The emergence of introns was a significant evolutionary leap that is a major distinguishing feature between prokaryotic and eukaryotic genomes. While historically introns were regarded merely as the sequences that are removed to produce spliced transcripts encoding functional products, increasingly data suggests that introns play important roles in the regulation of gene expression. Here, we use an intron-centric lens to review the role of introns in eukaryotic gene expression. First, we focus on intron architecture and how it may influence mechanisms of splicing. Second, we focus on the implications of spliceosomal snRNAs and their variants on intron splicing. Finally, we discuss how the presence of introns and the need to splice them influences transcription regulation. Despite the abundance of introns in the eukaryotic genome and their emerging role regulating gene expression, a lot remains unexplored. Therefore, here we refer to introns as the “dark matter” of the eukaryotic genome and discuss some of the outstanding questions in the field.

Developmental insults causing limb progenitor cell cycle defects or death tend to produce micromelic limbs with maintained segmentation. This suggests that the developing limb is plastic yet has a bias towards proximo-distal patterning. Here we use a minor spliceosome-deficient (U11-null) mouse forelimb, which has severe micromelia yet maintains proximo-distal segmentation, to decipher the mechanism(s) underlying this form of developmental robustness. We show that U11 loss triggers transcriptomic stalling upon spatially heterogenous mis-splicing of minor intron-containing genes. Through spatial transcriptomics, we detected a failure of the U11-null forelimb to separate its distal patterning program from its proximal differentiation program, which was supported by single-cell RNAseq-determined developmental delay of U11-null chondroprogenitors. Ultimately, these molecular and cellular deficits culminated in perturbed chondrogenesis, myogenesis, and axonogenesis. Taken together, we suggest that, upon sensing depletion of progenitors, the limb halts its transcriptional networks to pause its cellular trajectory, affording time to restructure its developmental program.