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/.

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.

Ribosomal RNA fragmentation into short RNAs (rRFs) is modulated in a sex- and population of origin-specific manner.

BACKGROUND: The advent of next generation sequencing (NGS) has allowed the discovery of short and long non-coding RNAs (ncRNAs) in an unbiased manner using reverse genetics approaches, enabling the discovery of multiple categories of ncRNAs and characterization of the way their expression is regulated. We previously showed that the identities and abundances of microRNA isoforms (isomiRs) and transfer RNA-derived fragments (tRFs) are tightly regulated, and that they depend on a person's sex and population origin, as well as on tissue type, tissue state, and disease type. Here, we characterize the regulation and distribution of fragments derived from ribosomal RNAs (rRNAs). rRNAs form a group that includes four (5S, 5.8S, 18S, 28S) rRNAs encoded by the human nuclear genome and two (12S, 16S) by the mitochondrial genome. rRNAs constitute the most abundant RNA type in eukaryotic cells. RESULTS: We analyzed rRNA-derived fragments (rRFs) across 434 transcriptomic datasets obtained from lymphoblastoid cell lines (LCLs) derived from healthy participants of the 1000 Genomes Project. The 434 datasets represent five human populations and both sexes. We examined each of the six rRNAs and their respective rRFs, and did so separately for each population and sex. Our analysis shows that all six rRNAs produce rRFs with unique identities, normalized abundances, and lengths. The rRFs arise from the 5'-end (5'-rRFs), the interior (i-rRFs), and the 3'-end (3'-rRFs) or straddle the 5' or 3' terminus of the parental rRNA (x-rRFs). Notably, a large number of rRFs are produced in a population-specific or sex-specific manner. Preliminary evidence suggests that rRF production is also tissue-dependent. Of note, we find that rRF production is not affected by the identity of the processing laboratory or the library preparation kit. CONCLUSIONS: Our findings suggest that rRFs are produced in a regimented manner by currently unknown processes that are influenced by both ubiquitous as well as population-specific and sex-specific factors. The properties of rRFs mirror the previously reported properties of isomiRs and tRFs and have implications for the study of homeostasis and disease.

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/.

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.

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.

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.

"Off-Spotter": very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs.

BACKGROUND: CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases) is a powerful component of the prokaryotic immune system that has been adapted for targeted genetic engineering in higher organisms. A key element of CRISPR/Cas is the "guide" RNA (gRNA) that is ~20 nucleotides (nts) in length and designed to be complementary to the intended target site. An integral requirement of the CRISPR/Cas system is that the target site be followed by a protospacer adjacent motif (PAM). Care needs to be exercised during gRNA design to avoid unintended ("off-target") interactions. RESULTS: We designed and implemented the Off-Spotter algorithm to assist with the design of optimal gRNAs. When presented with a candidate gRNA sequence and a PAM, Off-Spotter quickly and exhaustively identifies all genomic sites that satisfy the PAM constraint and are identical or nearly-identical to the provided gRNA. Off-Spotter achieves its extreme performance through purely algorithmic means and not through hardware accelerators such as graphical processing units (GPUs). Off-Spotter also allows the user to identify on-the-fly how many and which nucleotides of the gRNA comprise the "seed". Off-Spotter's output includes a histogram showing the number of potential off-targets as a function of the number of mismatches. The output also includes for each potential off-target the site's genomic location, a human genome browser hyperlink to the corresponding location, genomic annotation in the vicinity of the off-target, GC content, etc. CONCLUSION: Off-Spotter is very fast and flexible and can help in the design of optimal gRNAs by providing several PAM choices, a run-time definition of the seed and of the allowed number of mismatches, and a flexible output interface that allows sorting of the results, optional viewing/hiding of columns, etc. A key element of Off-Spotter is that it does not have a rigid definition of the seed: instead, the user can declare both the seed's location and extent on-the-fly. We expect that this flexibility in combination with Off-Spotter's speed and richly annotated output will enable experimenters to interactively and quickly explore different scenarios and gRNA possibilities.