Biology subjects

Czapiewski, R.

Publications and source records attributed to Czapiewski, R..

6 recordsLinked to original sources

bioRxiv2026

Cross-chemistry single-nucleus RNA-seq identifies gene length and CpG-island promoters as determinants of transcriptional noise.

Cell-to-cell transcriptional heterogeneity, or noise, is an intrinsic property of the transcriptome with implications for development, disease progression, and aging. Bulk RNA-seq masks this variability by averaging gene expression across cells, whereas single-cell RNA sequencing (scRNA-seq) resolves it. Nevertheless, separating biological noise from technical variance remains challenging, particularly across platforms with different chemistries. We benchmarked two widely adopted technologies, Evercode WT (SPLiT-seq, Parse Biosciences) and Chromium (10x Genomics), on human lymphoblastoid nuclei. Evercode WT achieved targeted sequencing depth and nuclei number far more reliably, and its random-hexamer priming yielded more intronic reads and non-coding RNA genes; Chromium recovered more cells and detected polyadenylated transcripts and cell-line markers more sensitively. Despite these opposing biases, the platforms showed comparable gene detection and strongly correlated expression profiles. Using datasets from both platforms, we defined a noise metric detrended from mean expression and showed that per-gene estimates were reproducible across chemistries. Noise was lower in G2M than in G1 and was most strongly associated with gene length rather than exonic length. Expression of genes with CpG-island promoters was less variable than that of those without. This study establishes a platform-independent basis for quantifying transcriptional noise and a framework for selecting an appropriate scRNA-seq platform.

genomics↗

bioRxiv2024

Nuclear RNA forms an interconnected network of transcription-dependent and tunable microgels

Living cells are organised through active, non-equilibrium processes, yet the physical principles governing their material states remain unclear. The cell nucleus has been described as either fluid-like or solid-like, but these behaviours have not been reconciled within a single framework. Here we show that nascent RNA and the multivalent RNA-binding protein SAF-A assemble into a nucleus-spanning microgel network maintained near the sol-gel transition. This active network exhibits scale- and time-dependent viscoelasticity and naturally reconciles solid-like and fluid-like responses. By integrating super-resolution imaging with mechanistic polymer modelling, we identify RNA production and degradation as control parameters that tune network connectivity. The model predicts enhanced susceptibility to modest perturbations near criticality, which we confirm experimentally by shifting the system between sol-like and gel-like regimes. Measurements of molecular transport, combined with simulations, reveal size-dependent diffusion and transient trapping within gel pores, establishing direct physical consequences of the near-critical state. Our results identify RNA metabolism as an energy-driven regulator of nuclear material properties and position the nucleus as a living example of an active microgel poised near criticality.

molecular biology↗

bioRxiv2022

Metabolic, Fibrotic, and Splicing Pathways Are All Altered in Emery-Dreifuss Muscular Dystrophy Spectrum Patients to Differing Degrees

Emery-Dreifuss muscular dystrophy (EDMD) is a genetically and clinically variable disorder. Previous attempts to use gene expression changes find its pathomechanism were unavailing, so we here engaged a functional pathway analysis. RNA-Seq was performed on cells from 10 patients diagnosed with an EDMD spectrum disease with different mutations in 7 genes. Upon comparing to controls, the pathway analysis revealed that multiple genes involved in fibrosis, metabolism, myogenic signaling, and splicing were affected in all patients. Splice variant analysis revealed alterations of muscle-specific variants for several important muscle genes. Deeper analysis of metabolic pathways revealed a reduction in glycolytic and oxidative metabolism and reduced numbers of mitochondria across a larger set of 14 EDMD patients and 7 controls. Intriguingly, the gene expression signatures segregated the patients into three subgroups whose distinctions could potentially relate to differences in clinical presentation. Finally, differential expression analysis of miRNAs changing in the patients similarly highlighted fibrosis, metabolism, and myogenic signaling pathways. This pathway approach revealed a clear EDMD signature that can both be used as the basis for establishing a biomarker panel specific to EDMD and direct further investigation into its pathomechanism. Furthermore, the segregation of specific gene changes into three distinct categories that appear to correlate with clinical presentation may be developed into prognostic biomarkers, though this will first require their testing in a wider set of patients with more clinical information.

