Small RNA profiling of KSHV-miRNA-expressing and KSHV-infected B cell lines

Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNA themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNAs accumulation. To this end, we used the Kaposi’s sarcoma herpesvirus, which encodes a cluster of twelve pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNA could be as high as 60-fold. Using high-throughput selective 2’-hydroxyl acylation analyzed by primer extension (hSHAPE), we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops for highly and lowly expressed miRNAs, which resulted in a perturbed accumulation of the mature miRNA. Examination of sRNA profiles in 3 independent B cell lines expressing KSHV miRNAs or infected with KSHV, without replicate