Biology subjects

Mikolajczyk, J.

Publications and source records attributed to Mikolajczyk, J..

3 recordsLinked to original sources

bioRxiv2026

Isotope-Free Mapping of protein:RNA Interactions Using fCRAC and trxtools

Defining high-confidence RNA interaction sites for specific proteins is essential to understand RNA biology, but existing methods face trade-offs between specificity, sensitivity, and experimental accessibility. Here, we present fluorescent Cross-linking and analysis of cDNAs (fCRAC), a mammalian-cell optimized update to the CRAC protocol. In fCRAC, a fluorescent adaptor is used, in place of radiolabeling, to visualize RNA-protein complexes during gel-purification. fCRAC retains the tandem affinity purification and stringent, denaturing conditions of classical CRAC, enabling nucleotide-resolution mapping of protein:RNA interactions with high signal to noise ratio. We initially tested fCRAC using RPP25L, a component the RNase MRP and RNase P complexes. RPP25L almost exclusively bound to predicted, single sites in the RNA components (RMRP and RPPH1), showing excellent selectivity with nucleotide resolution. To support analysis of UV cross-linking data for more complex targets, we developed the trxtools package and example pipeline for standardized processing, quality control, and analysis of data from fCRAC and related methods. We include tailored strategies for repetitive RNA classes, such as tRNA and rRNA, which can be challenging to analyze using other approaches. We applied fCRAC and trxtools to define the RNA interactome of human CYCLON/CCDC86, a nuclear protein previously implicated in oncogenesis. This revealed specific interactions with rRNA, tRNA and ncRNAs involved in pre-rRNA and pre-tRNA processing. HighlightsO_LINucleotide-resolution definition of RNAs interacting with specific proteins, including rRNA and tRNA C_LIO_LIStringent denaturing purifications and robust visualization steps, with no requirement for radioactive labelling C_LIO_LITrxtools provides an integrated analysis pipeline with approaches for analyzing both single and multi-copy RNA species C_LI

molecular biology↗

bioRxiv2026

CYCLON is a nucleolar protein that regulates pan-cancer cell fitness through ribosome biogenesis

Tumor progression is driven by cancer cell fitness, defined as the capacity of malignant cells to maintain growth, adapt to stress and withstand therapy Cellular fitness is fundamentally governed by nucleolar processes, which act as central regulators by integrating RNA processing with ribosome biogenesis to support protein synthesis and stress adaptation. The nuclear protein CYCLON, containing a large intrinsically disordered region (IDR) could be implicated in mediating biomolecular condensates and regulatory plasticity, which are key elements of nucleolar biology. CYCLON also emerged as a candidate regulator of cancer cell fitness, as it is frequently overexpressed across tumor types. Inducible silencing and cell biology approaches have shown that CYCLON maintains nucleolar integrity, controls nucleoli size and number, nucleolin and Ki-67 distribution and prevents nucleolar stress. CYCLON contributes to ribosome biogenesis by binding ribosomal RNA (rRNA) and regulating 32S and 21S pre-rRNA processing, ultimately influencing ribosomal subunit production and global protein synthesis. Its depletion impairs proliferation and clonogenic capacity by prolonging both interphase and mitosis, leading to slowed cell cycle progression. The impact of CYCLON on cellular fitness has been consistently observed across cancer models, reinforcing its essential role in the regulation of nucleolar biology.

cancer biology↗

bioRxiv2025

High-resolution mapping of human RNA polymerase III reveals transcription termination as a rate-limiting step

Transfer RNA (tRNA) molecules play a central role in the flow of genetic information, translating nucleic acid sequences into the functional protein portfolio of an organism. Despite the high abundance of tRNAs in cells, studying their biology remains challenging due to the repetitive nature of genomic sequences and the numerous modifications of mature molecules. tRNA genes (tDNAs) are transcribed exclusively by RNA polymerase III (RNAPIII), which has been recently linked to rare genetic disorders but also longevity. Here, we present the first mapping of actively transcribing RNAPIII in the human, using K562 cell line and UV-crosslinking followed by stringent purification. Our data reveals high variability in expression across tDNAs, common occurrences of transcriptional read-through, and unique transcription dynamics across transcription units, with the lowest kinetics associated with transcription termination. Further analysis revealed that release of the nascent transcript is a critical step in tRNA transcription. Unexpectedly, shorter terminators promote more efficient termination, which becomes the rate-limiting step for human RNAPIII transcription of highly expressed tDNAs. Our dataset provides insights into actively transcribing RNAPIII dynamics in human cells, allowing for a comprehensive analysis of genomic loci transcribed by RNAPIII. Our high resolution data and resulting kinetic information reveal that human RNAPIII contradicts the paradigm that initial steps of transcription are the main determinants of its output. This is significant as RNAPIII is commonly used to transcribe synthetic RNA constructs, such as short hairpin RNAs or guide RNAs used in gene editing technologies. With our study we offer a functional framework for the analysis of RNAPIII activity, addressing the need for comprehensive understanding of RNAPIII transcription fueled by a growing number of described genetic disorders caused by mutations in this enzyme.

molecular biology↗