MINRbase: a comprehensive database of nuclear- and mitochondrial-ribosomal-RNA-derived fragments (rRFs).

We describe the Mitochondrial and Nuclear rRNA fragment database (MINRbase), a knowledge repository aimed at facilitating the study of ribosomal RNA-derived fragments (rRFs). MINRbase provides interactive access to the profiles of 130 238 expressed rRFs arising from the four human nuclear rRNAs (18S, 5.8S, 28S, 5S), two mitochondrial rRNAs (12S, 16S) or four spacers of 45S pre-rRNA. We compiled these profiles by analyzing 11 632 datasets, including the GEUVADIS and The Cancer Genome Atlas (TCGA) repositories. MINRbase offers a user-friendly interface that lets researchers issue complex queries based on one or more criteria, such as parental rRNA identity, nucleotide sequence, rRF minimum abundance and metadata keywords (e.g. tissue type, disease). A 'summary' page for each rRF provides a granular breakdown of its expression by tissue type, disease, sex, ancestry and other variables; it also allows users to create publication-ready plots at the click of a button. MINRbase has already allowed us to generate support for three novel observations: the internal spacers of 45S are prolific producers of abundant rRFs; many abundant rRFs straddle the known boundaries of rRNAs; rRF production is regimented and depends on 'personal attributes' (sex, ancestry) and 'context' (tissue type, tissue state, disease). MINRbase is available at https://cm.jefferson.edu/MINRbase/.

The Typical tRNA Co-Expresses Multiple 5' tRNA Halves Whose Sequences and Abundances Depend on Isodecoder and Isoacceptor and Change with Tissue Type, Cell Type, and Disease.

Transfer RNA-derived fragments (tRFs) are noncoding RNAs that arise from either mature transfer RNAs (tRNAs) or their precursors. One important category of tRFs comprises the tRNA halves, which are generated through cleavage at the anticodon. A given tRNA typically gives rise to several co-expressed 5'-tRNA halves (5'-tRHs) that differ in the location of their 3' ends. These 5'-tRHs, even though distinct, have traditionally been treated as indistinguishable from one another due to their near-identical sequences and lengths. We focused on co-expressed 5'-tRHs that arise from the same tRNA and systematically examined their exact sequences and abundances across 10 different human tissues. To this end, we manually curated and analyzed several hundred human RNA-seq datasets from NCBI's Sequence Run Archive (SRA). We grouped datasets from the same tissue into their own collection and examined each group separately. We found that a given tRNA produces different groups of co-expressed 5'-tRHs in different tissues, different cell lines, and different diseases. Importantly, the co-expressed 5'-tRHs differ in their sequences, absolute abundances, and relative abundances, even among tRNAs with near-identical sequences from the same isodecoder or isoacceptor group. The findings suggest that co-expressed 5'-tRHs that are produced from the same tRNA or closely related tRNAs have distinct, context-dependent roles. Moreover, our analyses show that cell lines modeling the same tissue type and disease may not be interchangeable when it comes to experimenting with tRFs.

Roles of mitochondrial genetics in cancer metastasis.

The contributions of mitochondria to cancer have been recognized for decades. However, the focus on the metabolic role of mitochondria and the diminutive size of the mitochondrial genome compared to the nuclear genome have hindered discovery of the roles of mitochondrial genetics in cancer. This review summarizes recent data demonstrating the contributions of mitochondrial DNA (mtDNA) copy-number variants (CNVs), somatic mutations, and germline polymorphisms to cancer initiation, progression, and metastasis. The goal is to summarize accumulating data to establish a framework for exploring the contributions of mtDNA to neoplasia and metastasis.

Megakaryocyte-specific knockout of the Mir-99b/let7e/125a cluster lowers platelet count without altering platelet function.

The purpose of this research was to assess the effects of a microRNA (miRNA) cluster on platelet production. Human chromosome 19q13.41 harbors an evolutionarily conserved cluster of three miRNA genes (MIR99B, MIRLET7E, MIR125A) within 727 base-pairs. We now report that levels of miR-99b-5p, miR-let7e-5p and miR-125a-5p are strongly correlated in human platelets, and all are positively associated with platelet count, but not white blood count or hemoglobin level. Although the cluster regulates hematopoietic stem cell proliferation, the function of this genomic locus in megakaryocyte (MK) differentiation and platelet production is unknown. Furthermore, studies of individual miRNAs do not represent broader effects in the context of a cluster. To address this possibility, MK/platelet lineage-specific Mir-99b/let7e/125a knockout mice were generated. Compared to wild type littermates, cluster knockout mice had significantly lower platelet counts and reduced MK proplatelet formation, but no differences in MK numbers, ploidy, maturation or ultra-structural morphology, and no differences in platelet function. Compared to wild type littermates, knockout mice showed similar survival after pulmonary embolism. The major conclusions are that the effect of the Mir-99b/let7e/125a cluster is confined to a late stage of thrombopoiesis, and this effect on platelet number is uncoupled from platelet function.

