A genome-wide functional analysis of conserved intronic regions reveals essential roles for speckle-associated retained introns.

Hundreds of human introns harbor extended regions of high evolutionary conservation that have not been previously characterized. A survey of these sequences reveals that they are associated with intron retention and enriched in genes that function in RNA processing, chromatin remodeling and neuronal biology. Using a dual CRISPR-Cas editing approach, we targeted 2,600 of these regions for deletion and observed that a subset of these perturbations affects cell growth. Many of these "fitness" sequences affect intron retention and expression levels of their host genes. Deletions in nuclear speckle-associated retained introns in the FNBP4 and DDX5 genes further cause downstream effects on cell growth-related genes and intron retention, respectively. The intronic deletion in DDX5 additionally results in the accumulation of R-loops overlapping retained introns of speckle-proximal genes. Overall, the results highlight critical and multifaceted roles of highly conserved intronic sequences in the control of gene regulation, R-loop resolution, and cell growth.