Immunostimulatory short non-coding RNAs in the circulation of patients with tuberculosis infection.

Mycobacterium tuberculosis (Mtb) infection is among the world's deadliest infectious diseases. Developing effective treatments and biomarkers for tuberculosis requires a deeper understanding of its pathobiology and host responses. Here, we report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma samples from Mtb-infected patients. We achieved this by pre-treating plasma RNAs with T4 polynucleotide kinase to convert all RNA ends to those compatible with sncRNA sequencing. We discovered a global and drastic upregulation of plasma sncRNAs in Mtb-infected patients, with tRNA-derived sncRNAs representing the most dramatically elevated class. Most of these tRNA-derived sncRNAs originated from a limited subset of tRNAs, specifically from three tRNA isoacceptors, and exhibited skewed patterns to 5'-derived fragments, such as 5' halves, 5' tRNA fragments (tRFs), and internal tRFs (i-tRFs) from the 5' regions. Further, Mtb-infected patients displayed markedly upregulated and distinct profiles of both rRNA- and mRNA-derived sncRNAs. Some of these sncRNAs, which are abundant and specific to Mtb-infected patients, robustly activated human macrophages via Toll-like receptor 7 and induced cytokine production. This drastic accumulation of circulating, immunostimulatory sncRNAs in the plasma of Mtb-infected patients offers insights into the sncRNA-driven aspects of host immune response against infectious diseases and suggests a pool of potential therapeutic targets and biomarkers.

Structural insights into RNA bridging between HIV-1 Vif and antiviral factor APOBEC3G.

Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV's counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction.

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3' phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.

Making Invisible RNA Visible: Discriminative Sequencing Methods for RNA Molecules with Specific Terminal Formations.

Next generation sequencing of RNA molecules (RNA-seq) has become a common tool to characterize the expression profiles of RNAs and their regulations in normal physiological processes and diseases. Although increasingly accumulating RNA-seq data are widely available through publicly accessible sites, most of the data for short non-coding RNAs (sncRNAs) have been obtained for microRNA (miRNA) analyses by standard RNA-seq, which only capture the sncRNAs with 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) ends. The sncRNAs with other terminal formations such as those with a 5'-hydroxyl end (5'-OH), a 3'-phosphate (3'-P) end, or a 2',3'-cyclic phosphate end (2',3'-cP) cannot be efficiently amplified and sequenced by standard RNA-seq. Due to the invisibility in standard RNA-seq data, these non-miRNA-sncRNAs have been a hidden component in the transcriptome. However, as the functional significances of these sncRNAs have become increasingly apparent, specific RNA-seq methods compatible with various terminal formations of sncRNAs have been developed and started shedding light on the previously unrecognized sncRNAs that lack 5'-P/3'-OH ends. In this review, we summarize the expanding world of sncRNAs with various terminal formations and the strategic approaches of specific RNA-seq methods to distinctively characterize their expression profiles.

In vitro production and multiplex quantification of 2',3'-cyclic phosphate-containing 5'-tRNA half molecules.

RNA cleavages by many ribonucleases generate RNA molecules that contain a 2',3'-cyclic phosphate (cP) at their 3'-termini, and many cP-containing RNAs (cP-RNAs) are expressed as functional molecules in cells and tissues. 5'-tRNA half molecules are representative examples of functional cP-RNAs, playing important roles in various biological processes. We here show in vitro production of cP-containing 5'-tRNA half molecules that is able to prepare abundant synthetic cP-RNAs enough for functional analyses. Furthermore, we report a multiplex TaqMan RT-qPCR method which can simultaneously quantify multiple cP-containing 5'-tRNA half species. The method enabled us to efficiently quantify 5'-tRNA halves using samples with limited amounts, such as human plasma samples, revealing drastic enhancement of 5'-tRNA half levels at approximately 1,000-fold in patients infected with Mycobacterium tuberculosis. These in vitro production and multiplex quantification methods can be applied to any cP-RNAs, and they provide cost-effective, in-house techniques to accelerate expressional and functional characterizations of 5'-tRNA halves and other cP-RNAs.

RNase κ promotes robust piRNA production by generating 2',3'-cyclic phosphate-containing precursors.

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.

Infection-induced 5'-half molecules of tRNAHisGUG activate Toll-like receptor 7.

Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5'-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5'-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5'-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5'-tRNAHisGUG half molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5'-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5'-tRNA half species into EVs. The EV-5'-tRNAHisGUG half was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5'-tRNA half molecules was also observed in the plasma of patients infected with Mycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of "immune activators" to 5'-tRNA half molecules.

Oxidative stress enhances the expression of 2',3'-cyclic phosphate-containing RNAs.