Unraveling the role of microRNA/isomiR network in multiple primary melanoma pathogenesis.

Malignant cutaneous melanoma (CM) is a potentially lethal form of skin cancer whose worldwide incidence has been constantly increasing over the past decades. During their lifetime, about 8% of CM patients will develop multiple primary melanomas (MPMs), usually at a young age and within 3 years from the first tumor/diagnosis. With the aim of improving our knowledge on MPM biology and pathogenesis, we explored the miRNome of 24 single and multiple primary melanomas, including multiple tumors from the same patient, using a small RNA-sequencing approach. From a supervised analysis, 22 miRNAs were differentially expressed in MPM compared to single CM, including key miRNAs involved in epithelial-mesenchymal transition. The first and second melanoma from the same patient presented a different miRNA profile. Ten miRNAs, including miR-25-3p, 149-5p, 92b-3p, 211-5p, 125a-5p, 125b-5p, 205-5p, 200b-3p, 21-5p, and 146a-5p, were further validated in 47 single and multiple melanoma samples. Pathway enrichment analysis of miRNA target genes revealed a more differentiated and less invasive status of MPMs compared to CMs. Bioinformatic analyses at the miRNA isoform (isomiR) level detected a panel of highly expressed isomiRs belonging to miRNA families implicated in human tumorigenesis, including miR-200, miR-30, and miR-10 family. Moreover, we identified hsa-miR-125a-5p|0|-2 isoform as tenfold over-represented in melanoma than the canonical form and differentially expressed in MPMs arising in the same patient. Target prediction analysis revealed that the miRNA shortening could change the pattern of target gene regulation, specifically in genes implicated in cell adhesion and neuronal differentiation. Overall, we provided a putative and comprehensive characterization of the miRNA/isomiR regulatory network of MPMs, highlighting mechanisms of tumor development and molecular features differentiating this subtype from single melanomas.

IsoMiRmap: fast, deterministic and exhaustive mining of isomiRs from short RNA-seq datasets.

MOTIVATION: MicroRNA (miRNA) precursor arms give rise to multiple isoforms simultaneously called 'isomiRs.' IsomiRs from the same arm typically differ by a few nucleotides at either their 5' or 3' termini or both. In humans, the identities and abundances of isomiRs depend on a person's sex and genetic ancestry as well as on tissue type, tissue state and disease type/subtype. Moreover, nearly half of the time the most abundant isomiR differs from the miRNA sequence found in public databases. Accurate mining of isomiRs from deep sequencing data is thus important. RESULTS: We developed isoMiRmap, a fast, standalone, user-friendly mining tool that identifies and quantifies all isomiRs by directly processing short RNA-seq datasets. IsoMiRmap is a portable 'plug-and-play' tool, requires minimal setup, has modest computing and storage requirements, and can process an RNA-seq dataset with 50 million reads in just a few minutes on an average laptop. IsoMiRmap deterministically and exhaustively reports all isomiRs in a given deep sequencing dataset and quantifies them accurately (no double-counting). IsoMiRmap comprehensively reports all miRNA precursor locations from which an isomiR may be transcribed, tags as 'ambiguous' isomiRs whose sequences exist both inside and outside of the space of known miRNA sequences and reports the public identifiers of common single-nucleotide polymorphisms and documented somatic mutations that may be present in an isomiR. IsoMiRmap also identifies isomiRs with 3' non-templated post-transcriptional additions. Compared to similar tools, isoMiRmap is the fastest, reports more bona fide isomiRs, and provides the most comprehensive information related to an isomiR's transcriptional origin. AVAILABILITY AND IMPLEMENTATION: The codes for isoMiRmap are freely available at https://cm.jefferson.edu/isoMiRmap/ and https://github.com/TJU-CMC-Org/isoMiRmap/. IsomiR profiles for the datasets of the 1000 Genomes Project, spanning five population groups, and The Cancer Genome Atlas (TCGA), spanning 33 cancer studies, are also available at https://cm.jefferson.edu/isoMiRmap/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

On the expanding roles of tRNA fragments in modulating cell behavior.

The fragments that derive from transfer RNAs (tRNAs) are an emerging category of regulatory RNAs. Known as tRFs, these fragments were reported for the first time only a decade ago, making them a relatively recent addition to the ever-expanding pantheon of non-coding RNAs. tRFs are short, 16-35 nucleotides (nts) in length, and produced through cleavage of mature and precursor tRNAs at various positions. Both cleavage positions and relative tRF abundance depend strongly on context, including the tissue type, tissue state, and disease, as well as the sex, population of origin, and race/ethnicity of an individual. These dependencies increase the urgency to understand the regulatory roles of tRFs. Such efforts are gaining momentum, and comprise experimental and computational approaches. System-level studies across many tissues and thousands of samples have produced strong evidence that tRFs have important and multi-faceted roles. Here, we review the relevant literature on tRF biology in higher organisms, single cell eukaryotes, and prokaryotes.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

IsomiRs and tRNA-derived fragments are associated with metastasis and patient survival in uveal melanoma.

