Biology subjects

Kubicek, K.

Publications and source records attributed to Kubicek, K..

5 recordsLinked to original sources

bioRxiv2026

A negatively charged unstructured loop autoinhibits mammalian Dicer and supports fidelity of miRNA biogenesis.

Dicer RNases produce small RNAs for RNA interference (RNAi) and microRNA (miRNA) pathways. Although vertebrate Dicers share a conserved overall architecture with other eukaryotic Dicers, they are uniquely adapted for the production of gene-regulating miRNAs. We report here this adaptation extends beyond the structured core of the enzyme and relies on an intrinsically disordered region (IDR) conserved across jawed vertebrates. This negatively charged IDR supports fidelity of miRNA biogenesis while inhibiting RNAi. The IDR projects into a positively charged substrate-binding groove of Dicer and stabilizes a pre-dicing state, which effectively licenses authentic miRNA precursors. Electron cryo-microscopy shows that removal of the IDR shifts the equilibrium from the pre-dicing to the dicing state, thus increasing enzymatic activity and broadening substrate accessibility. In cells, deletion, replacement or variations of the IDR affect fidelity of microRNA biogenesis, change substrate specificity, and enhance RNAi. Altogether, our work highlights functional relevance of unstructured protein parts, which escape attention in structural analyses, and introduces an autoinhibitory IDR element mimicking negative RNA charge as a component of the molecular grammar governing IDRs in RNA-binding proteins.

molecular biology↗

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↗

bioRxiv2023

Brain map of aging-induced alterations in membrane ganglioside pattern

Recent efforts to develop comprehensive metabolome and proteome brain atlases for animal models have yielded significant progress. However, the ganglioside (GSs) profile of these models remains largely unexplored. As essential components of the brain, GSs play a crucial role in neuronal function. To address this gap in knowledge, we conducted an in-depth analysis of the young and adult rat brain, as well as its major brain regions, using ultra-high performance liquid chromatography and tandem mass spectrometry in positive and negative ion modes. We also analyzed GSs in cerebrospinal fluid (CSF) and serum from matched samples. Our findings indicate a shift in the ratio of a-series to b-series GSs with age, along with region-specific changes accompanied by aging. This could improve our understanding of brain aging and neurodegenerative diseases. Our study complements existing brain atlases of lipidome and protein expression and highlights the importance of further investigating the mechanisms underlying these GSs changes and the potential therapeutic implications of our findings.

neuroscience↗

bioRxiv2022

Molecular basis of indispensable accuracy of mammalian miRNA biogenesis

Mammalian Dicer is the gatekeeper into the essential gene-regulating miRNA pathway. What is committing mammalian Dicer to the miRNA pathway remains unknown. We report that Dicers highly conserved DExD/H helicase domain is the key structural element supporting accurate miRNA biogenesis. While ATPase activity of the domain is non-essential, its loss is lethal in mice. It is required during canonical miRNA biogenesis for efficient recognition, high-fidelity cleavage, and strand selection. Structure of Dicer-miRNA precursor complexes showed that the DExD/H domain acquired helicase-unrelated function defining Dicer conformations, which affect substrate loading and facilitate pre-selection of miRNA precursors. Dicer lacking the DExD/H domain favors conformations enabling reduced substrate selectivity and supporting RNA interference, a different small RNA pathway. Therefore, Dicers DExD/H domain ensures indispensable high-fidelity precursor processing of mammalian miRNAs, which constitutes a structural "mold" for adaptive miRNA evolution.

molecular biology↗

bioRxiv2018

Termination of non-coding transcription in yeast relies on both a CTD-interaction domain and a CTD-mimic in Sen1

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a risk that needs to be controlled to prevent the perturbation of gene expression. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy terminal domain (CTD) of RNA polymerase II and characterize structurally its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires the recognition of the CTD by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote Sen1 initial recruitment but rather the capacity of Sen1 to induce the release of paused RNAPII from the DNA. Our results shed light onto the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

genetics↗