Glomerular-tubular crosstalk via cold shock Y-box binding protein-1 in the kidney.

Glomerular-tubular crosstalk within the kidney has been proposed, but the paracrine signals enabling this remain largely unknown. The cold-shock protein Y-box binding protein 1 (YBX1) is known to regulate inflammation and kidney diseases but its role in podocytes remains undetermined. Therefore, we analyzed mice with podocyte specific Ybx1 deletion (Ybx1ΔPod). Albuminuria was increased in unchallenged Ybx1ΔPod mice, which surprisingly was associated with reduced glomerular, but enhanced tubular damage. Tubular toll-like receptor 4 (TLR4) expression, node-like receptor protein 3 (NLRP3) inflammasome activation and kidney inflammatory cell infiltrates were all increased in Ybx1ΔPod mice. In vitro, extracellular YBX1 inhibited NLRP3 inflammasome activation in tubular cells. Co-immunoprecipitation, immunohistochemical analyses, microscale cell-free thermophoresis assays, and blunting of the YBX1-mediated TLR4-inhibition by a unique YBX1-derived decapeptide suggests a direct interaction of YBX1 and TLR4. Since YBX1 can be secreted upon post-translational acetylation, we hypothesized that YBX1 secreted from podocytes can inhibit TLR4 signaling in tubular cells. Indeed, mice expressing a non-secreted YBX1 variant specifically in podocytes (Ybx1PodK2A mice) phenocopied Ybx1ΔPod mice, demonstrating a tubular-protective effect of YBX1 secreted from podocytes. Lipopolysaccharide-induced tubular injury was aggravated in Ybx1ΔPod and Ybx1PodK2A mice, indicating a pathophysiological relevance of this glomerular-tubular crosstalk. Thus, our data show that YBX1 is physiologically secreted from podocytes, thereby negatively modulating sterile inflammation in the tubular compartment, apparently by binding to and inhibiting tubular TLR4 signaling. Hence, we have uncovered an YBX1-dependent molecular mechanism of glomerular-tubular crosstalk.

RNA interaction format: a general data format for RNA interactions.

SUMMARY: RNA molecules play crucial roles in various biological processes. They mediate their function mainly by interacting with other RNAs or proteins. At present, information about these interactions is distributed over different resources, often providing the data in simple tab-delimited formats that differ between the databases. There is no standardized data format that can capture the nature of all these different interactions in detail. AVAILABILITY AND IMPLEMENTATION: Here, we propose the RNA interaction format (RIF) for the detailed representation of RNA-RNA and RNA-Protein interactions and provide reference implementations in C/C++, Python, and JavaScript. RIF is released under licence GNU General Public License version 3 (GNU GPLv3) and is available on https://github.com/RNABioInfo/rna-interaction-format.

Tailored machine learning models for functional RNA detection in genome-wide screens.

The in silico prediction of non-coding and protein-coding genetic loci has received considerable attention in comparative genomics aiming in particular at the identification of properties of nucleotide sequences that are informative of their biological role in the cell. We present here a software framework for the alignment-based training, evaluation and application of machine learning models with user-defined parameters. Instead of focusing on the one-size-fits-all approach of pervasive in silico annotation pipelines, we offer a framework for the structured generation and evaluation of models based on arbitrary features and input data, focusing on stable and explainable results. Furthermore, we showcase the usage of our software package in a full-genome screen of Drosophila melanogaster and evaluate our results against the well-known but much less flexible program RNAz.

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN.

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

Toward a Systematic Assessment of Sex Differences in Cystic Fibrosis.

(1) Background: Cystic fibrosis (CF) is a disease with well-documented clinical differences between female and male patients. However, this gender gap is very poorly studied at the molecular level. (2) Methods: Expression differences in whole blood transcriptomics between female and male CF patients are analyzed in order to determine the pathways related to sex-biased genes and assess their potential influence on sex-specific effects in CF patients. (3) Results: We identify sex-biased genes in female and male CF patients and provide explanations for some sex-specific differences at the molecular level. (4) Conclusion: Genes in key pathways associated with CF are differentially expressed between sexes, and thus may account for the gender gap in morbidity and mortality in CF.

Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.

CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq.

Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5' fragment with a 2',3' cyclic phosphate (2',3' cP) and a 3' fragment with a 5' hydroxyl (5' OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5' OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3' phosphate of the adapter and then ligates it directly to RNAs with 5' OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3' twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5' OH termini from total RNA.

Common Features in lncRNA Annotation and Classification: A Survey.

Long non-coding RNAs (lncRNAs) are widely recognized as important regulators of gene expression. Their molecular functions range from miRNA sponging to chromatin-associated mechanisms, leading to effects in disease progression and establishing them as diagnostic and therapeutic targets. Still, only a few representatives of this diverse class of RNAs are well studied, while the vast majority is poorly described beyond the existence of their transcripts. In this review we survey common in silico approaches for lncRNA annotation. We focus on the well-established sets of features used for classification and discuss their specific advantages and weaknesses. While the available tools perform very well for the task of distinguishing coding sequence from other RNAs, we find that current methods are not well suited to distinguish lncRNAs or parts thereof from other non-protein-coding input sequences. We conclude that the distinction of lncRNAs from intronic sequences and untranslated regions of coding mRNAs remains a pressing research gap.

cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3' cyclic phosphates and 5' hydroxyl ends.

Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate and a 3'-fragment with a 5'-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5'-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. [Figure: see text].

The Genome of the "Sea Vomit" Didemnum vexillum.

Tunicates are the sister group of vertebrates and thus occupy a key position for investigations into vertebrate innovations as well as into the consequences of the vertebrate-specific genome duplications. Nevertheless, tunicate genomes have not been studied extensively in the past, and comparative studies of tunicate genomes have remained scarce. The carpet sea squirt Didemnum vexillum, commonly known as "sea vomit", is a colonial tunicate considered an invasive species with substantial ecological and economical risk. We report the assembly of the D. vexillum genome using a hybrid approach that combines 28.5 Gb Illumina and 12.35 Gb of PacBio data. The new hybrid scaffolded assembly has a total size of 517.55 Mb that increases contig length about eightfold compared to previous, Illumina-only assembly. As a consequence of an unusually high genetic diversity of the colonies and the moderate length of the PacBio reads, presumably caused by the unusually acidic milieu of the tunic, the assembly is highly fragmented (L50 = 25,284, N50 = 6539). It is sufficient, however, for comprehensive annotations of both protein-coding genes and non-coding RNAs. Despite its shortcomings, the draft assembly of the "sea vomit" genome provides a valuable resource for comparative tunicate genomics and for the study of the specific properties of colonial ascidians.

Disturbance of phylogenetic layer-specific adaptation of human brain gene expression in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with typical neuropathological hallmarks, such as neuritic plaques and neurofibrillary tangles, preferentially found at layers III and V. The distribution of both hallmarks provides the basis for the staging of AD, following a hierarchical pattern throughout the cerebral cortex. To unravel the background of this layer-specific vulnerability, we evaluated differential gene expression of supragranular and infragranular layers and subcortical white matter in both healthy controls and AD patients. We identified AD-associated layer-specific differences involving protein-coding and non-coding sequences, most of those present in the subcortical white matter, thus indicating a critical role for long axons and oligodendrocytes in AD pathomechanism. In addition, GO analysis identified networks containing synaptic vesicle transport, vesicle exocytosis and regulation of neurotransmitter levels. Numerous AD-associated layer-specifically expressed genes were previously reported to undergo layer-specific switches in recent hominid brain evolution between layers V and III, i.e., those layers that are most vulnerable to AD pathology. Against the background of our previous finding of accelerated evolution of AD-specific gene expression, here we suggest a critical role in AD pathomechanism for this phylogenetic layer-specific adaptation of gene expression, which is most prominently seen in the white matter compartment.

LazyB: fast and cheap genome assembly.

