Biology subjects

Turowski, T. W.

Publications and source records attributed to Turowski, T. W..

7 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↗

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↗

bioRxiv2023

Transcription termination by RNA polymerase I

Transcription elongation is stochastic and driven by a Brownian ratchet mechanism, making it subject to changes in velocity. However, on regions occupied by multiple polymerases, notably the rDNA, DNA rotation plus torsion constrain polymerase molecules to proceed at the same rate generating "torsional entrainment". We report that release of entrainment, by co-transcriptional 3-end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5-exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by RNAPI subunit Rpa12. These activities were reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites, are targeted by both the exosome cofactor TRAMP and Rat1, promoting termination. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Biophysical modeling supported substantial ([~]10%) premature termination. HighlightsNascent pre-rRNA 3 cleavage promotes RNAPI deceleration and termination RNAPI undergoes early, start-site proximal termination at sites of polymerase pausing Biophysical modeling indicates [~]10% early termination - or [~]100 events per minute Model presented for overall organization of pre-rRNA transcription

molecular biology↗

bioRxiv2022

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the [~]190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs. HighlightsO_LIDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae. C_LIO_LIIncreasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy. C_LIO_LIThe tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain. C_LIO_LIComprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology. C_LI

synthetic biology↗

bioRxiv2021

A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first processing-specific human disorder to be described. HighlightsO_LIMutations in RMRP lncRNA impair pre-rRNA processing and T cell activation C_LIO_LIPatient derived fibroblasts show impaired pre-rRNA processing C_LIO_LICells with the most common disease-linked mutation have specific processing defects C_LIO_LICytoplasmic ribosomes and intact RNase MRP complexes are also reduced in these cells C_LI

cell biology↗

bioRxiv2020

Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His8, the C-terminus with His8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

cell biology↗

bioRxiv2020

Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5 bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/969709v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@f3f9fcorg.highwire.dtl.DTLVardef@1a133c8org.highwire.dtl.DTLVardef@4eae0dorg.highwire.dtl.DTLVardef@18502b8_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSStructures in the nascent RNA correlate with rapid elongation by RNAPI in vivo Stable RNA structures limit RNAPI backtracking in vitro GC content in the transcription bubble tunes transcription elongation rate Nascent transcript folding modulates dynamics of all three RNAPs in vivo

molecular biology↗