Sweet splicing.

Glucose is the main source of energy for cells. In this issue of Cell, a study now shows that glucose has additional non-energetic functions, acting as a biomolecular cue that regulates alternative splicing during epidermal differentiation. As keratinocytes differentiate, glucose associates with RNA-binding protein DDX21 and modulates its interaction properties, which modifies splicing decisions.

Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.

Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5'splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

Pseudouridylation: A new player in co-transcriptional splicing regulation.

Martinez et al. (2022) uncovered a novel function for the most abundant modified nucleoside in RNA. The study shows that uridines at splice sites and splicing regulatory motifs in the pre-mRNA may be converted to pseudouridine during transcription and impact splicing decisions.

Gene architecture directs splicing outcome in separate nuclear spatial regions.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

POINT technology illuminates the processing of polymerase-associated intact nascent transcripts.

Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.

RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing.

The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5' splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3' splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion.

Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.

Numerous long intervening noncoding RNAs (lincRNAs) are generated from the mammalian genome by RNA polymerase II (Pol II) transcription. Although multiple functions have been ascribed to lincRNAs, their synthesis and turnover remain poorly characterized. Here, we define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells. This is based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing (mNET-seq). Notably, mNET-seq patterns specific for different Pol II CTD phosphorylation states reveal weak co-transcriptional splicing and poly(A) signal-independent Pol II termination of lincRNAs as compared to pre-mRNAs. In addition, lincRNAs are mostly restricted to chromatin, since they are rapidly degraded by the RNA exosome. We also show that a lincRNA-specific co-transcriptional RNA cleavage mechanism acts to induce premature termination. In effect, functional lincRNAs must escape from this targeted nuclear surveillance process.

Smaug1 membrane-less organelles respond to AMPK and mTOR and affect mitochondrial function.

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.

Transcription and splicing dynamics during early Drosophila development.

Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

Racial inequalities in the development of multimorbidity of chronic conditions: results from a Brazilian prospective cohort.

BACKGROUND: The occurrence of multimorbidity and its impacts have differentially affected population subgroups. Evidence on its incidence has mainly come from high-income regions, with limited exploration of racial disparities. This study investigated the association between racial groups and the development of multimorbidity and chronic conditions in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). METHODS: Data from self-reported white, brown (pardos or mixed-race), and black participants at baseline of ELSA-Brasil (2008-2010) who were at risk for multimorbidity were analysed. The development of chronic conditions was assessed through in-person visits and self-reported diagnosis via telephone until the third follow-up visit (2017-2019). Multimorbidity was defined when, at the follow-up visit, the participant had two or more morbidities. Cumulative incidences, incidence rates, and adjusted incidence rate ratios (IRRs) were estimated using Poisson models. RESULTS: Over an 8.3-year follow-up, compared to white participants: browns had a 27% greater incidence of hypertension and obesity; and blacks had a 62% and 45% greater incidence, respectively. Blacks also had 58% more diabetes. The cancer incidence was greater among whites. Multimorbidity affected 41% of the participants, with a crude incidence rate of 57.5 cases per 1000 person-years (ranging from 56.3 for whites to 63.9 for blacks). Adjusted estimates showed a 20% higher incidence of multimorbidity in black participants compared to white participants (IRR: 1.20; 95% CI: 1.05-1.38). CONCLUSIONS: Significant racial disparities in the risk of chronic conditions and multimorbidity were observed. Many associations revealed a gradient increase in illness risk according to darker skin tones. Addressing fundamental causes such as racism and racial discrimination, alongside considering social determinants of health, is vital for comprehensive multimorbidity care. Intersectoral, equitable policies are essential for ensuring health rights for historically marginalized groups.

Causal agents of Stemphylium-induced foliar diseases of tomatoes and other Solanaceae hosts in Brazil.

