Farcas, A. | bioRxiv Science

bioRxiv2025

PRMT activity promotes global 3' UTR shortening in proliferating cells

Protein methyltransferase (PRMT)-catalysed arginine methylation is a widespread post-translational modification that regulates numerous RNA-binding proteins and frequently becomes dysregulated in cancer. While PRMT inhibitors have shown promise as an anti-cancer strategy, greater understanding of the downstream pathways linking arginine methylation to tumour-promoting phenotypes is needed to improve patient stratification and develop more effective therapeutic approaches. Here, we reveal arginine methylation as a critical regulator of alternative polyadenylation (APA) patterns that are fundamental to tumour progression. 3' RNA-sequencing assays uncover a rapid and global shift toward longer 3' UTR isoforms upon dual (symmetric and asymmetric) methylation (DMAi), impacting a broad range of cellular proliferation and signalling genes. Arginine methylation is required for sustaining proximal poly(A) site usage under high proliferative demand, as DMAi treatment blocks use of such sites in activated T cells, various cancer cell lines and patient-derived lung organoids. DMAi also counteracts the 3' UTR shortening caused by reduced CFIM25 expression, which normally promotes oncogenic isoforms. DMAi treatment affects APA in many of the same mRNAs as impaired cleavage and polyadenylation activity, and these mRNAs contain characteristic signatures such as high GC-content and long 3 UTRs. This systematic impact of PRMT activity on APA regulation broadens the potential utility of PRMT inhibitors as therapeutic agents for both cancer and immune-related diseases.

molecular biology↗

bioRxiv2024

10x GENOMICS GENE EXPRESSION FLEX IS A POWERFUL TOOL FOR SINGLE-CELL TRANSCRIPTOMICS OF XENOGRAFT MODELS

The 10x Genomics Gene Expression Flex protocol allows profiling of fixed or frozen material, greatly simplifying the logistics of sample collection, storage and transfer prior to single -cell sequencing. The method makes single-cell transcriptomics possible for existing fresh-frozen or FFPE tissue samples, but also facilitates the logistics of the sampling process, allowing instant preservation of samples. The technology relies on species-specific probes available for human and mouse. Nevertheless, processing of patient-derived (PDX) or cell line (CDX) xenografts, which contain mixed human and mouse cells, is currently not supported by this protocol due to the high degree of homology between the probe sets. Here we show that it is feasible to simultaneously profile populations containing both human and mouse cells by mixing the transcriptome probe sets of both species. Cellranger outputs a count table for each of the species allowing evaluation of the performance of the different probe sets. Cross-reactive probes are greatly outperformed by the specific probe hybridizations leading to a clear difference in the recovery of UMIs and unique genes per cell. Furthermore, we developed a pipeline that removes cross-reactive signal from the data and provides species-specific count tables for further downstream analysis. Hence, the 10x Genomics Gene Expression Flex protocol can be used to process xenograft samples without the need for separation of human and mouse cells by flow sorting and allows analysis of the human and mouse single-cell transcriptome from each sample. We anticipate it will be increasingly used for single-cell sequencing of cancer cell line and patient-derived xenografts, facilitating the preservation of the samples and allowing the interrogation of both the (human) xenograft and the (mouse) tumor microenvironment at single-cell resolution.

genomics↗