Identification of RNase L-Dependent, 3'-End-Modified, Viral Small RNAs in Sindbis Virus-Infected Mammalian Cells

Girardi, Erika; Chane-Woon-Ming, Béatrice; Messmer, Mélanie; Kaukinen, Pasi; Pfeffer, Sébastien

The fact that RNA fragments generated by RNase L are detected implies that some of them must be stabilized. Indeed, we discovered that the viral small RNA candidates appear to be modified on the 3= proximal nucleotide. We could extend this peculiarity to the global small RNA population by cloning and sequencing small RNAs after an oxidation treatment designed to prevent ligation of nonmodified RNAs. Our data indicate that this modification is most likely a 2=-O-methylation and seems to occur at the level of the viral genomic RNA, preferentially on adenosines. Thanks to a double approach of RTL-P and primer extension, we were able to map such a modification on position A 8333 of the SINV viral genome. Moreover, our sequencing data indicate that there are most likely many others given the enrichment in A residues at the 3= end of small RNAs cloned after oxidative treatment.