A paradigm for regulation at the effector interface with RNA-binding proteins

Abstract

RNA-binding proteins (RBPs) are key regulators of gene expression, but how RBPs convey regulatory instructions to the core effectors of RNA processing is unclear. We address this problem by dissecting the effector interface responsible for regulation by a developmentally essential RBP. We show that the sequence-specific RBP Unkempt, in addition to its RNA-binding domain, contains an extensive intrinsically disordered region (IDR) and a dimerization domain, all of which distinctly contribute to Unkempt's function. Within IDR, we identify an array of linear motifs that support direct contact with two central effectors, the CCR4-NOT complex and PABPC. The spatial arrangement, dual-purpose motifs, and obligate homodimerization allow for high-avidity effector interactions that are mandatory for the accuracy of RNA targeting, translational repression, and control of cell morphology by Unkempt. Our study establishes the molecular assembly and functional principles of an RBP-effector interface, with general implications for the evolution and function of RBP-operated regulatory networks.

Understanding species-specific and conserved RNA-protein interactionsin vivoandin vitro

“…To determine whether these in vitro binding patterns also correspond with in vivo regulatory patterns, we examined ribosome profiling data upon UNK induction 47 . UNK is a translational repressor 27 , thus UNK-regulated RNAs are predicted to have decreased translation as previously shown 27 .…”

“…To examine how binding changes across 100 vertebrates correlates with RNA regulation, we once again turned to ribosome profiling data 47 . As discussed above, we found correlations between UNK binding and evolutionary distance and sequence conservation ( Fig.…”

“…Note 1 ). iii) Due to difficulties in purifying full-length UNK protein, we have performed our experiments on the RBDs alone, which exhibit tight and specific binding to known UNK motifs (as shown above and previously 26 ); additional components of UNK, such as its disordered domain, may impact binding in unexpected ways 47 . iv) For practical reasons we have used human UNK protein for these studies.…”

“…Ribosome profiling data was obtained from Shah et al 47 . Genes bound in both human and mouse, human only, or mouse only (human not bound) were identified via iCLIP as described above.…”

“…To understand species-specific RNA binding, we used available individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) data 27 from a neuronal RBP, unkempt (UNK), in human and mouse. UNK regulates neuronal morphology, is a negative regulator of translation, and associates with polysomes 27,46,47 . We identified species-specific and shared UNK binding sites and found that 45% of UNK transcript binding was conserved across species.…”