Effects of Alternative Splicing-Specific Knockdown of Tjp1 α+ by Rbm47 on Tight Junctions Assembly during Blastocyst Development

Abstract

Tjp1 + is considered a crucial protein involved in the stepwise assembly of tight junctions (TJs) between compaction and blastocoel cavitation in early development. In this study, we investigated the specific role of Tjp1 + in TJ formation by employing an alternative splicing-specific knockdown of the Tjp1 + exon. To deplete Tjp1 + expression, we used siRNA targeting RNA-binding protein 47 (Rbm47), which induces the inclusion of the + exon in Tjp1 mRNA. The knockdown resulted in approximately 85% reduction in Rbm47 mRNA levels and 75% reduction in Tjp1 + mRNA levels in blastocysts. Surprisingly, despite this knockdown, blastocyst development and TJ permeability of trophectoderm were unaffected. Additionally, we observed an interaction between Tjp1 - and Ocln in Rbm47 knockdown blastocysts, suggesting a compensatory role of Tjp1 -. Overall, our findings indicate that Tjp1 + is not essential for the stepwise assembly of TJs and the completion of TJ biogenesis during blastocyst development in mice although a minimal amount of remaining Tjp1 + is sufficient for TJs assembly. Summary statementSelective loss of Tjp1 + mediated by Rbm47 knockdown did affect mouse blastocyst development, suggesting that Tjp1 + may not be crucial for stepwise TJs assembly during blastocyst development