Organ Protection by Caloric Restriction Depends on Activation of the De Novo NAD+ Synthesis Pathway.

SIGNIFICANCE STATEMENT: AKI is a major clinical complication leading to high mortality, but intensive research over the past decades has not led to targeted preventive or therapeutic measures. In rodent models, caloric restriction (CR) and transient hypoxia significantly prevent AKI and a recent comparative transcriptome analysis of murine kidneys identified kynureninase (KYNU) as a shared downstream target. The present work shows that KYNU strongly contributes to CR-mediated protection as a key player in the de novo nicotinamide adenine dinucleotide biosynthesis pathway. Importantly, the link between CR and NAD+ biosynthesis could be recapitulated in a human cohort. BACKGROUND: Clinical practice lacks strategies to treat AKI. Interestingly, preconditioning by hypoxia and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown. METHODS: Kynureninase (KYNU) knockout mice were generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and comparative transcriptome, proteome and metabolite analyses of murine kidneys pre- and post-ischemia-reperfusion injury in the context of CR or ad libitum diet were performed. In addition, acetyl-lysin enrichment and mass spectrometry were used to assess protein acetylation. RESULTS: We identified KYNU as a downstream target of CR and show that KYNU strongly contributes to the protective effect of CR. The KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway is necessary for CR-associated maintenance of NAD+ levels. This finding is associated with reduced protein acetylation in CR-treated animals, specifically affecting enzymes in energy metabolism. Importantly, the effect of CR on de novo NAD+ biosynthesis pathway metabolites can be recapitulated in humans. CONCLUSIONS: CR induces the de novo NAD+ synthesis pathway in the context of IRI and is essential for its full nephroprotective potential. Differential protein acetylation may be the molecular mechanism underlying the relationship of NAD+, CR, and nephroprotection.

Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.

RNA-binding proteins and their role in kidney disease.

RNA-binding proteins (RBPs) are of fundamental importance for post-transcriptional gene regulation and protein synthesis. They are required for pre-mRNA processing and for RNA transport, degradation and translation into protein, and can regulate every step in the life cycle of their RNA targets. In addition, RBP function can be modulated by RNA binding. RBPs also participate in the formation of ribonucleoprotein complexes that build up macromolecular machineries such as the ribosome and spliceosome. Although most research has focused on mRNA-binding proteins, non-coding RNAs are also regulated and sequestered by RBPs. Functional defects and changes in the expression levels of RBPs have been implicated in numerous diseases, including neurological disorders, muscular atrophy and cancers. RBPs also contribute to a wide spectrum of kidney disorders. For example, human antigen R has been reported to have a renoprotective function in acute kidney injury (AKI) but might also contribute to the development of glomerulosclerosis, tubulointerstitial fibrosis and diabetic kidney disease (DKD), loss of bicaudal C is associated with cystic kidney diseases and Y-box binding protein 1 has been implicated in the pathogenesis of AKI, DKD and glomerular disorders. Increasing data suggest that the modulation of RBPs and their interactions with RNA targets could be promising therapeutic strategies for kidney diseases.

CALINCA-A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease.

Diseases of the renal filtration unit-the glomerulus-are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury.

BACKGROUND: Although AKI lacks effective therapeutic approaches, preventive strategies using preconditioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. METHODS: To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. RESULTS: The gene expression signatures induced by both preconditioning strategies involve distinct common genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI. CONCLUSIONS: This comparative analysis of the gene expression signatures in preconditioning strategies shows overlapping patterns in caloric restriction and hypoxic preconditioning, pointing toward common molecular mechanisms. Our analysis identified a limited set of target genes not previously known to be associated with AKI; further study of their potential to provide the basis for novel preventive strategies is warranted. To allow for optimal interactive usability of the data by the kidney research community, we provide an online interface for user-defined interrogation of the gene expression datasets (http://shiny.cecad.uni-koeln.de:3838/IRaP/).

Activation of Hypoxia-Inducible Factor Signaling Modulates the RNA Protein Interactome in Caenorhabditis elegans.

The cellular response to hypoxia is crucial to organismal survival, and hypoxia-inducible factors (HIF) are the key mediators of this response. HIF-signaling is central to many human diseases and mediates longevity in the nematode. Despite the rapidly increasing knowledge on RNA-binding proteins (RBPs), little is known about their contribution to hypoxia-induced cellular adaptation. We used RNA interactome capture (RIC) in wild-type Caenorhabditis elegans and vhl-1 loss-of-function mutants to fill this gap. This approach identifies more than 1,300 nematode RBPs, 270 of which can be considered novel RBPs. Interestingly, loss of vhl-1 modulates the RBPome. This difference is not primarily explained by protein abundance suggesting differential RNA-binding. Taken together, our study provides a global view on the nematode RBPome and proteome as well as their modulation by HIF-signaling. The resulting RBP atlas is also provided as an interactive online data mining tool (http://shiny.cecad.uni-koeln.de:3838/celegans_rbpome).

A protein-RNA interaction atlas of the ribosome biogenesis factor AATF.

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.

Long Non-Coding RNAs in Kidney Disease.

Non-coding RNA species contribute more than 90% of all transcripts and have gained increasing attention in the last decade. One of the most recent members of this group are long non-coding RNAs (lncRNAs) which are characterized by a length of more than 200 nucleotides and a lack of coding potential. However, in contrast to this simple definition, lncRNAs are heterogenous regarding their molecular function-including the modulation of small RNA and protein function, guidance of epigenetic modifications and a role as enhancer RNAs. Furthermore, they show a highly tissue-specific expression pattern. These aspects already point towards an important role in cellular biology and imply lncRNAs as players in development, health and disease. This view has been confirmed by numerous publications from different fields in the last years and has raised the question as to whether lncRNAs may be future therapeutic targets in human disease. Here, we provide a concise overview of the current knowledge on lncRNAs in both glomerular and tubulointerstitial kidney disease.

The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells.

BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure.

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2 i6 A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2 i6 A from i6 A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.