Eukaryotic cells equip robust systems to respond to stress conditions. In stressed mammalian cells, angiogenin endoribonuclease cleaves anticodon-loops of tRNAs to generate tRNA halves termed tRNA-derived stress-induced RNAs (tiRNAs), which promote stress granule formation and regulate translation. The 5'-tiRNAs (5'-tRNA halves) contain a 2',3'-cyclic phosphate (cP) and thus belong to cP-containing RNAs (cP-RNAs). The cP-RNAs form a hidden layer of the transcriptome because standard RNA-seq cannot amplify and sequence them. In this study, we performed genome-wide analyses of short cP-RNA transcriptome in oxidative stress-exposed human cells. Using cP-RNA-seq that can specifically sequence cP-RNAs, we identified tiRNAs and numerous other cP-RNAs that are mainly derived from rRNAs and mRNAs. Although tiRNAs were produced from a wide variety of tRNA species, abundant species of tiRNAs were derived from a focal-specific subset of tRNAs. Regarding rRNA- and mRNA-derived cP-RNAs, determination of the processing sites of substrate RNAs revealed highly specific RNA cleavage events between pyrimidines and adenosine in generation of those cP-RNAs. Those cP-RNAs were derived from specific loci of substrate RNAs rather than from the overall region, implying that cP-RNAs are produced by regulated biogenesis pathways and not by random degradation events. We experimentally confirmed the identified sequences to be expressed as cP-RNAs in the cells, and their expressions were upregulated upon induction of oxidative stress. These analyses of the cP-RNA transcriptome unravel an abundant class of short ncRNAs that accumulate in cells under oxidative stress.

Genome-wide identification of short 2',3'-cyclic phosphate-containing RNAs and their regulation in aging.

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2',3'-cyclic phosphate (cP) at their 3'-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP's prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.

Exploration of CCA-added RNAs revealed the expression of mitochondrial non-coding RNAs regulated by CCA-adding enzyme.

Post-transcriptional non-template additions of nucleotides to 3'-ends of RNAs play important roles in the stability and function of RNA molecules. Although tRNA nucleotidyltransferase (CCA-adding enzyme) is known to add CCA trinucleotides to 3'-ends of tRNAs, whether other RNA species can be endogenous substrates of CCA-adding enzyme has not been widely explored yet. Herein, we used YAMAT-seq to identify non-tRNA substrates of CCA-adding enzyme. YAMAT-seq captures RNA species that form secondary structures with 4-nt protruding 3'-ends of the sequence 5'-NCCA-3', which is the hallmark structure of RNAs that are generated by CCA-adding enzyme. By executing YAMAT-seq for human breast cancer cells and mining the sequence data, we identified novel candidate substrates of CCA-adding enzyme. These included fourteen 'CCA-RNAs' that only contain CCA as non-genomic sequences, and eleven 'NCCA-RNAs' that contain CCA and other nucleotides as non-genomic sequences. All newly-identified (N)CCA-RNAs were derived from the mitochondrial genome and were localized in mitochondria. Knockdown of CCA-adding enzyme severely reduced the expression levels of (N)CCA-RNAs, suggesting that the CCA-adding enzyme-catalyzed CCA additions stabilize the expression of (N)CCA-RNAs. Furthermore, expression levels of (N)CCA-RNAs were severely reduced by various cellular treatments, including UV irradiation, amino acid starvation, inhibition of mitochondrial respiratory complexes, and inhibition of the cell cycle. These results revealed a novel CCA-mediated regulatory pathway for the expression of mitochondrial non-coding RNAs.

Generation of 2',3'-Cyclic Phosphate-Containing RNAs as a Hidden Layer of the Transcriptome.

Cellular RNA molecules contain phosphate or hydroxyl ends. A 2',3'-cyclic phosphate (cP) is one of the 3'-terminal forms of RNAs mainly generated from RNA cleavage by ribonucleases. Although transcriptome profiling using RNA-seq has become a ubiquitous tool in biological and medical research, cP-containing RNAs (cP-RNAs) form a hidden transcriptome layer, which is infrequently recognized and characterized, because standard RNA-seq is unable to capture them. Despite cP-RNAs' invisibility in RNA-seq data, increasing evidence indicates that they are not accumulated simply as non-functional degradation products; rather, they have physiological roles in various biological processes, designating them as noteworthy functional molecules. This review summarizes our current knowledge of cP-RNA biogenesis pathways and their catalytic enzymatic activities, discusses how the cP-RNA generation affects biological processes, and explores future directions to further investigate cP-RNA biology.

MINTbase v2.0: a comprehensive database for tRNA-derived fragments that includes nuclear and mitochondrial fragments from all The Cancer Genome Atlas projects.

