Role of the SAF-A/HNRNPU ATPase and RGG domains in X chromosome inactivation, nuclear dynamics, transcription, splicing, and cell proliferation

Abstract

The SAF-A/HNRNPU gene encodes an abundant nuclear protein conserved throughout vertebrates, and is mutated in individuals with HNRNPU syndrome, a neurological human disease. SAF-A is important for maintaining lncRNA localization, splicing, and gene expression state. The mechanistic role of SAF-A in each of these processes is coordinated by one or more of its functional domains, which include an N-terminal SAP domain, a central ATPase domain, and a series of C-terminal RGG repeats embedded in a low-complexity region. The SAP domain and RGG repeats define two nucleic acid interaction domains, with both capable of binding DNA or RNA. Here we use an allelic reconstitution strategy to investigate the role of the SAF-A ATPase domain and RGG repeats. We show that both the ATPase and RGG repeats control SAF-A nuclear dynamics, and present genetic evidence that SAF-A interacts with nascent transcripts through the RGG repeats. The SAF-A ATPase domain and RGG repeats were also required for maintaining XIST RNA and facultative heterochromatin marks on the inactive X chromosome, with distinct effects of mutations that block ATP binding and ATP hydrolysis. Analysis of transcriptome datasets revealed that the SAF-A ATPase domain and RGG repeats are both required for proper mRNA splicing, but not for gene expression. Importantly, we found that like the SAP domain, the SAF-A ATPase domain and RGG repeats are required for cell proliferation. Collectively, our findings highlight the importance of the SAF-A SAP, ATPase and RGG domains in essential functions of nuclear biology. These analyses will therefore inform our understanding of the disease state in HNRNPU syndrome.