Role of the SAF-A ATPase and RGG domains in X inactivation, transcription, splicing, and cell proliferation.
SAF-A/HNRNPU is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including a central ATPase domain and a C-terminal RGG domain. In this study we tested for the role of the ATP binding, ATP hydrolysis and RNA binding in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the ATPase and RGG domains are required to maintain XIST RNA localization and XIST-dependent histone modifications on the inactive X chromosome, to execute normal protein dynamics, and to maintain normal cell proliferation. We found that the ATPase and RGG domains are not required to maintain gene expression but play a major role in mRNA splicing. We propose a model whereby RNA binding, ATP binding and ATP hydrolysis promote SAF-A interaction with nascent RNA to promote normal mRNA splicing and XIST localization and function. Overall design: RPEa cells were engineered with a inducible SAF-A degron. These cell lines were then transduced with dox-inducible lentiviruses to express SAF-A WT, SAF-A Walker A, SAF-A Walker B, or SAF-A Delta RGG. SAF-A was depleted and rescue constructs were induced for 24 hours prior to harvesting total RNA.