DASHR: Database of Small Human Noncoding RNAs

Small non-coding RNAs (sncRNAs) are highly abundant RNAs, typically long, that act as key regulators of diverse cellular processes. Although thousands of sncRNA genes are known to exist in the human genome, no single database provides searchable, unified annotation, and expression information for full sncRNA transcripts and mature RNA products derived from these larger RNAs. Here, we present the Database of small human noncoding RNAs (DASHR) . DASHR contains the most comprehensive information to date on human sncRNA genes and mature sncRNA products. DASHR provides a simple user interface for researchers to view sequence and secondary structure, compare expression levels, and evidence of specific processing across all sncRNA genes and mature sncRNA products in various human tissues. DASHR annotation and expression data covers all major classes of sncRNAs including microRNAs (miRNAs), Piwi-interacting (piRNAs), small nuclear, nucleolar, cytoplasmic (sn-, sno-, scRNAs, respectively), transfer (tRNAs), and ribosomal RNAs (rRNAs). Currently, DASHR (v1.0) integrates 187 smRNA high-throughput sequencing (smRNA-seq) datasets with over 2.5 billion reads and annotation data from multiple public sources. DASHR contains annotations for ~48,000 human sncRNA genes and mature sncRNA products, 82% of which are expressed in one of more of the curated tissues. DASHR is available at http://lisanwanglab.org/DASHR

Profiling of RNAs from Human Islet-Derived Exosomes in a Model of Type 1 Diabetes

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing islet β cells. Biomarkers capable of identifying T1D risk and dissecting disease-related heterogeneity represent an unmet clinical need. Toward the goal of informing T1D biomarker strategies, we profiled coding and noncoding RNAs in human islet-derived exosomes and identified RNAs that were differentially expressed under proinflammatory cytokine stress conditions. Human pancreatic islets were obtained from cadaveric donors and treated with/without IL-1β and IFN-γ. Total RNA and small RNA sequencing were performed from islet-derived exosomes to identify mRNAs, long noncoding RNAs, and small noncoding RNAs. RNAs with a fold change ≥1.3 and a p-value <0.05 were considered as differentially expressed. mRNAs and miRNAs represented the most abundant long and small RNA species, respectively. Each of the RNA species showed altered expression patterns with cytokine treatment, and differentially expressed RNAs were predicted to be involved in insulin secretion, calcium signaling, necrosis, and apoptosis. Taken together, our data identify RNAs that are dysregulated under cytokine stress in human islet-derived exosomes, providing a comprehensive catalog of protein coding and noncoding RNAs that may serve as potential circulating biomarkers in T1D

Naprt expression regulation mechanisms: novel functions predicted by a bioinformatics approach

The nicotinate phosphoribosyltransferase (NAPRT) gene has gained relevance in the research of cancer therapeutic strategies due to its main role as a NAD biosynthetic enzyme. NAD metabolism is an attractive target for the development of anti-cancer therapies, given the high energy requirements of proliferating cancer cells and NAD-dependent signaling. A few studies have shown that NAPRT expression varies in different cancer types, making it imperative to assess NAPRT expression and functionality status prior to the application of therapeutic strategies targeting NAD. In addition, the recent finding of NAPRT extracellular form (eNAPRT) suggested the involvement of NAPRT in inflammation and signaling. However, the mechanisms regulating NAPRT gene expression have never been thoroughly addressed. In this study, we searched for NAPRT gene expression regulatory mechanisms in transcription factors (TFs), RNA binding proteins (RBPs) and microRNA (miRNAs) databases. We identified several potential regulators of NAPRT transcription activation, downregulation and alternative splicing and performed GO and expression analyses. The results of the functional analysis of TFs, RBPs and miRNAs suggest new, unexpected functions for the NAPRT gene in cell differentiation, development and neuronal biology.info:eu-repo/semantics/publishedVersio

Distinct Extracellular RNA Profiles in Different Plasma Components

Circulating extracellular RNAs (exRNAs) have great potential to serve as biomarkers for a wide range of diagnostic, therapeutic, and prognostic applications. So far, knowledge of the difference among different sources of exRNAs is limited. To address this issue, we performed a sequential physical and biochemical precipitation to collect four fractions (platelets and cell debris, the thrombin-induced precipitates, extracellular vesicles, and supernatant) from each of 10 plasma samples. From total RNAs of the 40 fractions, we prepared ligation-free libraries to profile full spectrum of all RNA species, without size selection and rRNA reduction. Due to complicated RNA composition in these libraries, we utilized a successive stepwise alignment strategy to map the RNA sequences to different RNA categories, including miRNAs, piwi-interacting RNAs, tRNAs, rRNAs, lincRNAs, snoRNAs, snRNAs, other ncRNAs, protein coding RNAs, and circRNAs. Our data showed that each plasma fraction had its own unique distribution of RNA species. Hierarchical cluster analyses using transcript abundance demonstrated similarities in the same plasma fraction and significant differences between different fractions. In addition, we observed various unique transcripts, and novel predicted miRNAs among these plasma fractions. These results demonstrate that the distribution of RNA species and functional RNA transcripts is plasma fraction-dependent. Appropriate plasma preparation and thorough inspection of different plasma fractions are necessary for an exRNA-based biomarker study

Workflows for the Large-Scale Assessment of miRNA Evolution: Birth and Death of miRNA Genes in Tunicates

As described over 20 years ago with the discovery of RNA interference (RNAi), double-stranded RNAs occupied key roles in regulation and as defense-line in animal cells. This thesis focuses on metazoan microRNAs (miRNAs). These small non-coding RNAs are distinguished from their small-interfering RNA (siRNA) relatives by their tightly controlled, efficient and flexible biogenesis, together with a broader flexibility to target multiple mRNAs by a seed imperfect base-pairing. As potent regulators, miRNAs are involved in mRNA stability and post-transcriptional regulation tasks, being a conserved mechanism used repetitively by the evolution, not only in metazoans, but plants and unicellular organisms. Through a comprehensive revision of the current animal miRNA model, the canonical pathway dominates the extensive literature about miRNAs, and served as a scaffold to understand the scenes behind the regulatory landscape performed by the cell. The characterization of a diverse set of non-canonical pathways has expanded this view, suggesting a diverse, rich and flexible regulatory landscape to generate mature miRNAs. The production of miRNAs, derived from isolated or clustered transcripts, is an efficient and highly conserved mechanism traced back to animals with high fidelity at family level. In evolutionary terms, expansions of miRNA families have been associated with an increasing morphological and developmental complexity. In particular, the Chordata clade (the ancient cephalochordates, highly derived and secondary simplified tunicates, and the well-known vertebrates) represents an interesting scenario to study miRNA evolution. Despite clearly conserved miRNAs along these clades, tunicates display massive restructuring events, including emergence of highly derived miRNAs. As shown in this thesis, model organisms or vertebrate-specific bias exist in current animal miRNA annotations, misrepresenting more diverse groups, such as marine invertebrates. Current miRNA databases, such as miRBase and Rfam, classified miRNAs under different definitions and possessed annotations that are not simple to be linked. As an alternative, this thesis proposes a method to curate and merge those annotations, making use of miRBase precursor/mature annotations and genomes together with Rfam predicted sequences. This approach generated structural models for shared miRNA families, based on the alignment of their correct-positioned mature sequences as anchors. In this process, the developed structural curation steps flagged 33 miRNA families from the Rfam as questionable. Curated Rfam and miRBase anchored-structural alignments provided a rich resource for constructing predictive miRNA profiles, using correspondent hidden Markov (HMMs) and covariance models (CMs). As a direct application, the use of those models is time-consuming, and the user has to deal with multiple iterations to achieve a genome-wide non-overlapping annotation. To resolve this, the proposed miRNAture pipeline provides an automatic and flexible solution to annotate miRNAs. It combines multiple homology approaches to generate the best candidates validated at sequence and structural levels. This increases the achievable sensitivity to annotate canonical miRNAs, and the evaluation against human annotation shows that clear false positive calls are rare and additional counterparts lie in retained-introns, transcribed lncRNAs or repeat families. Further development of miRNAture suggests an inclusion of multiple rules to distinguish non-canonical miRNA families. This thesis describes multiple homology approaches to annotate the genomic information from a non-model chordate: the colonial tunicate Didemnum vexillum. Detected high levels of genetic variance and unexpected levels of DNA degradation were evidenced through a comprehensive analysis of genome-assembly methods and gene annotation. Despite those challenges, it was possible to find candidate homeobox and skeletogenesis- related genes. On its own, the ncRNA annotation included expected conserved families, and an extensive search of the Rhabdomyosarcoma 2-associated transcript (RMST) lncRNA family traced-back at the divergence of deuterostomes. In addition, a complete study of the annotation thresholds suggested variations to detect miRNAs, later implemented on the miRNAture tool. This chapter is a showcase of the usual workflow that should follow comprehensive sequencing, assembly and annotation project, in the light of the increasing research approaching DNA sequencing. In the last 10 years, the remarkable increment in tunicate sequencing projects boosted the access to an expanded miRNA annotation landscape. In this way, a comprehensive homology approach annotated the miRNA complement of 28 deuterostome genomes (including current 16 reported tunicates) using miRNAture. To get proper structural models as input, corrected miRBase structural alignments served as a scaffold for building correspondent CMs, based on a developed genetic algorithm. By this means, this automatic approach selected the set of sequences that composed the alignments, generating 2492 miRNA CMs. Despite the multiple sources and associated heterogeneity of the studied genomes, a clustering approach successfully gathered five groups of similar assemblies and highlighted low quality assemblies. The overall family and loci reduction on tunicates is notorious, showing on average 374 microRNA (miRNA) loci, in comparison to other clades: Cephalochordata (2119), Vertebrata (3638), Hemichordata (1092) and Echinodermata (2737). Detection of 533 miRNA families on the divergence of tunicates shows an expanded landscape regarding currently miRNA annotated families. Shared sets of ancestral, chordates, Olfactores, and specific clade-specific miRNAs were uncovered using a phyloge- netic conservation criteria. Compared to current annotations, the family repertories were expanded in all cases. Finally, relying on the adjacent elements from annotated miRNAs, this thesis proposes an additional syntenic support to cluster miRNA loci. In this way, the structural alignment of miR-1497, originally annotated in three model tunicates, was expanded with a clear syntenic support on tunicates

