 27 of tRNA-Tyr-GUA. Surprisingly, TRMT1L depletion also impaired the deposition of acp3U and dihydrouridine on tRNA-Tyr-GUA, Cys-GCA, and Ala-CGC. TRMT1L knockout cells have a marked decrease in tRNA-Tyr-GUA levels, coinciding with a reduction in global translation rates and hypersensitivity to oxidative stress. Our results establish TRMT1L as the elusive methyltransferase catalyzing the m2 2G27 modification on tRNA Tyr, resolving a long-standing gap of knowledge and highlighting its potential role in a tRNA modification circuit crucial for translation regulation and stress response.

Sexually Antagonistic Mitonuclear Coevolution in Duplicate Oxidative Phosphorylation Genes.

Mitochondrial function is critical in eukaryotes. To maintain an adequate supply of energy, precise interactions must be maintained between nuclear- and mitochondrial-encoded gene products. Such interactions are paramount in chimeric enzymes such as the oxidative phosphorylation (OXPHOS) complexes. Mutualistic coevolution between the two genomes has therefore been suggested to be a critical, ubiquitous feature of eukaryotes that acts to maintain cellular function. However, mitochondrial genomes can also act selfishly and increase their own transmission at the expense of organismal function. For example, male-harming mutations are predisposed to accumulate in mitochondrial genomes due to their maternal inheritance ("mother's curse"). Here, we investigate sexually antagonistic mitonuclear coevolution in nuclear-encoded OXPHOS paralogs from mammals and Drosophila. These duplicate genes are highly divergent but must interact with the same set of mitochondrial-encoded genes. Many such paralogs show testis-specific expression, prompting previous hypotheses suggesting they may have evolved under selection to counteract male-harming mitochondrial mutations. We found increased rates of evolution in OXPHOS paralogs with testis-specific expression in mammals and Drosophila, supporting this hypothesis. However, further analyses suggested such patterns may be due to relaxed, not positive selection, especially in Drosophila. Structural data also suggest that mitonuclear interactions do not play a major role in the evolution of many OXPHOS paralogs in a consistent way. In conclusion, no single OXPHOS paralog met all our criteria for being under selection to counteract male-harming mitochondrial mutations. We discuss alternative explanations for the drastic patterns of evolution in these genes, including mutualistic mitonuclear coevolution, adaptive subfunctionalization after gene duplication, and relaxed selection on OXPHOS in male tissues.