Co-transcriptional splicing facilitates transcription of gigantic genes

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing. A small number of genes in our genome are characterized by excessively large introns, sometimes exceeding megabases. It remains poorly understood why such large introns exist or how they are processed during gene expression. By examining genes on Drosophila Y chromosome that contain gigantic introns, it is shown that the transcripts of these genes are co-transcriptionally spliced, and co-transcriptional splicing of these genes is critical for proper gene expression. Perturbation of splicing led to attenuation of transcription specifically in genes with gigantic introns, but not genes with average-sized introns. Whereas transcripts of genes with gigantic introns normally form separate nuclear domains, splicing perturbation led to intermingling of these domains, leading to a proposal that co-transcriptional splicing may function to keep transcript length short to avoid entanglement of transcripts, which may stall the progression of RNA polymerase II. Taken together, these results reveal a novel function of co-transcriptional splicing that specifically impacts the expression of genes with gigantic introns.