Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

Chang, Stewart T.; Thomas, Matthew J.; Sova, Pavel; Green, Richard R.; Palermo, Robert E.; Katze, Michael G.

A small number of annotations were enriched with target mR-NAs at frequencies greater than expected by chance (by one-sided Fisher's exact tests with P values of Ͻ0.05) ( Table 2 ; see also Table S3 in the supplemental material). These annotations included "transcription regulatory activity," which was associated with 111 upregulated target mRNAs at 24 hpi (Table 2 ). These mRNAs were in turn predicted to be targeted by 20 DE microRNAs (see Table S2 and Fig. S3A ). Another set of target mRNAs encoding transcriptional regulators (albeit with different specific annotation terms) was found to be downregulated (Table 2; see also Table S2 and Fig. S3B ). The presence of up-and downregulated microRNAs associated with multiple annotations related to transcriptional regulation suggest a role for microRNAs in regulating changes in gene expression during HIV infection. A subnetwork of upregulated mRNAs associated with DE downregulated microRNAs was found to include K(lysine) acetyltransferase 2B (KAT2B), a transcription factor known to contribute to HIV replication and known to be targeted posttranscriptionally by miR-17 and miR-20 (14) . This subnetwork showed that these and other microRNAs were predicted to target KAT2B as well as a broader set of mRNAs with functional connections to KAT2B (Fig. 3A ). Additional microRNAs in the subnetwork in-cluded miR-106a and miR-20b, two paralogs of miR-17 and miR-20, derived from the same host gene cluster (Fig. 3A) . Their inclusion in the subnetwork suggests they may also have been downregulated in the earlier experiment, implicating miR-17 and miR-20 (14) . In addition, a nonparalogous microRNA, miR-32, was also included in this subnetwork and may have regulated the same target mRNAs (Fig. 3A) . Together, these microRNAs may represent an overlapping, robust network of regulators upstream of mRNAs for transcriptional regulators that contribute to HIV replication.