Uveal melanoma (UVM) is the most common primary intraocular malignancy in adults. With over 50% of patients developing metastatic disease, there is an unmet need for improved diagnostic and therapeutic options. Efforts to understand the molecular biology of the disease have revealed several markers that correlate with patient prognosis, including the copy number of chromosome 3, genetic alterations in the BAP1, EIF1AX and SF3B1 genes, and other transcriptional features. Here, we expand upon previous reports by comprehensively characterizing the short RNA-ome in 80 primary UVM tumor samples. In particular, we describe a previously unseen complex network involving numerous regulatory molecules that comprise microRNA (miRNAs), novel UVM-specific miRNA loci, miRNA isoforms (isomiRs), and tRNA-derived fragments (tRFs). Importantly, we show that the abundance profiles of isomiRs and tRFs associate with various molecular phenotypes, metastatic disease, and patient survival. Our findings suggest deep involvement of isomiRs and tRFs in the disease etiology of UVM. We posit that further study and characterization of these novel molecules will improve understanding of the mechanisms underlying UVM, and lead to the development of new diagnostic and therapeutic approaches.

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.

tRNA Fragments Show Intertwining with mRNAs of Specific Repeat Content and Have Links to Disparities.

tRNA-derived fragments (tRF) are a class of potent regulatory RNAs. We mined the datasets from The Cancer Genome Atlas (TCGA) representing 32 cancer types with a deterministic and exhaustive pipeline for tRNA fragments. We found that mitochondrial tRNAs contribute disproportionally more tRFs than nuclear tRNAs. Through integrative analyses, we uncovered a multitude of statistically significant and context-dependent associations between the identified tRFs and mRNAs. In many of the 32 cancer types, these associations involve mRNAs from developmental processes, receptor tyrosine kinase signaling, the proteasome, and metabolic pathways that include glycolysis, oxidative phosphorylation, and ATP synthesis. Even though the pathways are common to multiple cancers, the association of specific mRNAs with tRFs depends on and differs from cancer to cancer. The associations between tRFs and mRNAs extend to genomic properties as well; specifically, tRFs are positively correlated with shorter genes that have a higher density in repeats, such as ALUs, MIRs, and ERVLs. Conversely, tRFs are negatively correlated with longer genes that have a lower repeat density, suggesting a possible dichotomy between cell proliferation and differentiation. Analyses of bladder, lung, and kidney cancer data indicate that the tRF-mRNA wiring can also depend on a patient's sex. Sex-dependent associations involve cyclin-dependent kinases in bladder cancer, the MAPK signaling pathway in lung cancer, and purine metabolism in kidney cancer. Taken together, these findings suggest diverse and wide-ranging roles for tRFs and highlight the extensive interconnections of tRFs with key cellular processes and human genomic architecture. SIGNIFICANCE: Across 32 TCGA cancer contexts, nuclear and mitochondrial tRNA fragments exhibit associations with mRNAs that belong to concrete pathways, encode proteins with particular destinations, have a biased repeat content, and are sex dependent.

Profiles of miRNA Isoforms and tRNA Fragments in Prostate Cancer.

MicroRNA (miRNA) isoforms ("isomiRs") and tRNA-derived fragments ("tRFs") are powerful regulatory non-coding RNAs (ncRNAs). In human tissues, both types of molecules are abundant, with expression patterns that depend on a person's race, sex and population origin. Here, we present our analyses of the Prostate Cancer (PRAD) datasets of The Cancer Genome Atlas (TCGA) from the standpoint of isomiRs and tRFs. This study represents the first simultaneous examination of isomiRs and tRFs in a large cohort of PRAD patients. We find that isomiRs and tRFs have extensive correlations with messenger RNAs (mRNAs). These correlations are disrupted in PRAD, which suggests disruptions of the regulatory network in the disease state. Notably, we find that the profiles of isomiRs and tRFs differ in patients belonging to different races. We hope that the presented findings can lay the groundwork for future research efforts aimed at elucidating the functional roles of the numerous and distinct members of these two categories of ncRNAs that are present in PRAD.

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/.

