Rapid adaptation of cellular metabolic rate to the MicroRNA complements of mammals and its relevance to the evolution of endothermy.

The metabolic efficiency of mammalian cells depends on the attenuation of intrinsic translation noise by microRNAs. We devised a metric of cellular metabolic rate (cMR), rMR/Mexp optimally fit to the number of microRNA families (mirFam), that is robust to variation in mass and sensitive to body temperature (Tb), consistent with the heat dissipation limit theory of Speakman and Król (2010). Using mirFam as predictor, an Ornstein-Uhlenbeck process of stabilizing selection, with an adaptive shift at the divergence of Boreoeutheria, accounted for 95% of the variation in cMR across mammals. Branchwise rates of evolution of cMR, mirFam and Tb concurrently increased 6- to 7-fold at the divergence of Boreoeutheria, independent of mass. Cellular MR variation across placental mammals was also predicted by the sum of model conserved microRNA-target interactions, revealing an unexpected degree of integration of the microRNA-target apparatus into the energy economy of the mammalian cell.

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Accurate microRNA annotation of animal genomes using trained covariance models of curated microRNA complements in MirMachine.

The annotation of microRNAs depends on the availability of transcriptomics data and expert knowledge. This has led to a gap between the availability of novel genomes and high-quality microRNA complements. Using >16,000 microRNAs from the manually curated microRNA gene database MirGeneDB, we generated trained covariance models for all conserved microRNA families. These models are available in our tool MirMachine, which annotates conserved microRNAs within genomes. We successfully applied MirMachine to a range of animal species, including those with large genomes and genome duplications and extinct species, where small RNA sequencing is hard to achieve. We further describe a microRNA score of expected microRNAs that can be used to assess the completeness of genome assemblies. MirMachine closes a long-persisting gap in the microRNA field by facilitating automated genome annotation pipelines and deeper studies into the evolution of genome regulation, even in extinct organisms.

Historical RNA expression profiles from the extinct Tasmanian tiger.

Paleogenomics continues to yield valuable insights into the evolution, population dynamics, and ecology of our ancestors and other extinct species. However, DNA sequencing cannot reveal tissue-specific gene expression, cellular identity, or gene regulation, which are only attainable at the transcriptional level. Pioneering studies have shown that useful RNA can be extracted from ancient specimens preserved in permafrost and historical skins from extant canids, but no attempts have been made so far on extinct species. We extract, sequence, and analyze historical RNA from muscle and skin tissue of a ∼130-year-old Tasmanian tiger (Thylacinus cynocephalus) preserved in desiccation at room temperature in a museum collection. The transcriptional profiles closely resemble those of extant species, revealing specific anatomical features such as slow muscle fibers or blood infiltration. Metatranscriptomic analysis, RNA damage, tissue-specific RNA profiles, and expression hotspots genome-wide further confirm the thylacine origin of the sequences. RNA sequences are used to improve protein-coding and noncoding annotations, evidencing missing exonic loci and the location of ribosomal RNA genes while increasing the number of annotated thylacine microRNAs from 62 to 325. We discover a thylacine-specific microRNA isoform that could not have been confirmed without RNA evidence. Finally, we detect traces of RNA viruses, suggesting the possibility of profiling viral evolution. Our results represent the first successful attempt to obtain transcriptional profiles from an extinct animal species, providing thought-to-be-lost information on gene expression dynamics. These findings hold promising implications for the study of RNA molecules across the vast collections of natural history museums and from well-preserved permafrost remains.

Direct observation of the evolution of cell-type-specific microRNA expression signatures supports the hematopoietic origin model of endothelial cells.

