BACKGROUND: Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS: Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS: Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS: Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.

Adenosine deaminases acting on RNA (ADARs) are the primary factors underlying adenosine to inosine (A-to-I) editing in metazoans. Here we report the first global study of ADAR1–RNA interaction in human cells using CLIP-seq. A large number of CLIP sites are observed in Alu repeats, consistent with ADAR1’s function in RNA editing. Surprisingly, thousands of other CLIP sites are located in non-Alu regions, revealing functional and biophysical targets of ADAR1 in the regulation of alternative 3′ UTR usage and miRNA biogenesis. We observe that binding of ADAR1 to 3′ UTRs precludes binding by other factors, causing 3′ UTR lengthening. Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression. These functions are dependent on ADAR1’s editing activity, at least for a subset of targets. Our study unfolds a broad landscape of the functional roles of ADAR1.

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3′ acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.

Seventeen miRNAs encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and their functions have begun to be characterized. Among these miRNAs, we report here that miR-K12-7 directly targets the replication and transcription activator (RTA) encoded by open reading frame 50. We found that miR-K12-7 targeted the RTA 3′ untranslated region (RTA3′UTR) in a seed sequence-dependent manner. miR-K12-7-5p derived from miR-K12-7 mediates the inhibition of RTA expression, and the mutation of the seed match site totally abrogated the inhibitory effect of miR-K12-7 on RTA3′UTR. The inhibition of RTA expression by miR-K12-7 was further confirmed in the latently KSHV-infected 293/Bac36 cell line through transient transfection of miR-K12-7 expression plasmid or specific inhibitor of miR-K12-7-5p, respectively. The transient transfection of miR-K12-7 into 293/Bac36 cells reduced RTA expression and the expression of the downstream early genes regulated by RTA, and also the production of progeny virus was significantly reduced after treatment with chemical inducers. Our study revealed that another miRNA, miR-K12-7-5p, targets the viral immediate early gene RTA and that this miRNA contributes to the maintenance of viral latency.

Type I interferon (IFN) signaling is the principal response mediating antiviral innate immunity. IFN transcription is dependent upon the activation of transcription factors IRF3/IRF7 and NF-κB. Many viral proteins have been shown as being capable of interfering with IFN signaling to facilitate evasion from the host innate immune response. Here, we report that a viral miRNA, miR-K12-11, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for the modulation of IFN signaling and acts through targeting I-kappa-B kinase epsilon (IKKɛ). Ectopic expression of miR-K12-11 resulted in decreased IKKɛ expression, while inhibition of miR-K12-11 was found to restore IKKɛ expression in KSHV-infected cells. Importantly, expression of miR-K12-11 attenuated IFN signaling by decreasing IKKɛ-mediated IRF3/IRF7 phosphorylation and by inhibiting the activation of IKKɛ-dependent IFN stimulating genes (ISGs), allowing miR-K12-11 suppression of antiviral immunity. Our data suggest that IKKɛ targeting by miR-K12-11 is an important strategy utilized by KSHV to modulate IFN signaling during the KSHV lifecycle, especially in latency. We also demonstrated that IKKɛ was able to enhance KSHV reactivation synergistically with the treatment of 12-O-tetradecanoylphorbol 13-acetate. Moreover, inhibition of miR-K12-11 enhanced KSHV reactivation induced by vesicular stomatitis virus infection. Taken together, our findings also suggest that miR-K12-11 can contribute to maintenance of KSHV latency by targeting IKKɛ.