Adaptive regulation of testis gene expression and control of male fertility by the Drosophila hairpin RNA pathway. [Corrected].

Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-ß in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs.

Correction: Correction: A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa.

[This corrects the article DOI: 10.1371/journal.ppat.1002924.].

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.

MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.

Formation, regulation and evolution of Caenorhabditis elegans 3'UTRs.

Post-transcriptional gene regulation frequently occurs through elements in mRNA 3' untranslated regions (UTRs). Although crucial roles for 3'UTR-mediated gene regulation have been found in Caenorhabditis elegans, most C. elegans genes have lacked annotated 3'UTRs. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3'UTRs. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3'UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3'UTRs have median length only one-sixth that of mammalian 3'UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation.

PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.

In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.

The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds.

Berninamycin is a member of the pyridine-containing thiopeptide class of antibiotics that undergoes massive posttranslational modifications from ribosomally generated preproteins. Berninamycin has a 2-oxazolyl-3-thiazolyl-pyridine core embedded in a 35-atom macrocycle rather than typical trithiazolylpyridine cores embedded in 26-atom and 29-atom peptide macrocycles. We describe the cloning of an 11-gene berninamycin cluster from Streptomyces bernensis UC 5144, its heterologous expression in Streptomyces lividans TK24 and Streptomyces venezuelae ATCC 10712, and detection of variant and incompletely processed scaffolds. Posttranslational maturation in S. lividans of both the wild-type berninamycin prepeptide (BerA) and also a T3A mutant generates macrocyclic compounds as well as linear variants, which have failed to form the pyridine and the macrocycle. Expression of the gene cluster in S. venezuelae generates a variant of the 35-atom skeleton of berninamycin, containing a methyloxazoline in the place of a methyloxazole within the macrocyclic framework.

Assemblathon 1: a competitive assessment of de novo short read assembly methods.

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.

A novel rhabdovirus associated with acute hemorrhagic fever in central Africa.

Deep sequencing was used to discover a novel rhabdovirus (Bas-Congo virus, or BASV) associated with a 2009 outbreak of 3 human cases of acute hemorrhagic fever in Mangala village, Democratic Republic of Congo (DRC), Africa. The cases, presenting over a 3-week period, were characterized by abrupt disease onset, high fever, mucosal hemorrhage, and, in two patients, death within 3 days. BASV was detected in an acute serum sample from the lone survivor at a concentration of 1.09 × 10(6) RNA copies/mL, and 98.2% of the genome was subsequently de novo assembled from ≈ 140 million sequence reads. Phylogenetic analysis revealed that BASV is highly divergent and shares less than 34% amino acid identity with any other rhabdovirus. High convalescent neutralizing antibody titers of >1:1000 were detected in the survivor and an asymptomatic nurse directly caring for him, both of whom were health care workers, suggesting the potential for human-to-human transmission of BASV. The natural animal reservoir host or arthropod vector and precise mode of transmission for the virus remain unclear. BASV is an emerging human pathogen associated with acute hemorrhagic fever in Africa.

The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs.

In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology. Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate approximately 21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila.

Five years later, with double the demographic data, naked mole-rat mortality rates continue to defy Gompertzian laws by not increasing with age.

The naked mole-rat (Heterocephalus glaber) is a mouse-sized rodent species, notable for its eusociality and long lifespan. Previously, we reported that demographic aging, i.e., the exponential increase of mortality hazard that accompanies advancing age in mammals and other organisms, does not occur in naked mole-rats (Ruby et al., 2018), a finding that has potential implications for human healthy aging. The demographic data supporting that conclusion had taken over three decades to accumulate, starting with the original rearing of H. glaber in captivity. This finding was controversial since many of the animals in that study were relatively young. In the 5 years following that study, we have doubled our quantity of demographic data. Here, we re-evaluated our prior conclusions in light of these new data and found them to be not only supported but indeed strengthened. We additionally provided insight into the social dynamics of captive H. glaber with data and analyses of body weight and colony size versus mortality. Finally, we provide a phylogenetically proximal comparator in the form of lifespan data from our Damaraland mole-rat (Fukomys damarensis) colony and demographic meta-analysis of those data along with published data from Ansell's mole-rat (Fukomys anselli). We found Fukomys mortality hazard to increase gradually with age, an observation with inferences on the evolution of exceptional lifespan among mole-rats and the ecological factors that may have accompanied that evolution.

Intronic microRNA precursors that bypass Drosha processing.