AIM: An extensive survey was done to clarify the prevalent Stemphylium species on Solanaceae plants across Brazil, and their host ranges. METHODS AND RESULTS: Eighty nine (89) Stemphylium isolates were obtained from naturally infected tomatoes as well as S. paniculatum, potato, eggplant, scarlet eggplant (Solanum aethiopicum var. gilo), Physalis angulata, and Capsicum species. Phylogenetic analyses encompassing the ITS-5.8S rDNA and glyceraldehyde-3-phosphate dehydrogenase genomic regions placed the isolates into two distinct groupings with either Stemphylium lycopersici or S. solani. Isolates of S. lycopersici (n = 81) were obtained infecting tomato, potato, eggplant, S. paniculatum, and P. angulata. Isolates of S. solani (n = 8) were detected in natural association with scarlet eggplant and tomato. Two isolates of S. lycopersici displayed a wide experimental host range in greenhouse bioassays, infecting accessions of 12 out of 18 species. Ocimum basilicum (Lamiaceae) was the only experimental host outside the Solanaceae family.

Bacterial Strains Isolated from Stingless Bee Workers Inhibit the Growth of Apis mellifera Pathogens.

Apis mellifera bees are an important resource for the local economy of various regions in Argentina and the maintenance of natural ecosystems. In recent years, different alternatives have been investigated to avoid the reduction or loss of colonies caused by pathogens and parasites such as Ascosphaera apis, Aspergillus flavus, and Paenibacillus larvae. We focused on bacterial strains isolated from the intestine of native stingless bees, to elucidate their antagonistic effect on diseases of A. mellifera colonies. For this purpose, worker bees of the species Tetragonisca fiebrigi, Plebeia spp., and Scaptotrigona jujuyensis were captured from the entrance to tree hives and transported to the laboratory, where their intestines were extracted. Twenty bacterial colonies were isolated from the intestines, and those capable of inhibiting enterobacteria in vitro and producing organic acids, proteases, and chitinases were selected. Four genera, Levilactobacillus, Acetobacter, Lactiplantibacillus, and Pantoea, were selected and identified by the molecular marker that codes for the 16S rRNA gene. For inhibition assays, cell suspensions and cell-free suspensions were performed. All treatments showed significant antibacterial effects, in comparison with the controls, against P. larvae and antifungal effects against A. apis and A. flavus. However, the mechanisms by which these bacteria inhibit the growth of these pathogens were not studied.

Sex and population differences in the cardiometabolic continuum: a machine learning study using the UK Biobank and ELSA-Brasil cohorts.

BACKGROUND: The temporal relationships across cardiometabolic diseases (CMDs) were recently conceptualized as the cardiometabolic continuum (CMC), sequence of cardiovascular events that stem from gene-environmental interactions, unhealthy lifestyle influences, and metabolic diseases such as diabetes, and hypertension. While the physiological pathways linking metabolic and cardiovascular diseases have been investigated, the study of the sex and population differences in the CMC have still not been described. METHODS: We present a machine learning approach to model the CMC and investigate sex and population differences in two distinct cohorts: the UK Biobank (17,700 participants) and the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) (7162 participants). We consider the following CMDs: hypertension (Hyp), diabetes (DM), heart diseases (HD: angina, myocardial infarction, or heart failure), and stroke (STK). For the identification of the CMC patterns, individual trajectories with the time of disease occurrence were clustered using k-means. Based on clinical, sociodemographic, and lifestyle characteristics, we built multiclass random forest classifiers and used the SHAP methodology to evaluate feature importance. RESULTS: Five CMC patterns were identified across both sexes and cohorts: EarlyHyp, FirstDM, FirstHD, Healthy, and LateHyp, named according to prevalence and disease occurrence time that depicted around 95%, 78%, 75%, 88% and 99% of individuals, respectively. Within the UK Biobank, more women were classified in the Healthy cluster and more men in all others. In the EarlyHyp and LateHyp clusters, isolated hypertension occurred earlier among women. Smoking habits and education had high importance and clear directionality for both sexes. For ELSA-Brasil, more men were classified in the Healthy cluster and more women in the FirstDM. The diabetes occurrence time when followed by hypertension was lower among women. Education and ethnicity had high importance and clear directionality for women, while for men these features were smoking, alcohol, and coffee consumption. CONCLUSIONS: There are clear sex differences in the CMC that varied across the UK and Brazilian cohorts. In particular, disadvantages regarding incidence and the time to onset of diseases were more pronounced in Brazil, against woman. The results show the need to strengthen public health policies to prevent and control the time course of CMD, with an emphasis on women.

