Keywords: g. alternative splicing; n. other (Co-transcriptional splicing)
Post-transcriptional processes of alternative splicing of RNA are essential in gene expression, critical for transcript diversity, eventually leading to cellular differentiation and specification. Alternative splicing is initiated during the initial embryonic stages when the key gene expression regulators are maternally deposited transcription factors (TFs). Yet, it is unknown how transcription factors (TFs) work with splicing factors to regulate alternative splicing. Also, how maternal factors shape the early embryonic transcriptome in a sex-specific manner is not well understood. Here, we show that the maternally deposited transcription factor CLAMP (Chromatin-linked adaptor for MSL proteins) is essential for sex-specific alternative splicing in early embryos of both sexes. We found that CLAMP binds along with the gene bodies of sex-specifically spliced genes in both sexes and differentially binds to RNA-binding protein components of the spliceosome complex in males and females. In females, CLAMP also modulates the chromatin environment at the sxl gene, the master regulator of sex-determination, affecting its splicing and thus downstream female-specific splicing events. In males, CLAMP influences the distribution of RNA-helicase Maleless (a component of spliceosome complex and Male Sex-lethal (MSL) complex in males) on chromatin, affecting male sex-specific splicing. We show that CLAMP is an RNA binding protein through iCLIP (individual-nucleotide resolution Cross-Linking and Immunoprecipitation) and RNA gel shift experiments and is important for the dynamics of formation of nuclear speckles of ribonucleoprotein origin that constitute alternative splicing complexes. Using CLAMP mutants, we demonstrate that the prion-like domain (PrLD) of CLAMP inhibits aggregation of RNA-binding splicing factors that renders them non-functional and is essential for survival. We hypothesize that the PrLD domain alters the phase transition properties of CLAMP. Thus, we define a new function for a maternally deposited TF in regulating sex-specific alternative splicing via interaction with chromatin and RNA-binding proteins, which influences the distribution of RNA-binding proteins on chromatin and between protein complexes. As a part of this study, we have also developed a computational pipeline called time2splice for the identification of alternative splicing events which change over time and between sexes.