A widespread Xrn1-resistant RNA motif composed of two short hairpins

Xrn1 is a major 59-39 exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 39 untranslated region of several beny- and cucumovirus RNAs harbors a so-called 9coremin9 motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence, to deduce putative structural features responsible for Xrn1-stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 59 proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 39 proximal hairpin loop sequence is variable. The sequence of the 9-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn-stalling motifs are more widespread among RNA viruses than previously realized.