Mechanisms Underlying Aberrant Splicing of Transcripts From the Intron-Less Coding Sequence of SPCA1

In protein expression systems, only the protein-coding sequence (CDS) of the gene is inserted in the vector, producing intron-less mRNA that is translated directly without splicing. We found that, on expression of the CDS of the Ca²⁺/Mn²⁺ pump SPCA1 using an expression system, the intron-less mRNA underwent aberrant splicing, resulting in unexpected SPCA1 truncated products. The primary aberrant splicing occurred between a 5' splice site at c.568 and a 3' splice site at c.2200. The isoform SPCA2 did not exhibit aberrant splicing, but the introduction of either the c.568 or c.2200 site into its CDS induced aberrant splicing. Analysis of chimeric SPCA2-SPCA1 CDSs and SPCA1 mutants demonstrated that the splicing between c.568 and c.2200 is influenced not only by cryptic splice sites but also by the flanking regulatory sequences. Furthermore, the splicing regulator MBNL1 promoted the aberrant splicing. Notably, insertion of a single intron into the SPCA1 CDS not only fails to suppress this process but can also activate novel cryptic splice sites. Our study suggests that aberrant splicing from a CDS in an expression vector is induced by the spatial proximity of cryptic splice sites and regulatory sequences that would normally be separated by introns in the genome.