Race Disparities in the Contribution of miRNA Isoforms and tRNA-Derived Fragments to Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a breast cancer subtype characterized by marked differences between White and Black/African-American women. We performed a systems-level analysis on datasets from The Cancer Genome Atlas to elucidate how the expression patterns of mRNAs are shaped by regulatory noncoding RNAs (ncRNA). Specifically, we studied isomiRs, that is, isoforms of miRNAs, and tRNA-derived fragments (tRF). In normal breast tissue, we observed a marked cohesiveness in both the ncRNA and mRNA layers and the associations between them. This cohesiveness was widely disrupted in TNBC. Many mRNAs become either differentially expressed or differentially wired between normal breast and TNBC in tandem with isomiR or tRF dysregulation. The affected pathways included energy metabolism, cell signaling, and immune responses. Within TNBC, the wiring of the affected pathways with isomiRs and tRFs differed in each race. Multiple isomiRs and tRFs arising from specific miRNA loci (e.g., miR-200c, miR-21, the miR-17/92 cluster, the miR-183/96/182 cluster) and from specific tRNA loci (e.g., the nuclear tRNAGly and tRNALeu, the mitochondrial tRNAVal and tRNAPro) were strongly associated with the observed race disparities in TNBC. We highlight the race-specific aspects of transcriptome wiring by discussing in detail the metastasis-related MAPK and the Wnt/ß-catenin signaling pathways, two of the many key pathways that were found differentially wired. In conclusion, by employing a data- and knowledge-driven approach, we comprehensively analyzed the normal and cancer transcriptomes to uncover novel key contributors to the race-based disparities of TNBC.Significance: This big data-driven study comparing normal and cancer transcriptomes uncovers RNA expression differences between Caucasian and African-American patients with triple-negative breast cancer that might help explain disparities in incidence and aggressive character. Cancer Res; 78(5); 1140-54. ©2017 AACR.

N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration.

BACKGROUND: Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. RESULTS: We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients' overall survival. CONCLUSIONS: The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific.

An miRNA Expression Signature for the Human Colonic Stem Cell Niche Distinguishes Malignant from Normal Epithelia.

Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial-mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778-90. ©2017 AACR.

Knowledge about the presence or absence of miRNA isoforms (isomiRs) can successfully discriminate amongst 32 TCGA cancer types.

Isoforms of human miRNAs (isomiRs) are constitutively expressed with tissue- and disease-subtype-dependencies. We studied 10 271 tumor datasets from The Cancer Genome Atlas (TCGA) to evaluate whether isomiRs can distinguish amongst 32 TCGA cancers. Unlike previous approaches, we built a classifier that relied solely on 'binarized' isomiR profiles: each isomiR is simply labeled as 'present' or 'absent'. The resulting classifier successfully labeled tumor datasets with an average sensitivity of 90% and a false discovery rate (FDR) of 3%, surpassing the performance of expression-based classification. The classifier maintained its power even after a 15× reduction in the number of isomiRs that were used for training. Notably, the classifier could correctly predict the cancer type in non-TCGA datasets from diverse platforms. Our analysis revealed that the most discriminatory isomiRs happen to also be differentially expressed between normal tissue and cancer. Even so, we find that these highly discriminating isomiRs have not been attracting the most research attention in the literature. Given their ability to successfully classify datasets from 32 cancers, isomiRs and our resulting 'Pan-cancer Atlas' of isomiR expression could serve as a suitable framework to explore novel cancer biomarkers.

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.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Post-transcriptional Regulation of BRCA2 through Interactions with miR-19a and miR-19b.

Breast cancer type 2, early onset susceptibility gene (BRCA2) is a major component of the homologous recombination DNA repair pathway. It acts as a tumor suppressor whose function is often lost in cancers. Patients with specific mutations in the BRCA2 gene often display discrete clinical, histopathological, and molecular features. However, a subset of sporadic cancers has wild type BRCA2 and display defects in the homology-directed repair pathway, which is the hallmark of 'BRCAness.' The mechanisms by which BRCAness arises are not well understood but post-transcriptional regulation of BRCA2 gene expression by microRNAs (miRNAs) may contribute to this phenotype. Here, we examine the post-transcriptional effects that some members of the six-miRNA cluster known as the miR-17/92 cluster have on the abundance of BRCA2's messenger RNA (mRNA) and protein. We discuss two interactions involving the miR-19a and miR-19b members of the cluster and the 3'UTR of BRCA2's mRNA. We investigated these miRNA:mRNA interactions in 15 cell lines derived from pancreatic, breast, colon, and kidney tissue. We show that over-expression of these two miRNAs results in a concomitant decrease of BRCA2's mRNA and protein expression in a subset of the tested cell lines. Additionally, using luciferase reporter assays we identified direct interactions between miR-19a/miR-19b and a miRNA response element (MRE) in BRCA2's 3'UTR. Our results suggest that BRCA2 is subject to a complex post-transcriptional regulatory program that has specific dependencies on the genetic and phenotypic background of cell types.