Aristeidis G. Telonis

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- And tissue-specific microRNAs

Significance MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology. Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Beyond the one-locus-one-miRNA paradigm: microRNA isoforms enable deeper insights into breast cancer heterogeneity

Here we describe our study of miRNA isoforms (isomiRs) in breast cancer (BRCA) and normal breast data sets from the Cancer Genome Atlas (TCGA) repository. We report that the full isomiR profiles, from both known and novel human-specific miRNA loci, are particularly rich in information and can distinguish tumor from normal tissue much better than the archetype miRNAs. IsomiR expression is also dependent on the patients race, exemplified by miR-183-5p, several isomiRs of which are upregulated in triple negative BRCA in white but not black women. Additionally, we find that an isomiRs 5′ endpoint and length, but not the genomic origin, are key determinants of the regulation of its expression. Overexpression of distinct miR-183-5p isomiRs in MDA-MB-231 cells followed by microarray analysis revealed that each isomiR has a distinct impact on the cellular transcriptome. Parallel integrative analysis of mRNA expression from BRCA data sets of the TCGA repository demonstrated that isomiRs can distinguish between the luminal A and luminal B subtypes and explain in more depth the molecular differences between them than the archetype molecules. In conclusion, our findings provide evidence that post-transcriptional studies of BRCA will benefit from transcending the one-locus-one-miRNA paradigm and taking into account all isoforms from each miRNA locus as well as the patients race.

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR’s role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

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

Abstract 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.

Plasma Metabolomic Profiling Suggests Early Indications for Predisposition to Latent Insulin Resistance in Children Conceived by ICSI

Background There have been increasing indications about an epigenetically-based elevated predisposition of assisted reproductive technology (ART) offspring to insulin resistance, which can lead to an unfavorable cardio-metabolic profile in adult life. However, the relevant long-term systematic molecular studies are limited, especially for the IntraCytoplasmic Sperm Injection (ICSI) method, introduced in 1992. In this study, we carefully defined a group of 42 prepubertal ICSI and 42 naturally conceived (NC) children. We assessed differences in their metabolic profile based on biochemical measurements, while, for a subgroup, plasma metabolomic analysis was also performed, investigating any relevant insulin resistance indices. Methods & Results Auxological and biochemical parameters of 42 6.8±2.1 yrs old ICSI-conceived and 42 age-matched controls were measured. Significant differences between the groups were determined using univariate and multivariate statistics, indicating low urea and low-grade inflammation markers (YKL-40, hsCRP) and high triiodothyronine (T3) in ICSI-children compared to controls. Moreover, plasma metabolomic analysis carried out for a subgroup of 10 ICSI- and 10 NC girls using Gas Chromatography-Mass Spectrometry (GC-MS) indicated clear differences between the two groups, characterized by 36 metabolites linked to obesity, insulin resistance and metabolic syndrome. Notably, the distinction between the two girl subgroups was accentuated when both their biochemical and metabolomic measurements were employed. Conclusions The present study contributes a large auxological and biochemical dataset of a well-defined group of pre-pubertal ICSI-conceived subjects to the research of the ART effect to the offsprings health. Moreover, it is the first time that the relevant usefulness of metabolomics was investigated. The acquired results are consistent with early insulin resistance in ICSI-offspring, paving the way for further systematic investigations. These data support that metabolomics may unravel metabolic differences before they become clinically or biochemically evident, underlining its utility in the ART research.

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.

MINTbase: a framework for the interactive exploration of mitochondrial and nuclear tRNA fragments

Motivation: It has been known that mature transfer RNAs (tRNAs) that are encoded in the nuclear genome give rise to short molecules, collectively known as tRNA fragments or tRFs. Recently, we reported that, in healthy individuals and in patients, tRFs are constitutive, arise from mitochondrial as well as from nuclear tRNAs, and have composition and abundances that depend on a person’s sex, population origin and race as well as on tissue, disease and disease subtype. Our findings as well as similar work by other groups highlight the importance of tRFs and presage an increase in the community’s interest in elucidating the roles of tRFs in health and disease. Results: We created MINTbase, a web-based framework that serves the dual-purpose of being a content repository for tRFs and a tool for the interactive exploration of these newly discovered molecules. A key feature of MINTbase is that it deterministically and exhaustively enumerates all possible genomic locations where a sequence fragment can be found and indicates which fragments are exclusive to tRNA space, and thus can be considered as tRFs: this is a very important consideration given that the genomes of higher organisms are riddled with partial tRNA sequences and with tRNA-lookalikes whose aberrant transcripts can be mistaken for tRFs. MINTbase is extremely flexible and integrates and presents tRF information from multiple yet interconnected vantage points (‘vistas’). Vistas permit the user to interactively personalize the information that is returned and the manner in which it is displayed. MINTbase can report comparative information on how a tRF is distributed across all anticodon/amino acid combinations, provides alignments between a tRNA and multiple tRFs with which the user can interact, provides details on published studies that reported a tRF as expressed, etc. Importantly, we designed MINTbase to contain all possible tRFs that could ever be produced by mature tRNAs: this allows us to report on their genomic distributions, anticodon/amino acid properties, alignments, etc. while giving users the ability to at-will investigate candidate tRF molecules before embarking on focused experimental explorations. Lastly, we also introduce a new labeling scheme that is tRF-sequence-based and allows users to associate a tRF with a universally unique label (‘tRF-license plate’) that is independent of a genome assembly and does not require any brokering mechanism. Availability and Implementation: MINTbase is freely accessible at http://cm.jefferson.edu/MINTbase/. Dataset submissions to MINTbase can be initiated at http://cm.jefferson.edu/MINTsubmit/. Contact: isidore.rigoutsos@jefferson.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Consequential considerations when mapping tRNA fragments.

We examine several of the choices that went into the design of tDRmapper, a recently reported tool for identifying transfer RNA (tRNA) fragments in deep sequencing data, evaluate them in the context of currently available knowledge, and discuss their potential impact on the output that the tool generates.

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

BackgroundNon-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.ResultsWe 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.ConclusionsThe 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.

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.