Biology subjects

Paulson, J.

Publications and source records attributed to Paulson, J..

8 recordsLinked to original sources

bioRxiv2025

A Yeast-Based High-Throughput Screening Platform for the Discovery of Novel pre-mRNA Splicing Modulators

Pre-mRNA splicing is a crucial process in eukaryotic gene expression, and splicing dysregulation has been linked to various diseases. However, very few small molecules have been discovered that can modulate spliced mRNA formation or inhibit the splicing machinery itself. This study presents a novel high-throughput screening (HTS) platform for identifying compounds that modulate splicing. Our platform comprises a two-tiered screening approach: a primary screen measuring growth inhibition in sensitized in S. cerevisiae (yeast) strains and a secondary screen that relies on production of a fluorescent protein as a readout for splicing inhibition. Using this approach, we identified 4 small molecules that cause accumulation of unspliced pre-mRNA in vivo in yeast. In addition, cancer cells expressing a myelodysplastic syndrome-associated splicing factor mutation (SRSF2P95H) are more sensitive to one of these compounds than those expressing the wild-type version of the protein. Transcriptome analyses showed that this compound causes widespread changes in gene expression in sensitive SRSF2P95H-expressing cells. Our results demonstrate the utility of using a yeast-based HTS to identify compounds capable of changing pre-mRNA splicing outcomes.

biochemistry↗

bioRxiv2025

A time-resolved atlas of histone modifications during mitotic entry

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms--particularly those involving phosphorylation and deacetylation of specific histone residues--have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

cell biology↗

bioRxiv2025

Enhanced fluorescence lifetime imaging microscopy denoising via principal component analysis

Fluorescence Lifetime Imaging Microscopy (FLIM) quantifies the autofluorescence lifetime to measure cellular metabolism, therapeutic efficacy, and disease progression. These dynamic processes are intrinsically heterogeneous, increasing the complexity of the signal analysis. Often noise reduction strategies that combine thresholding and non-selective data smoothing filters are applied. These can result in error introduction and data loss. To mitigate these issues, we develop noise-corrected principal component analysis (NC-PCA). This approach isolates the signal of interest by selectively identifying and removing the noise. To validate NC-PCA, a secondary analysis of FLIM images of patient-derived colorectal cancer organoids exposed to a range of therapeutics was performed. First, we demonstrate that NC-PCA decreases the uncertainty up to 4-fold in comparison to conventional analysis with no data loss. Then, using a merged data set, we show that NC-PCA, unlike conventional methods, identifies multiple metabolic states. Thus, NC-PCA provides an enabling tool to advance FLIM analysis across fields.

bioengineering↗

bioRxiv2025

Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

Mild replication interference is a consolidated strategy for cancer chemotherapy. Tolerance to mild replication stress (RS) relies on active fork slowing, mediated by transient fork reversal and RECQ1-assisted restart, and modulated by PARP1 and nuclear architectural components via yet-elusive mechanisms. We combined acute protein inactivation with cell biology and single-molecule approaches to investigate the role of Lamin A/C upon mild RS. We found that Lamin A/C dynamically interacts with replication factories throughout the nucleus and, together with its nucleoplasmic partner LAP2, is required to induce active fork slowing and maintain chromosome stability upon mild genotoxic treatments. Inactivating nucleoplasmic Lamin A/C reduces poly-ADP-ribosylation (PAR) levels at nascent DNA, triggering deregulated RECQ1-mediated restart of reversed forks. Moreover, we found that the heterochromatin mark H3K9me3, previously reported at stalled forks, also accumulates in response to mild RS. H3K9me3 accumulation requires Lamin A/C, which prevents its premature removal by the histone demethylase JMJD1A/KDM3A. H3K9me3 loss per se phenocopies Lamin A/C inactivation, reducing PAR levels and deregulating RECQ1 activity at forks. Hence, nucleoplasmic Lamin A/C, H3K9me3 and PARylation levels are crucial, mechanistically-linked modulators of fork slowing, remodelling and restart upon mild RS, with important implications for chemotherapy response and Lamin A/C deregulation in human disease.

