We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

Cotranscriptional cleavage mediated by a hammerhead ribozyme can affect alternative splicing if interposed between an exon and its intronic regulatory elements. This has been demonstrated using two different alternative splicing systems based on a-tropomyosin and fibronectin genes. We suggest that there is a requirement for intronic regulatory elements to be covalently attached to exons that are in turn tethered to the elongating polymerase. In the case of the alternatively spliced EDA exon of the fibronectin gene, we demonstrate that the newly identified intronic downstream regulatory element is associated with the splicing regulatory protein SRp20. Our results suggest that targeted hammerhead ribozyme cleavage within introns can be used as a tool to define splicing regulatory elements.

We previously showed that the insertion of a hammerhead ribozyme (Rz) in a critical intronic position between the EDA exon and a downstream regulatory element affects alternative splicing. Here we evaluate the effect of other intronic cotranscriptional cleavage events on alternative pre-mRNA processing using different ribozymes (Rz) and Microprocessor target sequences (MTSs). In the context of the fibronectin EDA minigene, intronic MTSs were cleaved very inefficiently and did not affect alternative splicing or the level of mature transcripts. On the contrary, all hammerhead Rz derivatives and hepatitis d Rz were completely cleaved before a splicing decision and able to affect alternative splicing. Despite the very efficient Rz-mediated cleavage, the levels of mature mRNA were only reduced to~40%. We show that this effect on mature transcripts occurs regardless of the type and intronic position of Rzs, or changes in alternative splicing and exon definition. Thus, we suggest that intron integrity is not strictly required for splicing but is necessary for efficient pre-mRNA biosynthesis.