BACKGROUND: Advances in genome sequencing over the last years have lead to a fundamental paradigm shift in the field. With steadily decreasing sequencing costs, genome projects are no longer limited by the cost of raw sequencing data, but rather by computational problems associated with genome assembly. There is an urgent demand for more efficient and and more accurate methods is particular with regard to the highly complex and often very large genomes of animals and plants. Most recently, "hybrid" methods that integrate short and long read data have been devised to address this need. RESULTS: LazyB is such a hybrid genome assembler. It has been designed specificially with an emphasis on utilizing low-coverage short and long reads. LazyB starts from a bipartite overlap graph between long reads and restrictively filtered short-read unitigs. This graph is translated into a long-read overlap graph G. Instead of the more conventional approach of removing tips, bubbles, and other local features, LazyB stepwisely extracts subgraphs whose global properties approach a disjoint union of paths. First, a consistently oriented subgraph is extracted, which in a second step is reduced to a directed acyclic graph. In the next step, properties of proper interval graphs are used to extract contigs as maximum weight paths. These path are translated into genomic sequences only in the final step. A prototype implementation of LazyB, entirely written in python, not only yields significantly more accurate assemblies of the yeast and fruit fly genomes compared to state-of-the-art pipelines but also requires much less computational effort. CONCLUSIONS: LazyB is new low-cost genome assembler that copes well with large genomes and low coverage. It is based on a novel approach for reducing the overlap graph to a collection of paths, thus opening new avenues for future improvements. AVAILABILITY: The LazyB prototype is available at https://github.com/TGatter/LazyB .

Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum.

Dictyostelium discoideum is a social amoeba, which on starvation develops from a single-cell state to a multicellular fruiting body. This developmental process is accompanied by massive changes in gene expression, which also affect non-coding RNAs. Here, we investigate how tRNAs as key regulators of the translation process are affected by this transition. To this end, we used LOTTE-seq to sequence the tRNA pool of D. discoideum at different developmental time points and analyzed both tRNA composition and tRNA modification patterns. We developed a workflow for the specific detection of modifications from reverse transcriptase signatures in chemically untreated RNA-seq data at single-nucleotide resolution. It avoids the comparison of treated and untreated RNA-seq data using reverse transcription arrest patterns at nucleotides in the neighborhood of a putative modification site as internal control. We find that nucleotide modification sites in D. discoideum tRNAs largely conform to the modification patterns observed throughout the eukaroytes. However, there are also previously undescribed modification sites. We observe substantial dynamic changes of both expression levels and modification patterns of certain tRNA types during fruiting body development. Beyond the specific application to D. discoideum our results demonstrate that the developmental variability of tRNA expression and modification can be traced efficiently with LOTTE-seq.

miRNAture-Computational Detection of microRNA Candidates.

Homology-based annotation of short RNAs, including microRNAs, is a difficult problem because their inherently small size limits the available information. Highly sensitive methods, including parameter optimized blast, nhmmer, or cmsearch runs designed to increase sensitivity inevitable lead to large numbers of false positives, which can be detected only by detailed analysis of specific features typical for a RNA family and/or the analysis of conservation patterns in structure-annotated multiple sequence alignments. The miRNAture pipeline implements a workflow specific to animal microRNAs that automatizes homology search and validation steps. The miRNAture pipeline yields very good results for a large number of "typical" miRBase families. However, it also highlights difficulties with atypical cases, in particular microRNAs deriving from repetitive elements and microRNAs with unusual, branched precursor structures and atypical locations of the mature product, which require specific curation by domain experts.

Alzheimer-related genes show accelerated evolution.

Alzheimer's disease (AD) is a neurodegenerative disorder of unknown cause with complex genetic and environmental traits. While AD is extremely prevalent in human elderly, it hardly occurs in non-primate mammals and even non-human-primates develop only an incomplete form of the disease. This specificity of AD to human clearly implies a phylogenetic aspect. Still, the evolutionary dimension of AD pathomechanism remains difficult to prove and has not been established so far. To analyze the evolutionary age and dynamics of AD-associated-genes, we established the AD-associated genome-wide RNA-profile comprising both protein-coding and non-protein-coding transcripts. We than applied a systematic analysis on the conservation of splice-sites as a measure of gene-structure based on multiple alignments across vertebrates of homologs of AD-associated-genes. Here, we show that nearly all AD-associated-genes are evolutionarily old and did not originate later in evolution than not-AD-associated-genes. However, the gene-structures of loci, that exhibit AD-associated changes in their expression, evolve faster than the genome at large. While protein-coding-loci exhibit an enhanced rate of small changes in gene structure, non-coding loci show even much larger changes. The accelerated evolution of AD-associated-genes indicates a more rapid functional adaptation of these genes. In particular AD-associated non-coding-genes play an important, as yet largely unexplored, role in AD. This phylogenetic trait indicates that recent adaptive evolution of human brain is causally involved in basic principles of neurodegeneration. It highlights the necessity for a paradigmatic change of our disease-concepts and to reconsider the appropriateness of current animal-models to develop disease-modifying strategies that can be translated to human.

