Nested introns in an intron: Evidence of multi-step splicing of a large intron in the human dystrophin pre-mRNA

The mechanisms by which huge human introns are spliced out precisely are poorly understood. We analyzed large intron 7 (110 199 nucleotides) generated from the human dystrophin (DMD) pre-mRNA by RT-PCR. We identified branching between the authentic 5′ splice site and the branch point; however, the sequences far from the branch site were not detectable. This RT-PCR product was resistant to exoribonuclease (RNase R) digestion, suggesting that the detected lariat intron has a closed loop structure but contains gaps in its sequence. Transient and concomitant generation of at least two branched fragments from nested introns within large intron 7 suggests internal nested splicing events before the ultimate splicing at the authentic 5′ and 3′ splice sites. Nested splicing events, which bring the authentic 5′ and 3′ splice sites into close proximity, could be one of the splicing mechanisms for the extremely large introns. ⺠Huge intron 7 spliced from endogenous dystrophin pre-mRNA was analyzed by RT-PCR. ⺠The excised lariat intron 7 had a closed loop but it included gaps in its sequence. ⺠Two presumed lariats generated from nested introns within intron 7 were detected. ⺠These RNA products were generated concomitantly with dystrophin pre-mRNA splicing. ⺠Proposed model: nested introns are spliced before ultimate splicing in huge intron.