C5aR1 signaling promotes region- and age-dependent synaptic pruning in models of Alzheimer's disease.

INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD. HIGHLIGHTS: C5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease.

The Management of Children and Adolescents with Overactive Bladder Refractory to Treatment with Parasacral Transcutaneous Electrical Nerve Stimulation.

BACKGROUND: Although parasacral TENS (pTENS) has been employed in various centers, there is a lack of studies on how children with overactive bladder (OAB) respond after failing to complete pTENS sessions. This study aimed to describe and assess treatments for OAB in children who did not respond to pTENS. MATERIAL AND METHODS: This retrospective case series examined patients aged 4-17 years. Patients were given subsequent treatment options, including: behavioral therapies; oxybutynin; imipramine; a combination of oxybutynin and imipramine; parasacral percutaneous electrical nerve stimulation (PENS); or a repeat course of pTENS. Outcomes were evaluated using the Dysfunctional Voiding Scoring System (DVSS) and the Visual Analogue Scale (VAS). RESULTS: Thirty children were included, with a median age of 7 years. Patients received one or more treatments. Of these, 70% underwent monotherapy. Among them, 57% experienced complete resolution of symptoms, 28% had partial resolution and were satisfied with the results, and 14% discontinued treatment. 30% out of the whole sample continued to experience bothersome symptoms. Complete response, according to initial subsequent, was achieved in: 54% with intensified behavioral therapies, 33% with oxybutynin, and 50% with imipramine alone. The median DVSS score decreased from 7.0 to 2.0 (p=0.025), while the median VAS score increased from 80 to 100 (p<0.001). CONCLUSION: Children with OAB refractory to pTENS who received structured subsequent treatments showed partial response in all cases, with complete symptom resolution in half of the patients. More intensive urotherapy, medications, or repeat pTENS in combination with oxybutinin can be effective for managing this challenging condition.

Active search signatures in a free-viewing task exploiting concurrent EEG and eye movements recordings.

Tasks we often perform in our everyday lives, such as reading or looking for a friend in the crowd, are seemingly straightforward but they actually require the orchestrated activity of several cognitive processes. Free-viewing visual search requires a plan to move our gaze on the different items, identifying them, and deciding on whether to continue with the search. Little is known about the electrophysiological signatures of these processes in free-viewing because there are technical challenges associated with eye movement artefacts. Here, we aimed to study how category information, as well as ecologically relevant variables such as the task performed, influence brain activity in a free-viewing paradigm. Participants were asked to observe/search from an array of faces and objects embedded in random noise. We concurrently recorded electroencephalogram and eye movements and applied a deconvolution analysis approach to estimate the contribution of the different elements embedded in the task. Consistent with classical fixed-gaze experiments and a handful of free-viewing studies, we found a robust categorical effect around 150 ms in occipital and occipitotemporal electrodes. We also report a task effect, more negative in posterior central electrodes in visual search compared with exploration, starting at around 80 ms. We also found significant effects of trial progression and an interaction with the task effect. Overall, these results generalise the characterisation of early visual face processing to a wider range of experiments and show how a suitable analysis approach allows to discern among multiple neural contributions to the signal, preserving key attributes of real-world tasks.

Mood and implicit confidence independently fluctuate at different time scales.