The evolution of specialized cell-types is a long-standing interest of biologists, but given the deep time-scales very difficult to reconstruct or observe. microRNAs have been linked to the evolution of cellular complexity and may inform on specialization. The endothelium is a vertebrate-specific specialization of the circulatory system that enabled a critical new level of vasoregulation. The evolutionary origin of these endothelial cells is unclear. We hypothesized that Mir-126, an endothelial cell-specific microRNA may be informative. We here reconstruct the evolutionary history of Mir-126. Mir-126 likely appeared in the last common ancestor of vertebrates and tunicates, which was a species without an endothelium, within an intron of the evolutionary much older EGF Like Domain Multiple (Egfl) locus. Mir-126 has a complex evolutionary history due to duplications and losses of both the host gene and the microRNA. Taking advantage of the strong evolutionary conservation of the microRNA among Olfactores, and using RNA in situ hybridization, we localized Mir-126 in the tunicate Ciona robusta. We found exclusive expression of the mature Mir-126 in granular amebocytes, supporting a long-proposed scenario that endothelial cells arose from hemoblasts, a type of proto-endothelial amoebocyte found throughout invertebrates. This observed change of expression of Mir-126 from proto-endothelial amoebocytes in the tunicate to endothelial cells in vertebrates is the first direct observation of the evolution of a cell-type in relation to microRNA expression indicating that microRNAs can be a prerequisite of cell-type evolution.

MicroRNAs are deeply linked to the emergence of the complex octopus brain.

Soft-bodied cephalopods such as octopuses are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled messenger RNAs and small RNAs in three cephalopod species including 18 tissues of the Octopus vulgaris. We show that the major RNA innovation of soft-bodied cephalopods is an expansion of the microRNA (miRNA) gene repertoire. These evolutionarily novel miRNAs were primarily expressed in adult neuronal tissues and during the development and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, notably, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains.

sRNAbench and sRNAtoolbox 2022 update: accurate miRNA and sncRNA profiling for model and non-model organisms.

The NCBI Sequence Read Archive currently hosts microRNA sequencing data for over 800 different species, evidencing the existence of a broad taxonomic distribution in the field of small RNA research. Simultaneously, the number of samples per miRNA-seq study continues to increase resulting in a vast amount of data that requires accurate, fast and user-friendly analysis methods. Since the previous release of sRNAtoolbox in 2019, 55 000 sRNAbench jobs have been submitted which has motivated many improvements in its usability and the scope of the underlying annotation database. With this update, users can upload an unlimited number of samples or import them from Google Drive, Dropbox or URLs. Micro- and small RNA profiling can now be carried out using high-confidence Metazoan and plant specific databases, MirGeneDB and PmiREN respectively, together with genome assemblies and libraries from 441 Ensembl species. The new results page includes straightforward sample annotation to allow downstream differential expression analysis with sRNAde. Unassigned reads can also be explored by means of a new tool that performs mapping to microbial references, which can reveal contamination events or biologically meaningful findings as we describe in the example. sRNAtoolbox is available at: https://arn.ugr.es/srnatoolbox/.

The limits of human microRNA annotation have been met.

Over the last few years, the number of microRNAs in the human genome has become a controversially debated issue. Several publications reported thousands of putative novel microRNAs not included in the curated microRNA gene database MirGeneDB and the repository miRBase. Recently, by using sequencing of ∼300 human tissues and cell lines, the human RNA atlas, an expanded inventory of human RNA annotations, was published, reporting thousands of putative microRNAs. We, the developers of established microRNA prediction tools and hosts of MirGeneDB, raise concerns about the frequently applied prediction and functional validation strategies, briefly discussing the drawbacks of false positive detections. By means of quantifying well-established biogenesis-derived features, we show that the reported novel microRNAs essentially represent false-positives and argue that the human microRNA complement, at about 550 microRNA genes, is already near complete. Output of available tools must be curated as false predictions will misguide scientists looking for biomarkers or therapeutic targets.

A comprehensive framework for analysis of microRNA sequencing data in metastatic colorectal cancer.

