Biology subjects

Klapstova, V.

Publications and source records attributed to Klapstova, V..

4 recordsLinked to original sources

bioRxiv2025

Mechanisms of transcription attenuation and condensation of RNA polymerase II by RECQ5

The elongation rates of RNA polymerase II (RNAPII) require precise control to prevent transcriptional stress, which can impede co-transcriptional pre-mRNA processing and contribute to many age- or disease-associated molecular changes (e.g., loss of proteostasis)1-5. Additionally mesoscale organization of transcription is thought to control the transcriptional rates6 and multiple factors have been reported to form biomolecular condensates and integrate RNAPII through the interaction with the C-terminal domain (CTD) of the largest subunit, RPB17-10. However, the structural organization of these condensates remains uncharacterized due to their small size and inherently dynamic nature. Here, we investigated the molecular mechanisms by which a general transcription factor - RECQ5 - associates with hyperphosphorylated RNAPII elongation complex (P-RNAPII EC) and controls translocation of RNAPII along genes. We combined biochemical reconstitution, electron cryomicroscopy, cryotomography, and coarse-grained simulations. We report two mechanisms by which RECQ5 modulates RNAPII transcription. At the atomic level, we demonstrate that RECQ5 uses the brake helix as a doorstop to control RNAPII translocation along DNA, attenuating transcription. At the mesoscale level, RECQ5 forms a condensate scaffold matrix, integrating P-RNAPII EC through a network of site-specific interactions, reinforcing the condensates structural integrity. Our integrative, multi-scale study provides insights into the structural basis of transcription attenuation and into the molecular architecture and biogenesis of a model RNAPII condensate.

biochemistry↗

bioRxiv2025

Distinct Mechanisms of Recognition of Phosphorylated RNAPII C-Terminal Domain by BRCT Repeats of the BRCA1-BARD1 Complex: Insights from Structural and Functional Analyses

Transcription competes with other DNA-dependent processes, such as DNA repair, for access to its substrate, DNA. However, the principles governing the interplay between these processes remain poorly understood. Evidence suggests that the BRCA1-BARD1 complex, a key player in the DNA damage response, may act as a mediator of this crosstalk. In this study, we investigated the molecular mechanism underpinning the interaction between RNA polymerase II (RNAPII) and the BRCA1-BARD1 complex, as well as its functional implications. Our findings reveal that the BRCT repeat of BRCA1 binds the Ser5-phosphorylated CTD of RNAPII, utilising a mechanism previously established for other BRCT ligands. Furthermore, we demonstrate that this interaction is critical for the organisation of RNAPII into condensates with liquid-like properties. Analysis of disease-associated variants within the BRCT repeats further supports the biological relevance of this condensation. Collectively, our results suggest that the BRCA1-BARD1 complex may coordinate transcription and DNA repair by facilitating the organisation of RNAPII into transcription factories.

biochemistry↗

bioRxiv2022

Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor

Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.

biochemistry↗

bioRxiv2019

Ancestral Mitochondrial Protein Secretion Machinery

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

evolutionary biology↗