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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. 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). 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. 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. ADAR1 is an adenosine deaminase that converts adenosine to inosine (A-to-I) mostly on Alu repeats in human RNA. Here by analysing transcriptome-wide ADAR1–RNA interactions, the authors show that ADAR1 also binds non- Alu sequences to regulate alternative 3′ UTR usage and miRNA biogenesis in the nucleus.

Understanding the function of rare non-coding variants represents a significant challenge. Using MapUTR, a screening method, we studied the function of rare 3′ UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare gnomAD variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated an interrogation of 11,929 somatic mutations, uncovering 3928 (33%) functional mutations in 155 cancer driver genes. Functional MapUTR variants were enriched in microRNA- or protein-binding sites and may underlie outlier gene expression in tumors. Further, we introduce untranslated tumor mutational burden (uTMB), a metric reflecting the amount of somatic functional MapUTR variants of a tumor and show its potential in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes ( MFN2 , FOSL2 , and IRAK1 ), demonstrating their cancer-driving potential. Our study elucidates the function of tens of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer. The function of rare non-coding variants remains challenging to decipher. Here, the authors developed MapUTR to uncover 10,524 functional rare 3’ UTR variants regulating mRNA abundance, many of which reside in cancer driver genes.

Background RNA editing generates modifications to the RNA sequences, thereby increasing protein diversity and shaping various layers of gene regulation. Recent studies have revealed global shifts in editing levels across many cancer types, as well as a few specific mechanisms implicating individual sites in tumorigenesis or metastasis. However, most tumor-associated sites, predominantly in noncoding regions, have unknown functional relevance. Results Here, we carry out integrative analysis of RNA editing profiles between epithelial and mesenchymal tumors, since epithelial-mesenchymal transition is a key paradigm for metastasis. We identify distinct editing patterns between epithelial and mesenchymal tumors in seven cancer types using TCGA data, an observation further supported by single-cell RNA sequencing data and ADAR perturbation experiments in cell culture. Through computational analyses and experimental validations, we show that differential editing sites between epithelial and mesenchymal phenotypes function by regulating mRNA abundance of their respective genes. Our analysis of RNA-binding proteins reveals ILF3 as a potential regulator of this process, supported by experimental validations. Consistent with the known roles of ILF3 in immune response, epithelial-mesenchymal differential editing sites are enriched in genes involved in immune and viral processes. The strongest target of editing-dependent ILF3 regulation is the transcript encoding PKR, a crucial player in immune and viral response. Conclusions Our study reports widespread differences in RNA editing between epithelial and mesenchymal tumors and a novel mechanism of editing-dependent regulation of mRNA abundance. It reveals the broad impact of RNA editing in cancer and its relevance to cancer-related immune pathways.

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs. Differential binding of RNA-binding proteins mediated by genetic variants (GVs) can influence posttranscriptional regulation. Here, the authors develop BEAPR, a computational approach to identify allele-specific binding events in eCLIP-Seq data.

Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing. Giovanni Quinones-Valdez et al. examined the role of over 200 RNA-binding proteins in mediating A-to-I RNA editing. They identified several RNA-binding proteins that regulate ADAR1 expression, interaction, or binding with Alu elements in a cell type-specific manner.

Drosophila neuropeptide F (NPF), a homolog of vertebrate neuropeptide Y, functions in feeding and coordination of behavioral changes in larvae and in modulation of alcohol sensitivity in adults, suggesting diverse roles for this peptide. To gain more insight into adult-specific NPF neuronal functions, we studied how npf expression is regulated in the adult brain. Here, we report that npf expression is regulated in both sex-nonspecific and male-specific manners. Our data show that male-specific npf (ms-npf) expression is controlled by the transformer (tra)-dependent sex-determination pathway. Furthermore, fruitless, one of the major genes functioning downstream of tra, is apparently an upstream regulator of ms-npf transcription. Males lacking ms-npf expression (through tra(F)-mediated feminization) or npf-ablated male flies display significantly reduced male courtship activity, suggesting that one function of ms-npf neurons is to modulate fruitless-regulated sexual behavior. Interestingly, one of the ms-npf neuronal groups belongs to the previously defined clock-controlling dorsolateral neurons. Such ms-npf expression in the dorsolateral neurons is absent in arrhythmic Clock(Jrk) and cycle⁰² mutants, suggesting that npf is under dual regulation by circadian and sex-determining factors. Based on these data, we propose that NPF also plays a role in clock-controlled sexual dimorphism in adult Drosophila.