genetics↗

bioRxiv2021

Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy

Little is known about the proteins that direct the highly conserved patterns of spatial genome organisation in fat. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope protein Tmem120a disrupts fat genome organisation, thus causing a novel lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a-/- mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifests upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein laminA mutations. Our data suggest TMEM120A may mediate/instigate novel categories of adipose tissue dysfunction across the adiposity spectrum and provide a new miRNA-based mechanism possibly driving the unexplained muscle hypertrophy in human lipodystrophy.

cell biology↗

bioRxiv2019

A Multistage Sequencing Strategy Pinpoints Many Novel and Candidate Disease Alleles for Orphan Disease Emery-Dreifuss Muscular Dystrophy and Supports Gene Misregulation as its Pathomechanism

Limitations of genome-wide approaches for genetically-heterogenous orphan diseases led us to develop a new approach to identify novel Emery-Dreifuss muscular dystrophy (EDMD) candidate genes. We generated a primer library to genes: (I) linked to EDMD, (II) mutated in related muscular dystrophies, (III) highlighted from limited exome sequencing, (IV) encoding muscle-specific nuclear membrane proteins. Sequencing 56 unlinked EDMD patients yielded confirmed or strong candidate alleles from all categories, accounting for most remaining unlinked patients. Known functions of newly-linked genes argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction through connectivity of candidates from the nuclear envelope to the plasma membrane.

genetics↗

bioRxiv2016

Host Vesicle Fusion Proteins VAPB, Rab11b and Rab18 Contribute to HSV-1 Infectivity by Facilitating Egress through the Nuclear Membrane

The herpesvirus process of primary envelopment and de-envelopment as viral particles exit the nucleus has been for many years one of the least understood steps in the virus life cycle. Though viral proteins such as pUL31, pUL34, pUS3 and others are clearly important, these are likely insufficient for efficient fusion with the nuclear membrane. We postulated that host nuclear membrane proteins involved in virus nuclear egress would move from the inner to outer nuclear membranes due to membrane fusion events in primary envelopment and de-envelopment and then diffuse into the endoplasmic reticulum. Membrane fractions were prepared enriched in the nuclear envelope or the endoplasmic reticulum with and without HSV-1 infection and analyzed by mass spectrometry, revealing several vesicle fusion proteins as candidates in the viral nuclear egress pathway. Knockdown of three of these, VAPB, Rab11b, and Rab18, significantly reduced titers of released virus while yielding nuclear accumulation of encapsidated particles. Antibody staining revealed that VAPB visually accumulates in the inner nuclear membrane during HSV-1 infection. VAPB also co-localizes at early time points with the viral pUL34 protein known to be involved in nuclear egress. Most strikingly, VAPB was also observed on HSV-1 virus particles by immunogold labelling electron microscopy. Thus, these data reveal several new host cell vesicle fusion proteins involved in viral nuclear egress.\n\nAuthor SummaryHuman herpesviruses are associated with common human diseases such as chicken pox, shingles and mononucleosis and infect a wide range of animals making them economically important pathogens for livestock. Herpes simplex virus 1 (HSV-1) is most commonly associated with cold sores, but is also the leading cause of blindness by infection in the Western world. All herpesviruses share many aspects of infection. As large nuclear replicating dsDNA viruses with capsid sizes too large to use the nuclear pores to exit the nucleus, they have evolved a complex mechanism for envelopment and de-envelopment of primary herpesvirus particles, but this critical step in the virus lifecycle remains poorly understood. We have identified several host cell vesicle fusion proteins, VAPB, Rab11b and Rab18 that appear to contribute to this step in the HSV-1 life cycle. VAPB accumulates at the nuclear envelope with the HSV-1 pUL34 protein important for viral nuclear egress. Knockdown of any of these vesicle fusion proteins reduces viral titers, further arguing that they are important for nuclear egress. As there appears to be a specific subset of vesicle fusion proteins involved in viral egress, they could possibly represent novel targets for therapeutic interventions.

cell biology↗