molecular biology↗

bioRxiv2025

IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

Intraflagellar transport (IFT) proteins are required for the assembly and function of primary cilia. They also regulate non-ciliary polarized vesicular traffic, such as T cell receptor recycling. We recently reported that lymphatic endothelial cells assemble primary cilia and express IFT proteins. Here, we report that IFT20 regulates vascular endothelial cadherin (VE-cadherin) localization at adherens junctions. IFT20 deletion caused discontinuous, button-like interendothelial junctions. This resulted in excessive lymphangiogenesis and impaired lymph drainage in mice. In vitro, VEGF-C treatment of IFT20 KD primary human dermal lymphatic endothelial cells caused accumulation of VE-cadherin in RAB5+ endosomes and enhanced and sustained VEGFR-3 signaling. Our findings are consistent with a model in which IFT20 promotes recycling of VE-cadherin to the adherens junction where it sequesters VEGFR-3 at the cell surface, thereby limiting pro-lymphangiogenic signaling. In the absence of IFT20, intercellular junctions are destabilized, pro-lymphangiogenic VEGFR-3 signaling is enhanced, and lymph transport is impaired by intracellular sequestration of VE-cadherin. This study elucidates the function of an IFT protein in lymphatic endothelial cells and provides mechanistic insight into the processes that regulate lymphatic endothelial cell-cell junctions and lymphangiogenic signaling.

cell biology↗

bioRxiv2020

Impact of Cancer-Associated Mutations in Hsh155/SF3b1 HEAT Repeats 9-12 on pre-mRNA Splicing in Saccharomyces cerevisiae

Mutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1-mutant disease phenotypes may have a complex origin on the spliceosome.

biochemistry↗

bioRxiv2018

metagenomeFeatures: An R package for working with 16S rRNA reference databases and marker-gene survey feature data.

We developed the metagenomeFeatures R Bioconductor package along with annotation packages for the three primary 16S rRNA databases (Greengenes, RDP, and SILVA) to facilitate working with 16S rRNA sequence databases and marker-gene survey feature data. The metagenomeFeatures package defines two classes, MgDb for working with 16S rRNA sequence databases, and mgFeatures for working with marker-gene survey feature data. The associated annotation packages provide a consistent interface to the different 16S rRNA databases facilitating database comparison and exploration. The mgFeatures represents a crucial step in the development of a common data structure for working with 16S marker-gene survey data in R.\n\nAvailabilityhttps://bioconductor.org/packages/release/bioc/html/metagenomeFeatures.html\n\nContactnolson@nist.gov

bioinformatics↗

bioRxiv2016

Tissue-aware RNA-Seq processing and normalization for heterogeneous and sparse data

Although ultrahigh-throughput RNA-Sequencing has become the dominant technology for genome-wide transcriptional profiling, the vast majority of RNA-Seq studies typically profile only tens of samples, and most analytical pipelines are optimized for these smaller studies. However, projects are generating ever-larger data sets comprising RNA-Seq data from hundreds or thousands of samples, often collected at multiple centers and from diverse tissues. These complex data sets present significant analytical challenges due to batch and tissue effects, but provide the opportunity to revisit the assumptions and methods that we use to preprocess, normalize, and filter RNA-Seq data - critical first steps for any subsequent analysis. We find analysis of large RNA-Seq data sets requires both careful quality control and that one account for sparsity due to the heterogeneity intrinsic in multi-group studies. An R package instantiating our method for large-scale RNA-Seq normalization and preprocessing, YARN, is available at bioconductor.org/packages/yarn.\n\nHighlightsO_LIOverview of assumptions used in preprocessing and normalization\nC_LIO_LIPipeline for preprocessing, quality control, and normalization of large heterogeneous data\nC_LIO_LIA Bioconductor package for the YARN pipeline and easy manipulation of count data\nC_LIO_LIPreprocessed GTEx data set using the YARN pipeline available as a resource\nC_LI

bioinformatics↗