5\u27-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5\u27-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, tRNA(HisGUG) has been known to uniquely contain an additional guanosine residue at the -1 position (G-1) of its 5\u27-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNA(HisGUG) 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 tRNA(HisGUG) 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\u27-tRNA(HisGUG) variants. A sequence library of sex hormone-induced 5\u27-tRNA halves (5\u27-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5\u27-tRNA(HisGUG) halves containing G-1, U-1, or G1 as 5\u27-terminal nucleotides. Although the detected 5\u27-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5\u27-half from the same BT-474 cells. The majority of mature tRNAs contained the -1 nucleotide, whereas the majority of 5\u27-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNA(HisGUG) molecules and provide insights into tRNA(HisGUG) maturation and the regulation of tRNA half production

Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences.

Recent advances in next-generation sequencing technologies have revealed that cellular functional RNAs are not always expressed as single entities with fixed terminal sequences but as multiple isoforms bearing complex heterogeneity in both length and terminal sequences, such as isomiRs, the isoforms of microRNAs. Unraveling the biogenesis and biological significance of heterogenetic RNA expression requires distinctive analysis of each RNA variant. Here, we report the development of dumbbell PCR (Db-PCR), an efficient and convenient method to distinctively quantify a specific individual small RNA variant. In Db-PCR, 5\u27- and 3\u27-stem-loop adapters are specifically hybridized and ligated to the 5\u27- and 3\u27-ends of target RNAs, respectively, by T4 RNA ligase 2 (Rnl2). The resultant ligation products with \u27dumbbell-like\u27 structures are subsequently quantified by TaqMan RT-PCR. We confirmed that high specificity of Rnl2 ligation and TaqMan RT-PCR toward target RNAs assured both 5\u27- and 3\u27-terminal sequences of target RNAs with single nucleotide resolution so that Db-PCR specifically detected target RNAs but not their corresponding terminal variants. Db-PCR had broad applicability for the quantification of various small RNAs in different cell types, and the results were consistent with those from other quantification method. Therefore, Db-PCR provides a much-needed simple method for analyzing RNA terminal heterogeneity

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

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

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5\u27-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5\u27-tRNA halves as well, suggesting a previously uncharacterized link between 5\u27-tRNA halves and td-piRNAs. An increase in levels of the 5\u27-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5\u27-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs

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

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 '-tRNA(HisGUG) 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 '-tRNA(HisGUG) 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

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

Nuclear and mitochondrial tRNA-lookalikes in the human genome.

We are interested in identifying and characterizing loci of the human genome that harbor sequences resembling known mitochondrial and nuclear tRNAs. To this end, we used the known nuclear and mitochondrial tRNA genes (the tRNA-Reference set) to search for tRNA-lookalikes and found many such loci at different levels of sequence conservation. We find that the large majority of these tRNA-lookalikes resemble mitochondrial tRNAs and exhibit a skewed over-representation in favor of some mitochondrial anticodons. Our analysis shows that the tRNA-lookalikes have infiltrated specific chromosomes and are preferentially located in close proximity to known nuclear tRNAs (z-score ≤ -2.54, P-value ≤ 0.00394). Examination of the transcriptional potential of these tRNA-lookalike loci using public transcript annotations revealed that more than 20% of the lookalikes are transcribed as part of either known protein-coding pre-mRNAs, known lncRNAs, or known non-protein-coding RNAs, while public RNA-seq data perfectly agreed with the endpoints of tRNA-lookalikes. Interestingly, we found that tRNA-lookalikes are significantly depleted in known genetic variations associated with human health and disease whereas the known tRNAs are enriched in such variations. Lastly, a manual comparative analysis of the cloverleaf structure of several of the transcribed tRNA-lookalikes revealed no disruptive mutations suggesting the possibility that these loci give rise to functioning tRNA molecules

Angiogenin mRNA Expression Levels in Prostate Cancer Tissue

Introduction: Prostate cancer is the most commonly diagnosed cancer in men and second leading cause of cancer deaths. Studies have shown that tRNA fragments are upregulated in prostate cancers and play important roles in carcinogenesis. This project looks at how tRNA cleaving enzyme angiogenin expression is regulated in prostate cancer tissues. Methods: Clinical data and mRNA expression levels of selected tRNA cleaving enzymes were extracted from the TCGA website. mRNAs were sequenced using IlluminaGA_RNASeqV2 at University of North Carolina. Results: 546 samples from 494 patients, with normal tissue from 53 patients were collected. ANG mRNA levels were lower in patients with higher Gleason scores(Intercept=1321.787362, regression coefficient= -87.05499452, R2=0.038). ANG mRNA levels were inconclusive in different clinical T grade(p=0.15), but were lower in higher pathologic T grade(intercept=1100.484695, x variable=-166.9047227, R2=0.038); ANG expression was lower in patients with nodal involvement versus without(539.56 vs 673.58, p=0.005). Discussion: Overall trend we found from the results were ANG mRNA expression levels are down regulated in patients that have more advanced disease versus early disease. This supports the hypothesis that ANG expression plays an interesting role in prostate cancer biology. This trend might be due to the negative feedback due to high levels of tRNA fragments however there is no single theory available to answer this question