Efficient RNA polymerase II pause release requires U2 snRNP function

Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery

Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells

Involvement of N4BP2L1, PLEKHA4, and BEGAIN genes in breast cancer and muscle cell development

Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 (N4BP2L1), pleckstrin homology domain-containing family A member 4 (PLEKHA4), and brain-enriched guanylate kinase-associated protein (BEGAIN) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development