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Muscleblind-like splicing regulators (MBNLs) activate or repress the inclusion of alternative splicing (AS) events, enabling the developmental transition of fetal mRNA splicing isoforms to their adult forms. Herein, we sought to elaborate the mechanism by which MBNLs mediate AS related to biological processes. We evaluated the functional role of DEAD-box (DDX) RNA helicases, DDX5 and DDX17 in MBNL-dependent AS regulation. Whole-transcriptome analysis and validation approaches revealed a handful of MBNLs-dependent AS events to be affected by DDX5 and DDX17 in mostly an opposite manner. The opposite expression patterns of these two groups of factors during muscle development and coordination of fetal-to-adult splicing transition indicate the importance of these proteins at early stages of development. The identified pathways of how the helicases modulate MBNL splicing activity include DDX5 and DDX17-dependent changes in the ratio of MBNL splicing isoforms and most likely changes in accessibility of MBNL-binding sites. Another pathway involves the mode of action of the helicases independent of MBNL activity. These findings lead to a deeper understanding of the network of interdependencies between RNA-binding proteins and constitute a valuable element in the discussion on developmental homeostasis and pathological states in which the studied protein factors play a significant role., (© 2023. The Author(s).)

Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type-specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution.

Estrogen and androgen receptors (ER and AR) play key roles in breast and prostate cancers, respectively, where they regulate the transcription of large arrays of genes. The activities of ER and AR are controlled by large networks of protein kinases and transcriptional coregulators, including Ddx5 and its highly related paralog Ddx17. The Ddx5 and Ddx17 RNA helicases are also splicing regulators. Here, we report that Ddx5 and Ddx17 are master regulators of the estrogen- and androgen-signaling pathways by controlling transcription and splicing both upstream and downstream of the receptors. First, Ddx5 and Ddx17 are required downstream of ER and AR for the transcriptional and splicing regulation of a large number of steroid hormone target genes. Second, Ddx5 and Ddx17 act upstream of ER and AR by controlling the expression, at the splicing level, of several key regulators of ER and AR activities. Of particular interest, we demonstrate that Ddx5 and Ddx17 control alternative splicing of the GSK3β kinase, which impacts on both ER and AR protein stability. We also provide a freely available online resource which gives information regarding splicing variants of genes involved in the estrogen- and androgen-signaling pathways.