ABSTRACT
Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor.
ABSTRACT
Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.
Subject(s)
Anticodon/chemistry , Methyltransferases/genetics , Mitochondria/genetics , RNA, Mitochondrial/chemistry , RNA, Transfer, Ser/chemistry , RNA, Transfer, Thr/chemistry , Anticodon/metabolism , Base Pairing , Cytosine/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Methylation , Methyltransferases/metabolism , Mitochondria/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Biosynthesis , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolism , RNA, Transfer, Ser/genetics , RNA, Transfer, Ser/metabolism , RNA, Transfer, Thr/genetics , RNA, Transfer, Thr/metabolism , Signal TransductionABSTRACT
High-yielding and stress-resistant crops are essential to ensure future food supply. Barley is an important crop to feed livestock and to produce malt, but the annual yield is threatened by pathogen infections. Pathogens can trigger an altered sugar partitioning in the host plant, which possibly leads to an advantage for the pathogen. Hampering these processes represents a promising strategy to potentially increase resistance. We analysed the response of the barley monosaccharide transporter HvSTP13 towards biotic stress and its potential use for plant protection. The expression of HvSTP13 increased on bacterial and fungal pathogen-associated molecular pattern (PAMP) application, suggesting a PAMP-triggered signalling that converged on the transcriptional induction of the gene. Promoter studies indicate a region that is probably targeted by transcription factors downstream of PAMP-triggered immunity pathways. We confirmed that the nonfunctional HvSTP13GR variant confers resistance against an economically relevant biotrophic rust fungus in barley. Our experimental setup provides basal prerequisites to further decode the role of HvSTP13 in response to biological stress. Moreover, in line with other studies, our experiments indicate that the alteration of sugar partitioning pathways, in a host-pathogen interaction, is a promising approach to achieve broad and durable resistance in plants.
