Biology subjects

Mackowiak, S. D.

Publications and source records attributed to Mackowiak, S. D..

3 recordsLinked to original sources

bioRxiv2021

SSAM-lite: a light-weight web app for rapid analysis of spatially resolved transcriptomics data

1The combination of a cells transcriptional profile and location defines its function in a spatial context. Spatially resolved transcriptomics (SRT) has emerged as the assay of choice for characterizing cells in situ. SRT methods can resolve gene expression up to single-molecule resolution. A particular computational problem with single-molecule SRT methods is the correct aggregation of mRNA molecules into cells. Traditionally, aggregating mRNA molecules into cell-based features begins with the identification of cells via segmentation of the nucleus or the cell membrane. However, recently a number of cell-segmentation-free approaches have emerged. While these methods have been demonstrated to be more performant than segmentation-based approaches, they are still not easily accessible since they require specialized knowledge of programming languages and access to large computational resources. Here we present SSAM-lite, a tool that provides an easy-to-use graphical interface to perform rapid and segmentation-free cell-typing of SRT data in a web browser. SSAM-lite runs locally and does not require computational experts or specialized hardware. Analysis of a tissue slice of the mouse somatosensory cortex took less than a minute on a laptop with modest hardware. Parameters can interactively be optimized on small portions of the data before the entire tissue image is analyzed. A server version of SSAM-lite can be run completely offline using local infrastructure. Overall, SSAM-lite is portable, lightweight, and easy to use, thus enabling a broad audience to investigate and analyze single-molecule SRT data. Availability and ImplementationSSAM-lite is an open-source browser-based web application with source code freely available on Github via https://github.com/HiDiHlabs/ssam-lite. Stable releases can be accessed via https://ssam-lite.bihealth.org and https://ssam-lite.netlify.app, and developmental releases can be accessed via https://dev--ssam-lite.netlify.app. The source code for a locally deployable server version, SSAM-lite-server, is available on GitHub via https://github.com/HiDiHlabs/ssam-lite-server. Both versions require a modern browser with JavaScript and WebGL support. Detailed user guides and documentation can be found at https://ssam-lite.readthedocs.io.

bioinformatics↗

bioRxiv2019

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure. Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome.

molecular biology↗

bioRxiv2019

Nuclear gene proximity and protein interactions shape transcript covariances in mammalian single cells

Single-cell RNA sequencing studies into gene co-expression patterns could yield important new regulatory and functional insights, but have so far been limited by the confounding effects of cell differentiation and the cell cycle. We apply a tailored experimental design that eliminates these confounders, and report >80,000 intrinsically covarying gene pairs in mouse embryonic stem cells. These covariances form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariances, and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariances. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex stoichiometry. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes on the same or even distinct chromosomes also provides substantial functional regulation in mammalian single cells.

systems biology↗