Mood is an important ingredient of decision-making. Human beings are immersed into a sea of ​​emotions where episodes of high mood alternate with episodes of low mood. While changes in mood are well characterized, little is known about how these fluctuations interact with metacognition, and in particular with confidence about our decisions. We evaluated how implicit measurements of confidence are related with mood states of human participants through two online longitudinal experiments involving mood self-reports and visual discrimination decision-making tasks. Implicit confidence was assessed on each session by monitoring the proportion of opt-out trials when an opt-out option was available, as well as the median reaction time on standard correct trials as a secondary proxy of confidence. We first report a strong coupling between mood, stress, food enjoyment, and quality of sleep reported by participants in the same session. Second, we confirmed that the proportion of opt-out responses as well as reaction times in non-opt-out trials provided reliable indices of confidence in each session. We introduce a normative measure of overconfidence based on the pattern of opt-out selection and the signal-detection-theory framework. Finally and crucially, we found that mood, sleep quality, food enjoyment, and stress level are not consistently coupled with these implicit confidence markers, but rather they fluctuate at different time scales: mood-related states display faster fluctuations (over one day or half-a-day) than confidence level (two-and-a-half days). Therefore, our findings suggest that spontaneous fluctuations of mood and confidence in decision making are independent in the healthy adult population.

Photochemoprevention of topical formulation containing purified fraction of Inga edulis leaves extract.

Natural antioxidants have attracted attention for their therapeutic use as photochemopreventive agents. Inga edulis leaves extract and its purified fraction have high polyphenolic content and high antioxidant capacity. In addition, they presented UV photostability and low citotoxicity in fibroblast cells. In this context, this study first aimed at development of topical formulation containing purified fraction of I. edulis extract and the evaluation of skin penetration of the compounds. Moreover, the photoprotective/photochemopreventive potential of the formulation containing I. edulis purified fraction were investigated in vitro and in vivo. The topical formulation containing 1% of the purified fraction of I. edulis increased the endogenous antioxidant potential of the skin, and vicenin-2 and myricetin compounds were able to penetrate the epidermis and dermis. Additionally, the purified fraction (25 and 50 mg/mL) showed a photoprotective effect against UVA and UVB radiation in L929 fibroblast cells. In vivo studies have shown that the formulation added with purified fraction provided an anti-inflammatory effect on the skin of animals after UVB exposure, since it was observed a reduction in MPO activity, IL-1ß and TNF-α cytokines, and CXCL1/KC chemokine concentrations. In conclusion, the purified fraction of I. edulis, rich in phenolic compounds, when incorporated in topical formulation, appears as an alternative to prevent skin damages induced by UV radiation, due to its antioxidant and anti-inflammatory properties.

Anthropometric trajectory in the course of life and occurrence of sarcopenia in men and women: results from the ELSA-Brasil cohort.

This study aimed to identify patterns of anthropometric trajectories throughout life and to analyse their association with the occurrence of sarcopenia in people from the Longitudinal Study of Adult Health (ELSA-Brasil). It is a cross-sectional study involving 9670 public servants, aged 38-79 years, who answered the call for new data collection and exams, conducted approximately 4 years after the study baseline (2012-2014). Data sequence analysis was used to identify patterns of anthropometric trajectory. A theoretical model was elaborated based on the directed acyclic graph (DAG) to select the variables of minimum adjustment in the analysis of the causal effect between trajectory and sarcopenia. Poisson regression with robust variance was adopted for data analysis. The patterns of change in the anthropometric trajectory were classified in stable weight (T1); change to normal weight (T2); change to excess weight (T3); weight fluctuation (T4) and change to low weight (T5). The prevalence of sarcopenia in men and women who changed the anthropometric path for the low weight was twice as large when compared to participants with a stable weight trajectory. A protective effect of the excess weight trajectory was observed for the occurrence of sarcopenia in them. The results pointed to the need for health policies that encourage the proper management of body components in order to prevent and control obesity, as well as to preserve the quantity and quality of skeletal muscle mass throughout life, especially in older adults.

Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.