Cis regulation within a cluster of viral microRNAs

MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the premiR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.

Automated summary

miRNAs are small regulatory RNAs involved in almost all biological processes, and little is known about the impact of their co-expression from clusters. This study focused on the regulatory mechanism controlling the expression of ten clustered pre-miRNAs from KSHV, identifying the importance of pre-miR-K1 and -K3 in the cleavage efficiency. Mutational analysis showed that the presence of a canonical pre-miRNA at the location is crucial, and the cluster is cleaved in a sequential manner, providing insights for inhibiting the expression of the whole set of miRNAs.