Although microRNAs (miRNAs) contribute to all hallmarks of cancer, miRNA dysregulation in metastasis remains poorly understood. The aim of this work was to reliably identify miRNAs associated with metastatic progression of colorectal cancer (CRC) using novel and previously published next-generation sequencing (NGS) datasets generated from 268 samples of primary (pCRC) and metastatic CRC (mCRC; liver, lung and peritoneal metastases) and tumor adjacent tissues. Differential expression analysis was performed using a meticulous bioinformatics pipeline, including only bona fide miRNAs, and utilizing miRNA-tailored quality control and processing. Five miRNAs were identified as up-regulated at multiple metastatic sites Mir-210_3p, Mir-191_5p, Mir-8-P1b_3p [mir-141-3p], Mir-1307_5p and Mir-155_5p. Several have previously been implicated in metastasis through involvement in epithelial-to-mesenchymal transition and hypoxia, while other identified miRNAs represent novel findings. The use of a publicly available pipeline facilitates reproducibility and allows new datasets to be added as they become available. The set of miRNAs identified here provides a reliable starting-point for further research into the role of miRNAs in metastatic progression.

MirGeneDB 2.1: toward a complete sampling of all major animal phyla.

We describe an update of MirGeneDB, the manually curated microRNA gene database. Adhering to uniform and consistent criteria for microRNA annotation and nomenclature, we substantially expanded MirGeneDB with 30 additional species representing previously missing metazoan phyla such as sponges, jellyfish, rotifers and flatworms. MirGeneDB 2.1 now consists of 75 species spanning over ∼800 million years of animal evolution, and contains a total number of 16 670 microRNAs from 1549 families. Over 6000 microRNAs were added in this update using ∼550 datasets with ∼7.5 billion sequencing reads. By adding new phylogenetically important species, especially those relevant for the study of whole genome duplication events, and through updating evolutionary nodes of origin for many families and genes, we were able to substantially refine our nomenclature system. All changes are traceable in the specifically developed MirGeneDB version tracker. The performance of read-pages is improved and microRNA expression matrices for all tissues and species are now also downloadable. Altogether, this update represents a significant step toward a complete sampling of all major metazoan phyla, and a widely needed foundation for comparative microRNA genomics and transcriptomics studies. MirGeneDB 2.1 is part of RNAcentral and Elixir Norway, publicly and freely available at http://www.mirgenedb.org/.

MicroRNAs as Indicators into the Causes and Consequences of Whole-Genome Duplication Events.

Whole-genome duplications (WGDs) have long been considered the causal mechanism underlying dramatic increases to morphological complexity due to the neo-functionalization of paralogs generated during these events. Nonetheless, an alternative hypothesis suggests that behind the retention of most paralogs is not neo-functionalization, but instead the degree of the inter-connectivity of the intended gene product, as well as the mode of the WGD itself. Here, we explore both the causes and consequences of WGD by examining the distribution, expression, and molecular evolution of microRNAs (miRNAs) in both gnathostome vertebrates as well as chelicerate arthropods. We find that although the number of miRNA paralogs tracks the number of WGDs experienced within the lineage, few of these paralogs experienced changes to the seed sequence, and thus are functionally equivalent relative to their mRNA targets. Nonetheless, in gnathostomes, although the retention of paralogs following the 1R autotetraploidization event is similar across the two subgenomes, the paralogs generated by the gnathostome 2R allotetraploidization event are retained in higher numbers on one subgenome relative to the second, with the miRNAs found on the preferred subgenome showing both higher expression of mature miRNA transcripts and slower molecular evolution of the precursor miRNA sequences. Importantly, WGDs do not result in the creation of miRNA novelty, nor do WGDs correlate to increases in complexity. Instead, it is the number of miRNA seed sequences in the genome itself that not only better correlate to instances in complexification, but also mechanistically explain why complexity increases when new miRNA families are established.

T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases.

