Characterization of Viral and Human RNAs Smaller than Canonical MicroRNAs

Recently identified small (20 to 40 bases) RNAs, such as microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) participate in important cellular pathways. In this report, we systematically characterized several novel features of human and viral RNA products smaller than miRNAs. We found that Kaposi sarcoma-associated herpesvirus K12-1 miRNA (23 bases) associates with a distinct, unusually small (17-base) RNA (usRNA) that can effectively downregulate a K12-1 miRNA target, human RAD21, suggesting that stable degradation-like products may also contribute to gene regulation. High-throughput sequencing reveals a diverse set of human miRNA-derived usRNAs and other non-miRNA-derived usRNAs. Human miRNA-derived usRNAs preferentially match to 5 ends of miRNAs and are also more likely to associate with the siRNA effector protein Ago2 than with Ago1. Many non-miRNA-derived usRNAs associate with Ago proteins and also frequently contain C-rich 3-specific motifs that are overrepresented in comparison to Piwi-interacting RNAs and transcription start site-associated RNAs. We postulate that approximately 30% of usRNAs could have evolved to participate in biological processes, including gene silencing. Recent studies have identified several classes of small RNAs, such as microRNAs (miRNAs), across a wide range of organisms (9). miRNAs can function as repressors (24) or activators (47) of gene expression. More than 500 human miRNAs are known, and many more are thought to exist (3). Advances in high-throughput sequencing have enabled the discovery of additional animal miRNAs and classes of small RNAs on an unprecedented scale across distantly related species, including animals, fungi, unicellular algae, plants, and viruses (9). In addition to miRNAs, recently identified metazoan small RNAs include endogenous small interfering RNAs (siRNAs) that mediate mRNA degradation (6, 13, 33, 45), hairpin RNAs that are thought to repress target transcripts in flies (32), Piwi-interacting RNAs (piRNAs) that modulate spermatogenesis in mammals (14, 22), piRNA-like repeat-associated siRNAs that regulate chromatin structure of retrotransposons in insect germ lines (21), transcription start site-associated RNAs (TSSa-RNAs), and the functionally uncharacterized piRNA-like 21U-RNAs harboring 5 uridines in worms (36). The various small-RNA classes frequently share sequence