Milevskiy, M. J. G. | Search bioRxiv

bioRxiv2025

Incorporating exon-exon junction reads enhances differential splicing detection

MotivationRNA sequencing (RNA-seq) is a gold standard technology for studying gene and transcript expression. Different transcripts from the same gene are usually determined by varying combinations of exons within the gene, formed by splicing events. One method of studying differential alternative splicing between groups in short-read RNA-seq experiments is through differential exon usage (DEU) analyses, which use exon-level read counts along with downstream statistical testing strategies. Popular pipelines for these analyses include Rsubread for read alignment and exon-level count quantification, and edgeR and limma for statistical testing of differential splicing. However, the standard Rsubread::featureCounts count summarization method does not consider exon-junction information, which may reduce the statistical power in differential splicing analyses. ResultsA new feature quantification approach is proposed to incorporate both exon and exon-junction reads into the popular Rsubread-edgeR and Rsubread-limma frameworks for detecting differential splicing events. This new Differential Exon-Junction Usage (DEJU) analysis pipeline demonstrates increased statistical power compared to existing popular methods while effectively controlling the false discovery rate. Availability and implementationData and code to reproduce the results shown in this article are available from https://github.com/TamPham271299/DEJU.

bioinformatics↗

bioRxiv2021

The minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers

Minor splicing is a second splicing system required for the correct expression of [~]700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38. Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3. We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

cancer biology↗