Biology subjects

Krebs, L.

Publications and source records attributed to Krebs, L..

3 recordsLinked to original sources

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↗

bioRxiv2022

Mammalian N1-adenosine PARylation is a reversible DNA modification

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. We conclude that N1-adenosine PARylation is a novel, reversible DNA modification.

molecular biology↗

bioRxiv2021

Endocytic vacuole formation by WASH-mediated endocytosis

Endocytosis is a fundamental cellular process facilitated by diverse mechanisms. Remarkably, several distinct clathrin-independent endocytic processes were identified and characterized following virus uptake into cells. For some, however, mechanistic execution and biological function remain largely unclear. This includes an endocytic process exploited by human papillomavirus type 16 (HPV16). Using HPV16, we examined how vesicles are formed by combining systematic cellular perturbations with electron and video microscopy. Cargo uptake occurred by uncoated, inward-budding pits. Mechanistically, vesicle scission was facilitated by actin polymerization controlled through the actin nucleation promoting factor WASH. While WASH typically functions in conjunction with the retromer complex on endosomes during retrograde trafficking, endocytic vesicle formation was largely independent of retromer itself and the heterodimeric membrane-bending SNX-BAR retromer adaptor, thereby uncovering a role of WASH in endocytosis in addition to its canonical role in intracellular membrane trafficking.

cell biology↗