View Program - 63rd Annual Drosophila Research Conference

Biomolecular condensates are membrane-less compartments formed by the biophysical phenomena of phase transition mainly through Protein-RNA interactions. Both transcriptional complexes and RNA processing units are reported to form such condensates. Several transcription factors and RNA binding proteins that contain IDR (Intrinsically Disordered Regions) have been reported to have a role in phase transition, and thus it is interesting to study how RNA-protein complexes can potentially form and dissolve such condensates. In the present study, we have explored the potential of DNA binding transcription factor protein CLAMP (Chromatin Linked Adaptor for MSL Proteins), which contains IDR, in Drosophila Melanogaster as a protein that can form bio-condensates. CLAMP recruits the MSL (Male Specific Lethal) Complex, an RNA-protein complex consisting of long non-coding RNAs roX1 and roX2 along with five other proteins, to the male X-chromosome resulting in the transcriptional upregulation of the male X-chromosome in a process known as dosage compensation. Therefore in this project, we studied the interaction between CLAMP and roX2 RNA to test the hypothesis that these two components might drive the process of dosage compensation via phase separation. Also, CLAMP regulates sex-specific splicing, a process regulated by ribonuceloprotein complexes called spliceosomes that coordinate co-transcriptional RNA processing via the mechanisim of alternative splicing. In flies and mammals many RNA-binding proteins are part of the alternative splicing spliceosome complex along with non-coding RNA components. In Drosophila, CLAMP binds to several of such RNA binding proteins that are reported to interact with a long non-coding RNA hsrω-n. Thus, we also explored CLAMP’s binding with hsrω-n to test CLAMP’s potential as a legitimate player in phase transition and formation of biomolecular splicing condensates. Using electron mobility shift assays (EMSA) we found that CLAMP and the CLAMP domain that contains the IDR physically bind to both roX2 and hsrω-n non-coding RNAs, reinforcing our hypothesis that CLAMP is a bifunctional protein with DNA/RNA binding capacity and has the potential of regulating transcription and co-transcriptional RNA processing by manipulating bio-physical properties of RNA-protein complexes.

Protein-RNA interaction drives co-transcriptional regulation and RNA processing