MicroRNAs (miRNAs) are approximately 22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs. In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a approximately 60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. From that duplex, the mature miRNA is incorporated into the silencing complex. Here we identify an alternative pathway for miRNA biogenesis, in which certain debranched introns mimic the structural features of pre-miRNAs to enter the miRNA-processing pathway without Drosha-mediated cleavage. We call these pre-miRNAs/introns 'mirtrons', and have identified 14 mirtrons in Drosophila melanogaster and another four in Caenorhabditis elegans (including the reclassification of mir-62). Some of these have been selectively maintained during evolution with patterns of sequence conservation suggesting important regulatory functions in the animal. The abundance of introns comparable in size to pre-miRNAs appears to have created a context favourable for the emergence of mirtrons in flies and nematodes. This suggests that other lineages with many similarly sized introns probably also have mirtrons, and that the mirtron pathway could have provided an early avenue for the emergence of miRNAs before the advent of Drosha.

Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures.

Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or 'evolutionary signatures', dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.

Genetics implicates overactive osteogenesis in the development of diffuse idiopathic skeletal hyperostosis.

Diffuse idiopathic skeletal hyperostosis (DISH) is a condition where adjacent vertebrae become fused through formation of osteophytes. The genetic and epidemiological etiology of this condition is not well understood. Here, we implemented a machine learning algorithm to assess the prevalence and severity of the pathology in ~40,000 lateral DXA scans in the UK Biobank Imaging cohort. We find that DISH is highly prevalent, above the age of 45, ~20% of men and ~8% of women having multiple osteophytes. Surprisingly, we find strong phenotypic and genetic association of DISH with increased bone mineral density and content throughout the entire skeletal system. Genetic association analysis identified ten loci associated with DISH, including multiple genes involved in bone remodeling (RUNX2, IL11, GDF5, CCDC91, NOG, and ROR2). Overall, this study describes genetics of DISH and implicates the role of overactive osteogenesis as a key driver of the pathology.

3D mouse pose from single-view video and a new dataset.

We present a method to infer the 3D pose of mice, including the limbs and feet, from monocular videos. Many human clinical conditions and their corresponding animal models result in abnormal motion, and accurately measuring 3D motion at scale offers insights into health. The 3D poses improve classification of health-related attributes over 2D representations. The inferred poses are accurate enough to estimate stride length even when the feet are mostly occluded. This method could be applied as part of a continuous monitoring system to non-invasively measure animal health, as demonstrated by its use in successfully classifying animals based on age and genotype. We introduce the Mouse Pose Analysis Dataset, the first large scale video dataset of lab mice in their home cage with ground truth keypoint and behavior labels. The dataset also contains high resolution mouse CT scans, which we use to build the shape models for 3D pose reconstruction.

An Automated, Home-Cage, Video Monitoring-based Mouse Frailty Index Detects Age-associated Morbidity in C57BL/6 and Diversity Outbred Mice.

Frailty indexes (FIs) provide quantitative measurements of nonspecific health decline and are particularly useful as longitudinal monitors of morbidity in aging studies. For mouse studies, frailty assessments can be taken noninvasively, but they require handling and direct observation that is labor-intensive to the scientist and stress inducing to the animal. Here, we implement, evaluate, and provide a refined digital FI composed entirely of computational analyses of home-cage video and compare it to manually obtained frailty scores in both C57BL/6 and genetically heterogeneous Diversity Outbred mice. We show that the frailty scores assigned by our digital index correlate with both manually obtained frailty scores and chronological age. Thus, we provide an automated tool for frailty assessment that can be collected reproducibly, at scale, without substantial labor cost.

Merochlorins A-D, cyclic meroterpenoid antibiotics biosynthesized in divergent pathways with vanadium-dependent chloroperoxidases.

Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Herein, we present the structures of four new meroterpenoid antibiotics, merochlorins A-D, produced by the marine bacterium Streptomyces sp. strain CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents. Draft genome sequencing, mutagenesis, and heterologous biosynthesis in the genome-minimized model actinomycete Streptomyces coelicolor provided the 57.6 kb merochlorin gene cluster that contains two genes encoding rare bacterial vanadium-dependent haloperoxidase (VHPO) genes. Pathway expression of two different fosmid clones that differ largely by the presence or absence of the VHPO gene mcl40 resulted in the differential biosynthesis of merochlorin C, suggesting that Mcl40 catalyzes an unprecedented 15-membered chloronium-induced macrocyclization reaction converting merochlorin D to merochlorin C.

Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor.

Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.

Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis.

A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen.