Biology subjects

Kuhle, B.

Publications and source records attributed to Kuhle, B..

4 recordsLinked to original sources

bioRxiv2026

Structure of the human mitochondrial RNA degradosomereveals a distinct mode of helicase-nuclease coupling

RNA degradation shapes cellular and organellar gene expression. In human mitochondria, this process is mediated by a dedicated degradosome comprising the helicase SUV3 and the exoribonuclease PNPase, but how these enzymes assemble and cooperate to degrade structured RNA has remained unknown. Here, we report the cryo-EM structure of the complete human mitochondrial RNA degradosome bound to RNA. The structure reveals an asymmetric heteropentamer composed of a SUV3 dimer and the trimeric PNPase. Degradosome assembly is accompanied by conformational rearrangements in PNPase that reshape the RNA-entry channel and generate an asymmetric S1-domain platform for SUV3 binding. This creates a continuous electropositive path from SUV3 to PNPase, suggesting how RNA may be guided during degradation. Together, these findings reveal a mode of helicase-nuclease coupling distinct from other RNA degradation machineries and provide a structural framework for understanding human mitochondrial RNA degradation, its regulation, and association with disease.

biochemistry↗

bioRxiv2026

Mechanism of human tRNA 3'CCA maturation

The non-templated addition of the 3CCA end is the final universal step of tRNA maturation. In humans, 3CCA addition on nuclear- (nu-tRNA) and mitochondria-encoded tRNAs (mt-tRNA) is catalyzed by a single CCA-adding enzyme, TRNT1, but its precise mechanism remains unknown. Here, we report structures of TRNT1 trapped at various stages during the 3CCA addition cycle on canonical and non-canonical mt-tRNAs bound to the mitochondrial tRNA maturation platform TRMT10C-SDR5C1. Combined with biochemical data, these structures demonstrate that 3CCA addition proceeds by a continuous polymerization and translocation mechanism, in which the growing RNA primer remodels the TRNT1 catalytic site to define the specificity of non-templated 3CCA addition. Moreover, they reveal a relaxed recognition mode that allows TRNT1 to mature both canonical nu-tRNA and non-canonical mt-tRNA substrates. Finally, biochemical analyses of disease-associated TRNT1 variants provide insights into their molecular pathogenesis. Taken together, these results provide a detailed mechanistic picture of human tRNA-3CCA maturation.

molecular biology↗

bioRxiv2024

Molecular basis for the interactions of eIF2β with eIF5, eIF2B, and 5MP1 and their regulation by CK2

The heterotrimeric GTPase eukaryotic translation initiation factor 2 (eIF2) delivers the initiator Met-tRNAi to the ribosomal translation preinitiation complex (PIC). eIF2{beta} has three lysine-rich repeats (K-boxes), important for binding to the GTPase-activating protein eIF5, the guanine nucleotide exchange factor eIF2B, and the regulator eIF5-mimic protein (5MP). Here, we combine X-ray crystallography with NMR to understand the molecular basis and dynamics of these interactions. The crystal structure of yeast eIF5-CTD in complex with eIF2{beta} K-box 3 reveals an extended binding site on eIF2{beta}, far beyond the K-box. We show that eIF2{beta} contains three distinct binding sites, centered on each of the K-boxes, and human eIF5, eIF2B{varepsilon}, and 5MP1 can bind to all three sites, while reducing each others affinities. Our results reveal how eIF2B speeds up the dissociation of eIF5 from eIF2-GDP to promote nucleotide exchange; and how 5MP1 can destabilize eIF5 binding to eIF2 and the PIC, to promote stringent start codon selection. All these affinities are increased by CK2 phosphomimetic mutations, highlighting the role of CK2 in both remodeling and stabilizing the translation apparatus.

biochemistry↗

bioRxiv2024

Molecular basis of human nuclear and mitochondrial tRNA 3'-processing

Eukaryotic transfer RNA (tRNA) precursors undergo sequential processing steps to become mature tRNAs. In humans, ELAC2 carries out 3-end processing of both nucleus-encoded (nu-tRNAs) and mitochondria-encoded tRNAs (mt-tRNAs). ELAC2 is self-sufficient for processing of nu-tRNAs, but requires TRMT10C and SDR5C1 to process most mt-tRNAs. Here, we show that TRMT10C-SDR5C1 specifically facilitate processing of structurally degenerate mt-tRNAs lacking the canonical elbow. Structures of ELAC2 in complex with TRMT10C, SDR5C1 and two divergent mt-tRNA substrates reveal two distinct mechanisms of pre-tRNA recognition. While canonical nu-tRNAs and mt-tRNAs are recognized by direct ELAC2-RNA interactions, processing of non-canonical mt-tRNAs depends on protein-protein interactions between ELAC2 and TRMT10C. These results provide the molecular basis for tRNA 3-processing in both the nucleus and mitochondria and explain the organelle-specific requirement for additional factors. Moreover, they suggest that TRMT10C-SDR5C1 evolved as a mitochondrial tRNA maturation platform to compensate for the structural erosion of mt-tRNAs in bilaterian animals.

biochemistry↗