Heterogeneous nuclear ribonucleoprotein hrp36 acts as an alternative splicing repressor in Litopenaeus vannamei Dscam.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are highly conserved from nematode to mammal because they play an important role in several aspects of pre-mRNA maturation, including RNA packaging and alternative splicing. In Drosophila, the hnRNP A1 homolog hrp36 regulates alternative splicing in several genes, including the Down syndrome cell adhesion molecule (Dscam), which produces tens of thousands of isoforms from one locus. In this study, the first hrp36 gene was identified and characterized from Litopenaeus vannamei (Lvhrp36). Its open reading frame (ORF) contains 1101 bp encoding 366 amino acids. The deduced Lvhrp36 protein includes two copies of the RNA recognition motif (RRM), a C-terminal glycine-rich domain (GRD), the highly degenerate RNP consensus sequences RNP-1 and RNP-2, and two RGG boxes. Tissue tropism analysis indicated that Lvhrp36 is expressed ubiquitously and at high levels in most tissues. dsRNA silencing of shrimp Lvhrp36 in vivo induced abnormal exon inclusions in LvDscam, especially in the Ig3 variable region. In the Ig3 region, a total of 14 different combinations were arranged in three different types of abnormal inclusion pattern. This compares to a single combination (one abnormal pattern) in Ig2 and three different combinations (one abnormal pattern) in Ig7. This is the first evidence to suggest that hrp36 may be involved in the regulation of the Ig7 variable region, and it is noteworthy because, at least in Drosophila, there are no hrp36 binding sequences in the Ig7 exon cluster. The above aberrant events were not observed in all of the Lvhrp36-silenced shrimp, and even when they occurred, the normal patterns of inclusion were far more common. We hypothesize that this continued prevalence of normal inclusions was probably due to other unsilenced proteins that were able to rescue Lvhrp36's functionality. Taken together, our results suggest that Lvhrp36 acts as a splicing repressor that regulates alternative splicing events in the Ig2, Ig3 and Ig7 variable regions of shrimp L. vannamei Dscam.