Human FASTK preferentially binds single-stranded and G-rich RNA
Fas-activated serine/threonine kinase (FASTK) is the founding member of the FASTKD protein family, which was shown to regulate the fate of mRNA molecules on multiple levels. The mitochondrial variant of FASTK co-localizes with mitochondrial RNA granules and regulates degradation of mitochondrial mRNAs, whereas the cytoplasmic and nuclear forms of FAST are involved in regulation of alternative splicing, cytoplasmic RNA granule formation and mRNA translation. Despite these multiple roles of FASTK in mRNA biology, the exact rules of RNA recognition by this protein remained undetermined. Here, we demonstrate direct RNA binding by purified human FASTK and show its preference for single-stranded G-rich sites and RNA G-quadruplexes. Addition of FASTK alone was sufficient to achieve protection of mitochondrial mRNAs from degradation by the degradosome. Structural characterization by SAXS showed that FASTK in solution is a monomer with an extended conformation. Point mutagenesis studies supported the structural predictions of an exposed RNA-binding interface in the central helical region, preceded by a smaller, flexibly attached, helical N-terminal domain. We provide the first such extensive in vitro characterization of the RNA binding properties for a representative of the FASTKD protein family, and suggest how these intrinsic properties may underly FASTK function in mRNA metabolism.