In Drosophila, transcriptional feedback loops contribute to intracellular timekeeping mechanisms responsible for daily rhythms. Pigment-dispersing factor (PDF) is the major neuropeptide produced by latero-ventral neurons (LNvs) that function as a central pacemaker for circadian locomotor activity rhythms. PDF synchronizes other clock neurons thereby playing an essential role in the maintenance and coordination of circadian locomotor rhythms. However, the underlying molecular mechanism of the LNvs-specific Pdf expression is not well understood. Here, using Pdf promoter-bashing experiment, we identified a cis-acting Pdf regulatory element (PRE) that is sufficient for driving Pdf expression in the LNvs. We have also identified a homeobox transcription factor, scarecrow (SCRO), as a direct binding factor to PRE. Furthermore, transgenic expression of scro in the clock neurons abolished Pdf expression and circadian locomotor activity rhythms, and such repressive function requires DNA-binding homeodomain, but none of the other conserved domains. scro is predominantly expressed in the optic lobe and various clusters of cells in other areas of the central nervous system. A homozygous scro-null mutant generated by CRIPSR is lethal during embryonic and early larval development, suggesting that scro plays a vital role during early development.

The abundance and stimulus-responsiveness of mature mRNA is thought to be determined by nuclear synthesis, processing, and cytoplasmic decay. However, the rate and efficiency of moving mRNA to the cytoplasm almost certainly contributes, but has rarely been measured. Here, we investigated mRNA export rates for innate immune genes. We generated high spatio-temporal resolution RNA-seq data from endotoxin-stimulated macrophages and parameterized a mathematical model to infer kinetic parameters with confidence intervals. We find that the effective chromatin-to-cytoplasm export rate is gene-specific, varying 100-fold: for some genes, less than 5% of synthesized transcripts arrive in the cytoplasm as mature mRNAs, while others show high export efficiency. Interestingly, effective export rates do not determine temporal gene responsiveness, but complement the wide range of mRNA decay rates; this ensures similar abundances of short- and long-lived mRNAs, which form successive innate immune response expression waves. The UCLA Ribonomics group reports that the nuclear export efficiency of innate immune mRNAs varies over a hundred-fold range such that for many genes only a small fraction of the newly synthesized premRNA reaches the cytoplasm. They show that nuclear export and cytoplasmic decay rates are correlated thereby ensuring similar expression levels of short-lived and long-lived mRNAs.

A group of small ventrolateral neurons (s-LNv's) are the principal pacemaker for circadian locomotor rhythmicity of Drosophila melanogaster, and the pigment-dispersing factor (Pdf) neuropeptide plays an essential role as a clock messenger within these neurons. In our comparative studies on Pdf-associated circadian rhythms, we found that daily locomotor activity patterns of D. virilis were significantly different from those of D. melanogaster. Activities of D. virilis adults were mainly restricted to the photophase under light:dark cycles and subsequently became arrhythmic or weakly rhythmic in constant conditions. Such activity patterns resemble those of Pdf01 mutant of D. melanogaster. Intriguingly, endogenous D. virilis Pdf (DvPdf) expression was not detected in the s-LNv-like neurons in the adult brains, implying that the Pdf01-like behavioral phenotypes of D. virilis are attributed in part to the lack of DvPdf in the s-LNv-like neurons. Heterologous transgenic analysis showed that cis-regulatory elements of the DvPdf transgene are capable of directing their expression in all endogenous Pdf neurons including s-LNv's, as well as in non-Pdf clock neurons (LNd's and fifth s-LNv) in a D. melanogaster host. Together these findings suggest a significant difference in the regulatory mechanisms of Pdf transcription between the two species and such a difference is causally associated with species-specific establishment of daily locomotor activity patterns.