Molecular basis of the autoregulatory mechanism of motor neuron-related splicing factor 30

Motor neuron-related splicing factor 30 (SPF30, also known as SMNDC1) is a paralog of the survival motor neuron protein that regulates the expression of various genes by affecting mRNA splicing. SPF30 has an autoregulatory mechanism that controls its expression. However, the detailed molecular mechanisms determining cellular levels of SPF30 remain unclear. Here, we demonstrated that SPF30 expression was controlled via the negative autoregulatory feedback, whereby increased SPF30 expression caused the inclusion of cassette exon within intron 2 and/or the generation of a newly spliced variant with exon 4a (produced by splicing 17 bases upstream of the canonical intron 3 and exon 4 junction). Altered transcripts with cassette exon or exon 4a were subjected to nonsense-mediated mRNA decay, leading to reduced SPF30 mRNA levels. Conversely, the loss of SPF30 protein resulted in a drastic reduction in the alternative exon 4a splice site usage compared to cassette exon inclusion, suggesting that alternative splicing at exon 4a contributes more to adjusting SPF30 expression levels. An in vivo splicing assay designed to reflect the usage of alternative exon 4a splice site demonstrated that a short stretch of sequence within exon 4 of SPF30 mRNA was required for the alternative splicing at exon 4a. In addition, the C terminal region of SPF30, particularly the latter part of α-helix and a kink-like structure, was crucial for the autoregulatory mechanism by enabling binding to exon 4a-containing RNA. These results reveal the molecular basis of the autoregulatory mechanism underlying SPF30 gene expression.