BACKGROUND: Colorectal liver metastasis (CLM) is a leading cause of colorectal cancer mortality, and the response to immune checkpoint inhibition (ICI) in microsatellite-stable CRC has been disappointing. Administration of cytotoxic chemotherapy may cause increased density of tumor-infiltrating T cells, which has been associated with improved response to ICI. This study aimed to quantify and characterize T-cell infiltration in CLM using T-cell receptor (TCR) repertoire sequencing. Eighty-five resected CLMs from patients included in the Oslo CoMet study were subjected to TCR repertoire sequencing. Thirty-five and 15 patients had received neoadjuvant chemotherapy (NACT) within a short or long interval, respectively, prior to resection, while 35 patients had not been exposed to NACT. T-cell fractions were calculated, repertoire clonality was analyzed based on Hill evenness curves, and TCR sequence convergence was assessed using network analysis. RESULTS: Increased T-cell fractions (10.6% vs. 6.3%) were detected in CLMs exposed to NACT within a short interval prior to resection, while modestly increased clonality was observed in NACT-exposed tumors independently of the timing of NACT administration and surgery. While private clones made up >90% of detected clones, network connectivity analysis revealed that public clones contributed the majority of TCR sequence convergence. CONCLUSIONS: TCR repertoire sequencing can be used to quantify T-cell infiltration and clonality in clinical samples. This study provides evidence to support chemotherapy-driven T-cell clonal expansion in CLM in a clinical context.

MapToCleave: High-throughput profiling of microRNA biogenesis in living cells.

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

In Silico Analysis of Micro-RNA Sequencing Data.

High-throughput sequencing for micro-RNAs (miRNAs) to obtain expression estimates is a central method of molecular biology. Surprisingly, there are a number of different approaches to converting sequencing output into micro-RNA counts. Each has their own strengths and biases that impact on the final data that can be obtained from a sequencing run. This chapter serves to make the reader aware of the trade-offs one must consider in analyzing small RNA sequencing data. It then compares two methods, miRge2.0 and the sRNAbench and the steps utilized to output data from their tools.

Digging into bivalve miRNAomes: between conservation and innovation.

Bivalves are a diverse mollusc group of economic and ecological importance. An evident resilience to pollution, parasites and extreme environments makes some bivalve species important models for studying adaptation and immunity. Despite substantial progress in sequencing projects of bivalves, information on non-coding genes and gene-regulatory aspects is still lacking. Here, we review the current repertoire of bivalve microRNAs (miRNAs), important regulators of gene expression in Metazoa. We exploited available short non-coding RNA (sncRNA) data for Pinctada martensii, Crassostrea gigas, Corbicula fluminea, Tegillarca granosa and Ruditapes philippinarum, and we produced new sncRNA data for two additional bivalves, the Mediterranean mussel Mytilus galloprovincialis and the blood clam Scapharca broughtonii. We found substantial heterogeneity and incorrect annotations of miRNAs; hence, we reannotated conserved miRNA families using recently established criteria for bona fide microRNA annotation. We found 106 miRNA families missing in the previously published bivalve datasets and 89 and 87 miRNA complements were identified in the two additional species. The overall results provide a homogeneous and evolutionarily consistent picture of miRNAs in bivalves and enable future comparative studies. The identification of two bivalve-specific miRNA families sheds further light on the complexity of transcription and its regulation in bivalve molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

A comparative analysis of heart microRNAs in vertebrates brings novel insights into the evolution of genetic regulatory networks.

