Pumilio proteins license viral noncoding RNA engagement with target mRNAs

Noncoding RNAs commonly rely on RNA-binding proteins (RBPs) to execute their regulatory functions, but whether RBPs can instead facilitate target recognition by noncoding RNAs is poorly understood. Here, we identify a non-canonical function for Pumilio proteins PUM1 and PUM2 in gene regulation as cofactors of the viral small nuclear RNAs HSUR1 and HSUR2 encoded by Herpesvirus saimiri. HSUR1 and HSUR2 function as microRNA and RBP adaptors that bind target mRNAs to downregulate their expression. We show that HSUR1 and HSUR2 contain conserved Pumilio response elements (PREs) through which they associate with PUM1 and PUM2, without measurably altering PUM protein activity or subcellular distribution in infected cells. Instead, PUM binding is required for HSUR-mediated mRNA repression. Mutation of the PREs abolishes PUM association and HSUR-mediated repression without disrupting HSUR association with microRNAs, whereas tethering PUM1 or PUM2 directly to PRE-deficient HSURs restores repression. Furthermore, HSURs unable to bind PUMs fail to associate with target mRNAs, demonstrating that PUM binding is required for productive HSUR-mRNA engagement. These findings reveal that PUM proteins can function as enabling factors for noncoding RNA target recognition rather than as downstream effectors of repression, expanding the functions of these RBPs in RNA-mediated gene regulation.