Effective resolution of inflammation following acute lung infection or injury is critical for restoring immune and tissue homeostasis to ensure functional recovery. Prolonged or unresolved inflammation can impair lung repair, promote fibrosis, and contribute to pulmonary dysfunction. While systemic steroid signalling is known to modulate general immune responses, the specific role of immune cell-mediated steroidogenesis in regulating lung inflammation and repair remains unknown. Here, we show that immune cell de novo steroidogenesis is essential for resolving inflammation and promoting recovery in a murine model of acute lung injury. During the resolution phase, steroid-synthesizing immune cells, predominantly basophils, are enriched in the lung. Mice with immune cell-specific ablation of de novo steroidogenesis exhibit exacerbated lung injury, impaired resolution of inflammation, and defective tissue repair. These findings reveal a previously unrecognized immunoregulatory function of immune cell-derived steroids and identify immune cell steroidogenesis as a potential therapeutic target for promoting resolution and recovery in inflammatory lung diseases. ### Competing Interest Statement The authors have declared no competing interest. This work is supported by CRUK Career Development Fellowship (RCCFEL\100095), NSF-BIO/UKRI-BBSRC project grant (BB/V006126/1), and MRC project grant (MR/V028995/1).

Transcription occurs as irregular bursts in a very wide range of systems, including numerous different species and many genes within these. In this review, we examine the underlying theories, discuss how these relate to experimental measurements, and explore some of the discrepancies that have emerged among various studies. Finally, we consider more recent works that integrate novel concepts, such as the involvement of biomolecular condensates in enhancer-promoter interactions and their effects on the dynamics of transcriptional bursting.

Cell cycle analysis of CLL and B cells. Purified B and CLL cells are stained for CD5, CD19 and DNA content (vybrant dye). The gating strategy for flow cytometry is shown for B (A) and CLL cells (B). (C) The graph shows mean+SD for cells in G1, S and G2/M phase of the cell cycle (CLL n=7; B cells n=5)

Despite the recent rise of RNA-seq datasets combining single-cell (sc) resolution with 4-thiouridine (4sU) labelling, analytical methods exploiting their power to dissect transcriptional bursting are lacking. Here, we present a mathematical model and Bayesian inference implementation to facilitate genome-wide joint parameter estimation and confidence quantification (R package: burstMCMC). We demonstrate that, unlike conventional scRNA-seq, 4sU scRNA-seq resolves temporal parameters and furthermore boosts inference of dimensionless parameters via a synergy between single-cell resolution and 4sU labelling. We apply our method to published 4sU scRNA-seq data and linked with ChIP-seq data, we uncover previously obscured associations between different parameters and histone modifications.

Microhomologies within repair junctions from substrate #7 in CLL and B cells. The most frequent sequences (frequency >100) are aligned and microhomologies given as colored letters. ssDNA overhangs are marked by colored areas.

Transcription is a complex phenomenon that permits the conversion of genetic information into phenotype by means of an enzyme called RNA polymerase, which erratically moves along and scans the DNA template. We perform Bayesian inference over a paradigmatic mechanistic model of non-equilibrium statistical physics, i.e. the asymmetric exclusion processes in the hydrodynamic limit, assuming a Gaussian process prior for the polymerase progression rate as a latent variable. Our framework allows us to infer the speed of polymerases during transcription given their spatial distribution, while avoiding the explicit inversion of the system's dynamics. The results, which show processing rates strongly varying with genomic position and minor role of traffic-like congestion, may have strong implications for the understanding of gene expression.

Sequences of the 9 DNA substrates used for the DNA repair assay. Restriction sites for linearization are indicated. Primer binding sites are colored red, unique 6bp motifs used for substrate identification of resected junctions are colored green.

Realtime RT-PCR on Lig1, XRCC1 and PARP1 on cDNA from CLL cells, B cells and Mec1 cells. Transcript levels are shown as relative abundance, normalized to the respective mean in B cells.

Supplementary table S1. Patient information. Supplementary table S2. Plasmid assay data for all inter- and intramolecular junctions in CLL and B cells. Supplementary table S3. Plasmid assay data for the most frequent intramolecular junctions in CLL and B cells. Supplementary table S4. Plasmid assay data for the most frequent inter- and intramolecular junctions in CLL and B cells including sequences. Supplementary table S5. Number of reads from plasmid assays corresponding to amplicons amplified by forward and reverse primer pairs.

Most functional eukaryotic mRNAs contain a 5′ 7-methylguanosine (m 7 G) cap. Although capping is essential for many biological processes including mRNA processing, export and translation, the fate of uncapped transcripts has not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that although the degradation of cap-deficient mRNA is dominant, the levels of hundreds of non-capped mRNAs increase upon depletion of the capping enzymes. Overall, the abundance of non-capped mRNAs is inversely correlated to the expression levels, altogether resembling the effects observed in cells lacking the cytoplasmic 5′−3′ exonuclease Xrn1 and indicating differential degradation fates of non-capped mRNAs. The inactivation of the nuclear 5′−3′ exonuclease Rat1 does not rescue the non-capped mRNA levels indicating that Rat1 is not involved in their degradation and consequently, the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the cap presence is essential to initiate the Xrn1-dependent degradation of mRNAs underpinning the role of 5′ cap in the Xrn1-dependent buffering of the cellular mRNA levels.

