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

Area of Science:

• Molecular Biology
• Gene Expression
• Protein Synthesis

Background:

• Protein expression systems typically use only the protein-coding sequence (CDS) for direct mRNA translation.
• Intron-less mRNA is expected to bypass the splicing process entirely.

Purpose of the Study:

• To investigate the phenomenon of aberrant splicing in intron-less mRNA during protein expression.
• To identify the mechanisms and factors contributing to unexpected splicing events in CDS.

Main Methods:

• Expression of Ca2+/Mn2+ pump SPCA1 CDS in a protein expression system.
• Analysis of mRNA splicing patterns and protein products.
• Introduction of specific splice sites and regulatory sequences into SPCA2 CDS.
• Construction and analysis of chimeric SPCA2-SPCA1 CDSs and SPCA1 mutants.
• Assessment of the role of MBNL1 in aberrant splicing.

Main Results:

• Aberrant splicing was observed in SPCA1 CDS, producing truncated proteins.
• The primary aberrant splicing event occurred between specific 5' (c.568) and 3' (c.2200) splice sites.
• SPCA2 did not show aberrant splicing, but introducing these sites induced it.
• Splicing was influenced by cryptic splice sites, flanking regulatory sequences, and the splicing regulator MBNL1.
• Intron insertion into SPCA1 CDS did not suppress but could activate new cryptic splice sites.

Conclusions:

• Aberrant splicing in expression vectors can arise from the proximity of cryptic splice sites and regulatory sequences within the CDS.
• These elements, normally separated by introns, can trigger splicing when placed together in an intron-less context.
• Understanding these mechanisms is crucial for accurate protein expression and avoiding unintended protein variants.