MCPIP1 ribonuclease can bind and cleave AURKA mRNA in MYCN-amplified neuroblastoma cells.

The role of the inflammation-silencing ribonuclease, MCPIP1 (monocyte chemoattractant protein-induced protein 1), in neoplasia continuous to emerge. The ribonuclease can cleave not only inflammation-related transcripts but also some microRNAs (miRNAs) and viral RNAs. The suppressive effect of the protein has been hitherto suggested in breast cancer, clear cell renal cell carcinoma, osteosarcoma, and neuroblastoma. Our previous results have demonstrated a reduced levels of several oncogenes, as well as inhibited growth of neuroblastoma cells upon MCPIP1 overexpression. Here, we investigate the mechanisms underlying the suppression of MYCN proto-oncogene, bHLH transcription factor (MYCN)-amplified neuroblastoma cells overexpressing the MCPIP1 protein. We showed that the levels of several transcripts involved in cell cycle progression decreased in BE(2)-C and KELLY cells overexpressing MCPIP1 in a ribonucleolytic activity-dependent manner. However, RNA immunoprecipitation indicated that only AURKA mRNA (encoding for Aurora A kinase) interacts with the ribonuclease. Furthermore, the application of a luciferase assay suggested MCPIP1-dependent destabilization of the transcript. Further analyses demonstrated that the entire conserved region of AURKA seems to be indispensable for the interaction with the MCPIP1 protein. Additionally, we examined the effect of the ribonuclease overexpression on the miRNA expression profile in MYCN-amplified neuroblastoma cells. However, no significant alterations were observed. Our data indicate a key role of the binding and cleavage of the AURKA transcript in an MCPIP1-dependent suppressive effect on neuroblastoma cells.

Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing.

BACKGROUND: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive. RESULTS: Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3'-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. CONCLUSIONS: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

Are spliced ncRNA host genes distinct classes of lncRNAs?

Many small nucleolar RNAs and many of the hairpin precursors of miRNAs are processed from long non-protein-coding host genes. In contrast to their highly conserved and heavily structured payload, the host genes feature poorly conserved sequences. Nevertheless, there is mounting evidence that the host genes have biological functions beyond their primary task of carrying a ncRNA as payload. So far, no connections between the function of the host genes and the function of their payloads have been reported. Here we investigate whether there is evidence for an association of host gene function or mechanisms with the type of payload. To assess this hypothesis we test whether the miRNA host genes (MIRHGs), snoRNA host genes (SNHGs), and other lncRNA host genes can be distinguished based on sequence and/or structure features unrelated to their payload. A positive answer would imply a functional and mechanistic correlation between host genes and their payload, provided the classification does not depend on the presence and type of the payload. A negative answer would indicate that to the extent that secondary functions are acquired, they are not strongly constrained by the prior, primary function of the payload. We find that the three classes can be distinguished reliably when the classifier is allowed to extract features from the payloads. They become virtually indistinguishable, however, as soon as only sequence and structure of parts of the host gene distal from the snoRNAs or miRNA payload is used for classification. This indicates that the functions of MIRHGs and SNHGs are largely independent of the functions of their payloads. Furthermore, there is no evidence that the MIRHGs and SNHGs form coherent classes of long non-coding RNAs distinguished by features other than their payloads.

Unusual Occurrence of Two Bona-Fide CCA-Adding Enzymes in Dictyostelium discoideum.

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3'-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome.