Mitochondria are essential molecular machinery for the maintenance of cellular energy supply by the oxidative phosphorylation system (OXPHOS). Mitochondrial transcription factor B1 (TFB1M) is a dimethyltransferase that maintains mitochondrial homeostasis by catalyzing dimethylation of two adjacent adenines located in helix45 (h45) of 12S rRNA. This m62A modification is indispensable for the assembly and maturation of human mitochondrial ribosomes. However, both the mechanism of TFB1M catalysis and the precise function of TFB1M in mitochondrial homeostasis are unknown. Here we report the crystal structures of a ternary complex of human (hs) TFB1M–h45–S-adenosyl-methionine and a binary complex hsTFB1M–h45. The structures revealed a distinct mode of hsTFB1M interaction with its rRNA substrate and with the initial enzymatic state involved in m62A modification. The suppression of hsTFB1M protein level or the overexpression of inactive hsTFB1M mutants resulted in decreased ATP production and reduced expression of components of the mitochondrial OXPHOS without affecting transcription of the corresponding genes and their localization to the mitochondria. Therefore, hsTFB1M regulated the translation of mitochondrial genes rather than their transcription via m62A modification in h45.

We report a novel proton-detected MAS solid-state NMR strategy based on N-N proton assisted recoupling to detect the inter-nucleotide NHN hydrogen bonds within the Watson-Crick base pairs of micro-crystallized dimeric RNA and to confirm the kissing-loop structure. This would contribute to advances in the structural determination of RNA using solid-state NMR.

In bacteria, small non-coding RNAs (sRNAs) could function in gene regulations under variable stress responses. DsrA is an ∼90-nucleotide Hfq-dependent sRNA found in Escherichia coli. It regulates the translation and degradation of multiple mRNAs, such as rpoS, hns, mreB and rbsD mRNAs. However, its functional structure and particularly how it regulates multiple mRNAs remain obscure. Using NMR, we investigated the solution structures of the full-length and isolated stem–loops of DsrA. We first solved the NMR structure of the first stem–loop (SL1), and further studied the melting process of the SL1 induced by the base-pairing with the rpoS mRNA and the A-form duplex formation of the DsrA/rpoS complex. The secondary structure of the second stem–loop (SL2) was also determined, which contains a lower stem and an upper stem with distinctive stability. Interestingly, two conformational states of SL2 in dynamic equilibrium were observed in our NMR spectra, suggesting that the conformational selection may occur during the base-pairing between DsrA and mRNAs. In summary, our study suggests that the conformational plasticity of DsrA may represent a special mechanism sRNA employed to deal with its multiple regulatory targets of mRNA.

Eukaryotes contain two sets of genomes: the nuclear genome and the mitochondrial genome. The mitochondrial genome transcripts 13 mRNAs that encode 13 essential proteins for the oxidative phosphorylation complex, 2 rRNAs (12s rRNA and 16s rRNA), and 22 tRNAs. The proper assembly and maturation of the mitochondrial ribosome (mitoribosome) are critical for the translation of the 13 key proteins and the function of the mitochondrion. Human ribosome-binding factor A (hsRBFA) is a mitoribosome assembly factor that binds with helix 28, helix 44 and helix 45 of 12S rRNA and facilitates the transcriptional modification of 12S rRNA during the mitoribosomal biogenesis. Previous research mentioned that the malfunction of hsRBFA will induce the instability of mitoribosomes and affect the function of mitochondria, but the mechanisms underlying the interaction between hsRBFA and 12S rRNA and its influence on mitochondrial function are still unknown. In this study, we found that hsRBFA binds with double strain RNA (dsRNA) through its whole N-terminus (Nt) instead of the KH-like domain alone, which is different from the other homologous. Furthermore, we mapped the key residues that affected the RNA binding and maturation of mitoribosomes in vitro. Finally, we investigated how these residues affect mitochondrial functions in detail and systematically.