Cap-adjacent nucleotides of animal, protist and viral mRNAs can be O-methylated at the 2' position of the ribose (cOMe). The functions of cOMe in animals, however, remain largely unknown. Here we show that the two cap methyltransferases (CMTr1 and CMTr2) of Drosophila can methylate the ribose of the first nucleotide in mRNA. Double-mutant flies lack cOMe but are viable. Consistent with prominent neuronal expression, they have a reward learning defect that can be rescued by conditional expression in mushroom body neurons before training. Among CMTr targets are cell adhesion and signaling molecules. Many are relevant for learning, and are also targets of Fragile X Mental Retardation Protein (FMRP). Like FMRP, cOMe is required for localization of untranslated mRNAs to synapses and enhances binding of the cap binding complex in the nucleus. Hence, our study reveals a mechanism to co-transcriptionally prime mRNAs by cOMe for localized protein synthesis at synapses.

Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders, such as Prader-Willi syndrome (PWS), which results from the deletion of an imprinted locus on paternally inherited chromosome 15. We analyzed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5' snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance, while upregulated genes are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data reveal the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provide a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature, which is important for neuronal differentiation and development.

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe . This association is RNase sensitive and it correlates with Pol II transcription and mRNA expression levels. While changes in Pol II occupancy were detected at only some genes in a Upf1-deficient ( upf1Δ ) strain, there is an increased Ser2 Pol II signal at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1Δ cells are hypersensitive to the transcription elongation inhibitor 6-azauracil and display Pol II abnormalities suggestive of Pol II hyperphosphorylation. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1Δ. These data envisage that by operating on the nascent transcript Upf1 might influence Pol II phosphorylation and transcription.

Transcription of many genes in metazoans is subject to polymerase pausing, which is the transient stop of transcriptionally engaged polymerases. This is known to mainly occur in promoter-proximal regions but it is not well understood. In particular, a genome- wide measurement of pausing times at high resolution has been lacking. We present here the time-variant precision nuclear run-on and sequencing (TV-PRO-seq) assay, an extension of the standard PRO-seq that allows us to estimate genome-wide pausing times at single-base resolution. Its application to human cells demonstrates that, proximal to promoters, polymerases pause more frequently but for shorter times than in other genomic regions. Comparison with single-cell gene expression data reveals that the polymerase pausing times are longer in highly expressed genes, while transcriptionally noisier genes have higher pausing frequencies and slightly longer pausing times. Analyses of histone modifications suggest that the marker H3K36me3 is related to the polymerase pausing.

BACKGROUND Idiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess, and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype. METHODS In fasted, matched IIH (n = 97) and control (n = 43) patients, we assessed glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case-control study. RESULTS We demonstrate an insulin- and leptin-resistant phenotype in IIH in excess of that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed toward depot-specific lipogenesis. CONCLUSION These data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improve cardiovascular outcomes. FUNDING This study was supported by the UK NIHR (NIHR-CS-011-028), the UK Medical Research Council (MR/K015184/1), Diabetes UK, Wellcome Trust (104612/Z/14/Z), the Sir Jules Thorn Award, and the Midlands Neuroscience Teaching and Research Fund.

ABSTRACTThe regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

Experience powerfully influences neuronal function and cognitive performance, but the cellular and molecular events underlying the experience-dependent enhancement of mental ability have remained elusive. In particular, the mechanisms that couple the external environment to the genomic changes underpinning this improvement are unknown. To address this, we have used male mice harboring an inactivating mutation of mitogen- and stress-activated protein kinase 1 (MSK1), a brain-derived neurotrophic factor (BDNF)-activated enzyme downstream of the mitogen-activated protein kinase (MAPK) pathway. We show that MSK1 is required for the full extent of experience-induced improvement of spatial memory, for the expansion of the dynamic range of synapses, exemplified by the enhancement of hippocampal long-term potentiation (LTP) and long-term depression (LTD), and for the regulation of the majority of genes influenced by enrichment. In addition, and unexpectedly, we show that experience is associated with an MSK1-dependent downregulation of key MAPK and plasticity-related genes, notably of EGR1/Zif268 and Arc/Arg3.1, suggesting the establishment of a novel genomic landscape adapted to experience. By coupling experience to homeostatic changes in gene expression MSK1, represents a prime mechanism through which the external environment has an enduring influence on gene expression, synaptic function, and cognition. SIGNIFICANCE STATEMENT Our everyday experiences strongly influence the structure and function of the brain. Positive experiences encourage the growth and development of the brain and support enhanced learning and memory and resistance to mood disorders such as anxiety. While this has been known for many years, how this occurs is not clear. Here, we show that many of the positive aspects of experience depend on an enzyme called mitogen- and stress-activated protein kinase 1 (MSK1). Using male mice with a mutation in MSK1, we show that MSK1 is necessary for the majority of gene expression changes associated with experience, extending the range over which the communication between neurons occurs, and for both the persistence of memory and the ability to learn new task rules.

RNA editing—primarily conversion of adenosine to inosine (A > I)—is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy.

Protocols for preparing RNA sequencing (RNA-seq) libraries, most prominently “Smart-seq” variations, introduce global biases that can have a significant impact on the quantification of gene expression levels. This global bias can lead to drastic over- or under-representation of RNA in non-linear length-dependent fashion due to enzymatic reactions during cDNA production. It is currently not corrected by any RNA-seq software, which mostly focus on local bias in coverage along RNAs. This paper describes LiBiNorm, a simple command line program that mimics the popular htseq-count software and allows diagnostics, quantification, and global bias removal. LiBiNorm outputs gene expression data that has been normalized to correct for global bias introduced by the Smart-seq2 protocol. In addition, it produces data and several plots that allow insights into the experimental history underlying library preparation. The LiBiNorm package includes an R script that allows visualization of the main results. LiBiNorm is the first software application to correct for the global bias that is introduced by the Smart-seq2 protocol. It is freely downloadable at http://www2.warwick.ac.uk/fac/sci/lifesci/research/libinorm.