BACKGROUND: During vertebrate evolution, the heart has undergone remarkable changes that lead to morphophysiological differences in the fully formed heart of these species, such as chamber septation, heart rate frequency, blood pressure, and cardiac output volume. Despite these differences, the heart developmental process is guided by a core gene set conserved across vertebrates. Nonetheless, the regulatory mechanisms controlling the expression of genes involved in heart development and maintenance are largely uncharted. MicroRNAs (miRNAs) have been described as important regulatory elements in several biological processes, including heart biology. These small RNA molecules are broadly conserved in sequence and genomic context in metazoans. Mutations may occur in miRNAs and/or genes that contribute to the establishment of distinct repertoires of miRNA-target interactions, thereby favoring the differential control of gene expression and, consequently, the origin of novel phenotypes. In fact, several studies showed that miRNAs are integrated into genetic regulatory networks (GRNs) governing specific developmental programs and diseases. However, studies integrating miRNAs in vertebrate heart GRNs under an evolutionary perspective are still scarce. RESULTS: We comprehensively examined and compared the heart miRNome of 20 species representatives of the five major vertebrate groups. We found 54 miRNA families with conserved expression and a variable number of miRNA families with group-specific expression in fishes, amphibians, reptiles, birds, and mammals. We also detected that conserved miRNAs present higher expression levels and a higher number of targets, whereas the group-specific miRNAs present lower expression levels and few targets. CONCLUSIONS: Both the conserved and group-specific miRNAs can be considered modulators orchestrating the core and peripheral genes of heart GRNs of vertebrates, which can be related to the morphophysiological differences and similarities existing in the heart of distinct vertebrate groups. We propose a hypothesis to explain evolutionary differences in the putative functional roles of miRNAs in the heart GRNs analyzed. Furthermore, we present new insights into the molecular mechanisms that could be helping modulate the diversity of morphophysiology in the heart organ of vertebrate species.

Differential expression of microRNAs and tRNA fragments mediate the adaptation of the liver fluke Fasciola gigantica to its intermediate snail and definitive mammalian hosts.

The tropical liver fluke Fasciola gigantica affects livestock and humans in many Asian countries, large parts of Africa, and parts of Europe. Despite the public health and economic impacts of F. gigantica, understanding of F. gigantica biology and how the complex lifecycle of this liver fluke is transcriptionally regulated remain unknown. Here, we tested the hypothesis that the regulatory small non-coding RNAs (sncRNAs), microRNAs (miRNAs) and tRNA-derived fragments (tRFs) play roles in the adaptation of F. gigantica to its intermediate and definitive hosts. We sequenced sncRNAs of eight lifecycle stages of F. gigantica. In total, 56 miRNAs from 33 conserved families and four Fasciola-specific miRNAs were identified. Expression analysis of miRNAs suggested clear stage-related patterns. By leveraging the existing transcriptomic data, we predicted a miRNA-based regulation of metabolism, transport, growth and developmental processes. Also, by comparing miRNA complement of F. gigantica with that of Fasciola hepatica, we detected a high level of conservation and identified differences in some miRNAs, which can be used to distinguish the two species. Moreover, we found that tRFs at each lifecycle stage were predominantly derived by tRNA-Lys and tRNA-Gly at 5' half sites, but relatively high expression was related to the buffalo-infecting stages. Taken together, we provided a comprehensive overview of the dynamic transcriptional changes of small RNAs that occur during the developmental stages of F. gigantica. This global analysis of F. gigantica lifecycle stages revealed new roles of miRNAs and tRFs in parasite development and will facilitate future research into understanding of fasciolosis pathobiology.

Encyclopedia of tools for the analysis of miRNA isoforms.

RNA sequencing data sets rapidly increase in quantity. For microRNAs (miRNAs), frequently dozens to hundreds of billion reads are generated per study. The quantification of annotated miRNAs and the prediction of new miRNAs are leading computational tasks. Now, the increased depth of coverage allows to gain deeper insights into the variability of miRNAs. The analysis of isoforms of miRNAs (isomiRs) is a trending topic, and a range of computational tools for the analysis of isomiRs has been developed. We provide an overview on 27 available computational solutions for the analysis of isomiRs. These include both stand-alone programs (17 tools) and web-based solutions (10 tools) and span a publication time range from 2010 to 2020. Seven of the tools were published in 2019 and 2020, confirming the rising importance of the topic. While most of the analyzed tools work for a broad range of organisms or are completely independent of a reference organism, several tools have been tailored for the analysis of human miRNA data or for plants. While 14 of the tools are general analysis tools of miRNAs, and isomiR analysis is one of their features, the remaining 13 tools have specifically been developed for isomiR analysis. A direct comparison on 20 deep sequencing data sets for selected tools provides insights into the heterogeneity of results. With our work, we provide users a comprehensive overview on the landscape of isomiR analysis tools and in that support the selection of the most appropriate tool for their respective research task.