Reducing body weight to improve metabolic health and related comorbidities is a primary goal in treating obesity^1,2. However, maintaining weight loss is a considerable challenge, especially as the body seems to retain an obesogenic memory that defends against body weight changes^3,4. Overcoming this barrier for long-term treatment success is difficult because the molecular mechanisms underpinning this phenomenon remain largely unknown. Here, by using single-nucleus RNA sequencing, we show that both human and mouse adipose tissues retain cellular transcriptional changes after appreciable weight loss. Furthermore, we find persistent obesity-induced alterations in the epigenome of mouse adipocytes that negatively affect their function and response to metabolic stimuli. Mice carrying this obesogenic memory show accelerated rebound weight gain, and the epigenetic memory can explain future transcriptional deregulation in adipocytes in response to further high-fat diet feeding. In summary, our findings indicate the existence of an obesogenic memory, largely on the basis of stable epigenetic changes, in mouse adipocytes and probably other cell types. These changes seem to prime cells for pathological responses in an obesogenic environment, contributing to the problematic ‘yo-yo’ effect often seen with dieting. Targeting these changes in the future could improve long-term weight management and health outcomes.

The RNA-binding proteins Roquin-1 and Roquin-2 redundantly control gene expression and cell-fate decisions. Here, we show that Roquin not only interacts with stem–loop structures, but also with a linear sequence element present in about half of its targets. Comprehensive analysis of a minimal response element of the Nfkbid 3′-UTR shows that six stem–loop structures cooperate to exert robust and profound post-transcriptional regulation. Only binding of multiple Roquin proteins to several stem–loops exerts full repression, which redundantly involved deadenylation and decapping, but also translational inhibition. Globally, most Roquin targets are regulated by mRNA decay, whereas a small subset, including the Nfat5 mRNA, with more binding sites in their 3′-UTRs, are also subject to translational inhibition. These findings provide insights into how the robustness and magnitude of Roquin-mediated regulation is encoded in complex cis-elements.

The danger signal extracellular calcium is pathophysiologically increased in the synovial fluid of patients with rheumatoid arthritis (RA). Calcium activates the NLRP3-inflammasome via the calcium-sensing receptor in monocytes/macrophages primed by lipopolysaccharide, and this effect is mediated by the uptake of calciprotein particles (CPPs) formed out of calcium, phosphate, and fetuin-A. Aim of the study was to unravel the influence of calcium on monocytes when the priming signal is not present. Monocytes were isolated from the blood of healthy controls and RA patients. Macrophages were characterized using scRNA-seq, DNA microarray, and proteomics. Imaging flow cytometry was utilized to study intracellular events. Here we show that extracellular calcium and CPPs lead to the differentiation of monocytes into calcium-macrophages when the priming signal is absent. Additional growth factors are not needed, and differentiation is triggered by calcium-dependent CPP-uptake, lysosomal alkalization due to CPP overload, and TFEB- and STAT3-dependent increased transcription of the lysosomal gene network. Calcium-macrophages have a needle-like shape, are characterized by excessive, constitutive SPP1/osteopontin production and a strong pro-inflammatory cytokine response. Calcium-macrophages differentiated out of RA monocytes show a stronger manifestation of this phenotype, suggesting the differentiation process might lead to the pro-inflammatory macrophage response seen in the RA synovial membrane.

Many repetitive DNA elements are transcribed at appreciable expression levels. Mapping the corresponding RNA sequencing reads back to a reference genome is notoriously difficult and error-prone task, however. This is in particular true if chemical modifications introduce systematic mismatches, while at the same time the genomic loci are only approximately identical, as in the case of tRNAs. We therefore developed a dedicated mapping strategy to handle RNA-seq reads that map to tRNAs relying on a modified target genome in which known tRNA loci are masked and instead intronless tRNA precursor sequences are appended as artificial ‘chromosomes’. In a first pass, reads that overlap the boundaries of mature tRNAs are extracted. In the second pass, the remaining reads are mapped to a tRNA-masked target that is augmented by representative mature tRNA sequences. Using both simulated and real life data we show that our best-practice workflow removes most of the mapping artefacts introduced by simpler mapping schemes and makes it possible to reliably identify many of chemical tRNA modifications in generic small RNA-seq data. Using simulated data the FDR is only 2%. We find compelling evidence for tissue specific differences of tRNA modification patterns. The workflow is available both as a bash script and as a Galaxy workflow from https://github.com/AnneHoffmann/tRNA-read-mapping. fabian@tbi.univie.ac.at Supplementary data are available at Bioinformatics online.

Transfer RNAs belong to the most abundant type of ribonucleic acid in the cell, and detailed investigations revealed correlations between alterations in the tRNA pool composition and certain diseases like breast cancer. However, currently available methods do not sample the entire tRNA pool or lack specificity for tRNAs. A specific disadvantage of such methods is that only full-length tRNAs are analyzed, while tRNA fragments or incomplete cDNAs due to RT stops at modified nucleosides are lost. Another drawback in certain approaches is that the tRNA fraction has to be isolated and separated from high molecular weight RNA, resulting in considerable labor costs and loss of material. Based on a hairpin-shaped adapter oligonucleotide selective for tRNA transcripts, we developed a highly specific protocol for efficient and comprehensive high-throughput analysis of tRNAs that combines the benefits of existing methods and eliminates their disadvantages. Due to a 3ʹ-TGG overhang, the adapter is specifically ligated to the tRNA 3ʹ-CCA end. Reverse transcription prior to the ligation of a second adapter allows to include prematurely terminated cDNA products, increasing the number of tRNA reads. This strategy renders this approach a powerful and universal tool to analyze the tRNA pool of cells and organisms under different conditions in health and disease.

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3′-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3′-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3′-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.