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval

Identification of transcriptomic signature in cellular senescence and characterization of circulating small non-coding RNA during human aging

Accumulation of cellular senescence always forebodes the initialization of aging and cancer. It is an irreversible process that leads to cell cycle arrest while senescent cells still own metabolic viability to affect tissue homeostasis. Senescent cells not only accelerate individual aging process, they are also the driver of age-related diseases such as cancer, osteoarthritis, atherosclerosis, and Alzheimer’s diseases. Senescent phenotype shows heterogeneity in different cell lines under diverse triggers, and blurred traits with non-senescent cells make it difficult to identify senescence precisely. It is very necessary to identify robust shared markers, senescence-specific pathways and biological processes across different senescence models. Recently the emerging role of non-coding RNA in senescence and aging has been noticed due to its ability to control cell cycle at post-transcriptional level. Usually the highly proactive secretome from senescent cells, termed the senescence-associated secretory phenotype (SASP), can result in age-related process through intercellular communication, whereas only a number of factors have been identified in very specific scenarios and the role of secreted extracellular RNAs (exRNAs) is not well understood. Detection of exRNAs protected by EV membrane uncovered the fact that most of extracellular mRNAs are fragmentation, along with small non-coding RNAs (sncRNAs), such as miRNAs, piRNA and tRNA fragments. Therefore it is promising to uncover the role of extracellular sncRNA in aging related dysfunction during cell-cell interaction.To better understand the nature of cellular senescence and its corresponding human aging process at transcriptome level, RNA sequencing data from different cell types and senescence inductions were collected, and significantly shared gene markers and pathways among multiple senescence models were determined through meta-analysis and machine learning-based logistic regression methods. Extensionally, the function of identified senescence associated long non-coding RNAs (lncRNAs) during cell cycle were verified through short interfering RNAs (siRNAs) knock-down treatment in lung fibroblasts (IMR-90). In parallel, the abundance of extracellular sncRNAs from healthy people aged 20 to 99 was quantified using 446 small RNA sequencing datasets. The expressional trends of each sncRNA subspecies were detected with age and a sncRNAs-based age predictors was established using high performance ensemble machine learning strategy

Endolysosomal inhibition induces the autophagy-dependent secretion of autophagosomal cargo

Autophagy is traditionally viewed as a degradative pathway in which cytoplasmic material is sequestered in double-membrane organelles called autophagosomes and delivered to the lysosome for breakdown. However, autophagy-related proteins (ATGs) have also been implicated in promoting unconventional secretion of proteins. We uncovered a role for the ATG conjugation system in the loading and secretion of proteins in small extracellular vesicles (EVs) separate from the canonical degradative autophagy pathway. To determine the interrelationships between degradative and secretory autophagy, I utilized a proteomic strategy to evaluate the secretome in response to pharmacologically inhibiting endolysosomal acidification, thereby blocking autophagic degradation. In contrast to secretion from cells with baseline autophagic activity, the secretome in response to endolysosomal inhibition is significantly enriched with autophagy-related proteins and autophagic cargoes. This suggests that impaired autophagic degradation in cells results in the redirection of degradative cargo to be secreted outside of the cell. To further investigate this pathway, I characterized the secretion of the autophagy regulator LC3 and its interacting cargo receptors (p62, NBR1, OPTN, NDP52), which selectively deliver cargo to the growing autophagosome but have not previously been implicated in secretory autophagy. These autophagy cargo receptors are secreted as unprotected EV-associated protein in an autophagosome formation-dependent manner, indicating a distinct mechanism requiring classical autophagosome formation for the secretion of these proteins. Furthermore, genetic knockdown of two separate SNARE proteins mediating autophagosome-lysosome fusion increases secretion of LC3 and autophagy cargo receptors. Overall, these findings establish a new relationship between degradative autophagy and secretory autophagy, in which the impaired degradation of autophagic cargo due to the lack of autophagosome-lysosome fusion results in a concomitant increase in EV-associated secretion of cargo receptors