Synergistic DNA and RNA binding of the Hox transcription factor Ultrabithorax coordinates splicing and shapes in vivo homeotic functions

The dual interaction of many transcription factors (TFs) with both DNA and RNA is an underexplored issue that could fundamentally reshape our understanding of gene regulation. We address this central issue by investigating the RNA binding activity of the Drosophila Hox TF Ultrabithorax (Ubx) in alternative splicing and morphogenesis. Relying on molecular and genetic interactions, we uncover a homodimerization-dependent mechanism by which Ubx regulates splicing. Notably, this mechanism enables the decoupling of Ubx-DNA and -RNA binding activity in splicing. We identify a critical residue for Ubx-RNA binding and demonstrate the essential role of Ubx-RNA binding ability for its homeotic functions. Overall, we uncover a unique mechanism for Ubx-mediated splicing and underscore the critical contribution of synergistic DNA/RNA binding for its morphogenetic functions. These findings advance our understanding of co-transcriptional regulation and highlight the significance of TF-DNA/RNA synergistic function in shaping gene regulatory networks in living organisms. Total RNAs were extracted from three independent replicates from Drosophila S2R+ cells expressing 150ng of GFPnls, myc-UbxWT or myc-UbxN51A or co-expressing 150+150ng of myc-Ubx(WT+N51A) or myc-Ubx(WT+N51A-K58A) (Gal4-UAS, actin promoter) using Qiagen RNA extraction kit (RNeasy). RNA quality and integrity were assessed using BioAnalyzer 2100TM (Agilent Technologies). Material handling and mRNA-Seq directional libraries were performed by Novogene with poly(A) selection according to the manufacturer's protocol (Illumina). Sequencing was performed with NovaSeq X Plus Series (8 G raw data per sample) with a read length of 150 bp and paired-end reads. Replicates were validated by FastQC report, and Principal Component Analysis (PCA) was performed to verify the sample similarity. The data were generated and uploaded on the Gene Expression Omnibus according to the ENCODE guideline (see Data Availability section).