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

Given the cleavage properties of RNase L (see next section), it was unlikely that the identified small RNAs were direct products of this enzyme. We therefore looked for other factors implicated in RNA degradation that could affect SvsRNA accumulation. We used RNAi to downregulate the expression of the major cytoplasmic 5=-3= exoribonuclease Xrn1 (Fig. 2D ). As expected, the capped viral genome was not affected by downregulation of Xrn1 (Fig. 2E) . However, the levels of SvsRNA-1, -2, and -3 and their precursors were stabilized (Fig. 2F) . We then looked at the effect of combining knockdown of these factors (see Fig. S6 in the supplemental material). We found that the double knockdown of both RNase L and Dicer did not affect the accumulation of SvsRNA-1 compared to the single RNase L knockdown, which confirms that Dicer does not play a role (Fig. S6A) . On the contrary, the accumulation of SvsRNA-1 upon double knockdown of both RNase L and Xrn1 increased compared to the single RNase L knockdown (Fig. S6A ). This finding suggests that both RNase L and Xrn1 can act upon viral RNAs and have opposite activities in terms of generation and stabilization of the SvsRNAs. Xrn1 might act upstream of RNase L in degrading, for example, uncapped viral RNAs that would otherwise be a substrate for RNase L; alternatively, it could directly act upon the RNase L products. The former hypothesis is more likely though because Xrn1 has a preference for 5= phosphorylated RNAs, whereas RNase L leaves a 5= OH after cleavage. We also performed a high-molecular-weight Northern blot analysis to look at the effects of the various knockdowns on the accumulation of the genomic and subgenomic viral RNAs.