Sharing sensitive data in life sciences: an overview of centralized and federated approaches.

Biomedical data are generated and collected from various sources, including medical imaging, laboratory tests and genome sequencing. Sharing these data for research can help address unmet health needs, contribute to scientific breakthroughs, accelerate the development of more effective treatments and inform public health policy. Due to the potential sensitivity of such data, however, privacy concerns have led to policies that restrict data sharing. In addition, sharing sensitive data requires a secure and robust infrastructure with appropriate storage solutions. Here, we examine and compare the centralized and federated data sharing models through the prism of five large-scale and real-world use cases of strategic significance within the European data sharing landscape: the French Health Data Hub, the BBMRI-ERIC Colorectal Cancer Cohort, the federated European Genome-phenome Archive, the Observational Medical Outcomes Partnership/OHDSI network and the EBRAINS Medical Informatics Platform. Our analysis indicates that centralized models facilitate data linkage, harmonization and interoperability, while federated models facilitate scaling up and legal compliance, as the data typically reside on the data generator's premises, allowing for better control of how data are shared. This comparative study thus offers guidance on the selection of the most appropriate sharing strategy for sensitive datasets and provides key insights for informed decision-making in data sharing efforts.

The Data Use Ontology to streamline responsible access to human biomedical datasets.

Human biomedical datasets that are critical for research and clinical studies to benefit human health also often contain sensitive or potentially identifying information of individual participants. Thus, care must be taken when they are processed and made available to comply with ethical and regulatory frameworks and informed consent data conditions. To enable and streamline data access for these biomedical datasets, the Global Alliance for Genomics and Health (GA4GH) Data Use and Researcher Identities (DURI) work stream developed and approved the Data Use Ontology (DUO) standard. DUO is a hierarchical vocabulary of human and machine-readable data use terms that consistently and unambiguously represents a dataset's allowable data uses. DUO has been implemented by major international stakeholders such as the Broad and Sanger Institutes and is currently used in annotation of over 200,000 datasets worldwide. Using DUO in data management and access facilitates researchers' discovery and access of relevant datasets. DUO annotations increase the FAIRness of datasets and support data linkages using common data use profiles when integrating the data for secondary analyses. DUO is implemented in the Web Ontology Language (OWL) and, to increase community awareness and engagement, hosted in an open, centralized GitHub repository. DUO, together with the GA4GH Passport standard, offers a new, efficient, and streamlined data authorization and access framework that has enabled increased sharing of biomedical datasets worldwide.

RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia.

In hypoxic stress conditions, glycolysis enzymes assemble into singular cytoplasmic granules called glycolytic (G) bodies. G body formation in yeast correlates with increased glucose consumption and cell survival. However, the physical properties and organizing principles that define G body formation are unclear. We demonstrate that glycolysis enzymes are non-canonical RNA binding proteins, sharing many common mRNA substrates that are also integral constituents of G bodies. Targeting nonspecific endoribonucleases to G bodies reveals that RNA nucleates G body formation and maintains its structural integrity. Consistent with a phase separation mechanism of biogenesis, recruitment of glycolysis enzymes to G bodies relies on multivalent homotypic and heterotypic interactions. Furthermore, G bodies fuse in vivo and are largely insensitive to 1,6-hexanediol, consistent with a hydrogel-like composition. Taken together, our results elucidate the biophysical nature of G bodies and demonstrate that RNA nucleates phase separation of the glycolysis machinery in response to hypoxic stress.

Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress.

Glycolysis is upregulated under conditions such as hypoxia and high energy demand to promote cell proliferation, although the mechanism remains poorly understood. We find that hypoxia in Saccharomyces cerevisiae induces concentration of glycolytic enzymes, including the Pfk2p subunit of the rate-limiting phosphofructokinase, into a single, non-membrane-bound granule termed the "glycolytic body" or "G body." A yeast kinome screen identifies the yeast ortholog of AMP-activated protein kinase, Snf1p, as necessary for G-body formation. Many G-body components identified by proteomics are required for G-body integrity. Cells incapable of forming G bodies in hypoxia display abnormal cell division and produce inviable daughter cells. Conversely, cells with G bodies show increased glucose consumption and decreased levels of glycolytic intermediates. Importantly, G bodies form in human hepatocarcinoma cells in hypoxia. Together, our results suggest that G body formation is a conserved, adaptive response to increase glycolytic output during hypoxia or tumorigenesis.

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

MORC-1 Integrates Nuclear RNAi and Transgenerational Chromatin Architecture to Promote Germline Immortality.

Germline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans, nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood. We discovered that morc-1 is essential for transgenerational fertility and acts as an effector of endo-siRNAs. Unexpectedly, morc-1 is dispensable for siRNA inheritance but is required for target silencing and maintenance of siRNA-dependent chromatin organization. A forward genetic screen identified mutations in met-1, which encodes an H3K36 methyltransferase, as potent suppressors of morc-1(-) and nuclear RNAi mutant phenotypes. Further analysis of nuclear RNAi and morc-1(-) mutants revealed a progressive, met-1-dependent enrichment of H3K36me3, suggesting that robust fertility requires repression of MET-1 activity at nuclear RNAi targets. Without MORC-1 and nuclear RNAi, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality.

A Novel Long Non-Coding RNA in the hTERT Promoter Region Regulates hTERT Expression.