MINTbase is a repository that comprises nuclear and mitochondrial tRNA-derived fragments ('tRFs') found in multiple human tissues. The original version of MINTbase comprised tRFs obtained from 768 transcriptomic datasets. We used our deterministic and exhaustive tRF mining pipeline to process all of The Cancer Genome Atlas datasets (TCGA). We identified 23 413 tRFs with abundance of ≥ 1.0 reads-per-million (RPM). To facilitate further studies of tRFs by the community, we just released version 2.0 of MINTbase that contains information about 26 531 distinct human tRFs from 11 719 human datasets as of October 2017. Key new elements include: the ability to filter tRFs on-the-fly by minimum abundance thresholding; the ability to filter tRFs by tissue keywords; easy access to information about a tRF's maximum abundance and the datasets that contain it; the ability to generate relative abundance plots for tRFs across cancer types and convert them into embeddable figures; MODOMICS information about modifications of the parental tRNA, etc. Version 2.0 of MINTbase contains 15x more datasets and nearly 4x more distinct tRFs than the original version, yet continues to offer fast, interactive access to its contents. Version 2.0 is available freely at http://cm.jefferson.edu/MINTbase/.

Dumbbell-PCR for Discriminative Quantification of a Small RNA Variant.

Cellular RNAs are often expressed as multiple isoforms of complex heterogeneity in both length and terminal sequences. IsomiRs, the isoforms of microRNAs, are such an example. Distinct quantification of each RNA variant is necessary to unravel the biogenesis mechanism and biological significance of heterogenetic RNA expression. Here we describe Dumbbell-PCR (Db-PCR), a TaqMan RT-PCR-based method that distinctively quantifies a specific small RNA variant with single-nucleotide resolution at terminal sequences. Db-PCR enables the quantitative analysis of RNA terminal heterogeneity without performing Next-Generation Sequencing.

Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells.

Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function.

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes.

5'-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5'-halves.

Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the -1 position (G-1) of its 5'-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G-1, U-1, A-1, or C-1 or lacking the -1 nucleotide (starting from G1). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNAHisGUG containing U-1 as well as the one containing G-1 Moreover, tRNA lacking the -1 nucleotide was also detected, thus indicating the heterogeneous expression of 5'-tRNAHisGUG variants. A sequence library of sex hormone-induced 5'-tRNA halves (5'-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5'-tRNAHisGUG halves containing G-1, U-1, or G1 as 5'-terminal nucleotides. Although the detected 5'-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5'-half from the same BT-474 cells. The majority of mature tRNAs contained the -1 nucleotide, whereas the majority of 5'-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.

A novel HSD17B10 mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression.

We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.

tRNA-Derived Short Non-coding RNA as Interacting Partners of Argonaute Proteins.

The advent of next-generation sequencing technologies has not only accelerated findings on various novel non-coding RNA (ncRNA) species but also led to the revision of the biological significance and versatility of fundamental RNA species with canonical function, such as transfer RNAs (tRNAs). Although tRNAs are best known as adapter components of translational machinery, recent studies suggest that tRNAs are not always end products but can further serve as a source for short ncRNAs. In many organisms, various tRNA-derived ncRNA species are produced from mature tRNAs or their precursor transcripts as functional molecules involved in various biological processes beyond translation. In this review, we focus on the tRNA-derived ncRNAs associated with Argonaute proteins and summarize recent studies on their conceivable biogenesis factors and on their emerging roles in gene expression regulation as regulatory RNAs.

Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers.

Sex hormones and their receptors play critical roles in the development and progression of the breast and prostate cancers. Here we report that a novel type of transfer RNA (tRNA)-derived small RNA, termed Sex HOrmone-dependent TRNA-derived RNAs (SHOT-RNAs), are specifically and abundantly expressed in estrogen receptor (ER)-positive breast cancer and androgen receptor (AR)-positive prostate cancer cell lines. SHOT-RNAs are not abundantly present in ER(-) breast cancer, AR(-) prostate cancer, or other examined cancer cell lines from other tissues. ER-dependent accumulation of SHOT-RNAs is not limited to a cell culture system, but it also occurs in luminal-type breast cancer patient tissues. SHOT-RNAs are produced from aminoacylated mature tRNAs by angiogenin-mediated anticodon cleavage, which is promoted by sex hormones and their receptors. Resultant 5'- and 3'-SHOT-RNAs, corresponding to 5'- and 3'-tRNA halves, bear a cyclic phosphate (cP) and an amino acid at the 3'-end, respectively. By devising a "cP-RNA-seq" method that is able to exclusively amplify and sequence cP-containing RNAs, we identified the complete repertoire of 5'-SHOT-RNAs. Furthermore, 5'-SHOT-RNA, but not 3'-SHOT-RNA, has significant functional involvement in cell proliferation. These results have unveiled a novel tRNA-engaged pathway in tumorigenesis of hormone-dependent cancers and implicate SHOT-RNAs as potential candidates for biomarkers and therapeutic targets.

Structure of the Vif-binding domain of the antiviral enzyme APOBEC3G.

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the α1-ß1, ß2-α2 and ß4-α4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development.