A novel antisense transcript was identified in the human telomerase reverse transcriptase (hTERT) promoter region, suggesting that the hTERT promoter is bidirectional. This transcript, named hTERT antisense promoter-associated (hTAPAS) RNA, is a 1.6 kb long non-coding RNA. hTAPAS transcription is initiated 167 nucleotides upstream of the hTERT transcription start site and is present in both the nucleus and the cytoplasm. Surprisingly, we observed that a large fraction of the hTERT polyadenylated RNA is localized in the nucleus, suggesting this might be an additional means of regulating the cellular abundance of hTERT protein. Both hTAPAS and hTERT are expressed in immortalized B-cells and human embryonic stem cells but are not detected in normal somatic cells. hTAPAS expression inversely correlates with hTERT expression in different types of cancer samples. Moreover, hTAPAS expression is not promoted by an hTERT promoter mutation (-124 C>T). Antisense-oligonucleotide mediated knockdown of hTAPAS results in an increase in hTERT expression. Conversely, ectopic overexpression of hTAPAS down regulates hTERT expression, suggesting a negative role in hTERT gene regulation. These observations provide insights into hTAPAS as a novel player that negatively regulates hTERT expression and may be involved in telomere length homeostasis.

Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Bioinformatic Analysis.

Protein-RNA interactions are integral components of posttranscriptional gene regulatory processes including mRNA processing and assembly of cellular architectures. Dysregulation of RNA-binding protein (RBP) expression or disruptions in RBP-RNA interactions underlie a variety of human pathologies and genetic diseases including cancer and neurodegenerative diseases (reviewed in (Cooper et al., Cell 136(4):777-793, 2009; Darnell, Cancer Res Treat 42(3):125-129, 2010; Lukong et al., Trends Genet 24 (8):416-425, 2008)). Recent studies have uncovered only a small proportion of the extensive RBP-RNA interactome in any organism (Baltz et al., Mol Cell 46(5):674-690, 2012; Castello et al., Cell 149(6):1393-1406, 2012; Freeberg et al., Genome Biol 14(2):R13, 2013; Hogan et al., PLoS Biol 6(10):e255, 2008; Mitchell et al., Nat Struct Mol Biol 20(1):127-133, 2013; Tsvetanova et al. PLoS One 5(9): pii: e12671, 2010; Schueler et al., Genome Biol 15(1):R15, 2014; Silverman et al., Genome Biol 15(1):R3, 2014). To expand our understanding of how RBP-RNA interactions govern RNA-related processes, we developed gPAR-CLIP-seq (global photoactivatable-ribonucleoside-enhanced cross-linking and precipitation followed by deep sequencing) for capturing and sequencing all regions of the Saccharomyces cerevisiae transcriptome bound by RBPs (Freeberg et al., Genome Biol 14(2):R13, 2013). This chapter describes a pipeline for bioinformatic analysis of gPAR-CLIP-seq data. The first half of this pipeline can be implemented by running locally installed programs or by running the programs using the Galaxy platform (Blankenberg et al., Curr Protoc Mol Biol. Chapter 19:Unit 19 10 11-21, 2010; Giardine et al., Genome Res 15 (10):1451-1455, 2005; Goecks et al., Genome Biol 11(8):R86, 2010). The second half of this pipeline can be implemented by user-generated code in any language using the pseudocode provided as a template.

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1.

MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway.

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

BACKGROUND: Autophagy as a conserved lysosomal/vacuolar degradation and recycling pathway is important in normal development and physiology, and defects in this process are linked to many kinds of disease. Because too much or too little autophagy can be detrimental, the process must be tightly regulated both temporally and in magnitude. Two parameters that affect this regulation are the size and the number of autophagosomes; however, although we know that the amount of Atg8 affects the size of autophagosomes, the mechanism for regulating their number has not been elucidated. The transcriptional induction and repression of the autophagy-related (ATG) genes is one crucial aspect of autophagy regulation, but the transcriptional regulators that modulate autophagy are not well characterized. RESULTS: We detected increased expression levels of ATG genes, and elevated autophagy activity, in cells lacking the transcriptional regulator Pho23. Using transmission electron microscopy, we found that PHO23 null mutant cells contain significantly more autophagosomes than the wild-type. By RNA sequencing transcriptome profiling, we identified ATG9 as one of the key targets of Pho23, and our studies with strains expressing modulated levels of Atg9 show that the amount of this protein directly correlates with the frequency of autophagosome formation and the level of autophagy activity. CONCLUSIONS: Our results identified Pho23 as a master transcriptional repressor for autophagy that regulates the frequency of autophagosome formation through its negative regulation of ATG9.

A conserved upstream motif orchestrates autonomous, germline-enriched expression of Caenorhabditis elegans piRNAs.

Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.

Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae.

BACKGROUND: Protein-RNA interactions are integral components of nearly every aspect of biology, including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. RESULTS: Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the untranslated, polyadenylated transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTRs) covering 72% of protein-coding transcripts encoded in the genome, confirming 3' UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3' UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. CONCLUSIONS: Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo.

piRNAs and siRNAs collaborate in Caenorhabditis elegans genome defense.

Caenorhabditis elegans piRNAs promote genome surveillance by triggering siRNA-mediated silencing of nonself DNA in competition with licensing programs that support endogenous gene expression.