Mammalian RNA complexity is regulated through interactions of RNA-binding proteins (RBPs) with their target transcripts. High-throughput sequencing together with UV-crosslinking and ...immunoprecipitation (HITS-CLIP) is able to globally map RBP-binding footprint regions at a resolution of ~30-60 nucleotides. Here we describe a systematic way to analyze HITS-CLIP data to identify exact crosslink sites, and thereby determine protein-RNA interactions at single-nucleotide resolution. We found that reverse transcriptase used in CLIP frequently skips the crosslinked amino-acid-RNA adduct, resulting in a nucleotide deletion. Genome-wide analysis of these crosslinking-induced mutation sites (CIMS) in HITS-CLIP data for Nova and Argonaute (Ago) proteins in mouse brain tissue revealed deletions in ~8-20% of mRNA tags, which mapped to Nova and Ago binding sites on mRNA or miRNA. CIMS analysis provides a general and more precise means of mapping protein-RNA interactions than currently available methods and insight into the biochemical properties of such interactions in living tissues.
The neurodegenerative disease spinal muscular atrophy (SMA) is caused by deficiency in the survival motor neuron (SMN) protein. Currently approved SMA treatments aim to restore SMN, but the potential ...for SMN expression beyond physiological levels is a unique feature of adeno-associated virus serotype 9 (AAV9)-SMN gene therapy. Here, we show that long-term AAV9-mediated SMN overexpression in mouse models induces dose-dependent, late-onset motor dysfunction associated with loss of proprioceptive synapses and neurodegeneration. Mechanistically, aggregation of overexpressed SMN in the cytoplasm of motor circuit neurons sequesters components of small nuclear ribonucleoproteins, leading to splicing dysregulation and widespread transcriptome abnormalities with prominent signatures of neuroinflammation and the innate immune response. Thus, long-term SMN overexpression interferes with RNA regulation and triggers SMA-like pathogenic events through toxic gain-of-function mechanisms. These unanticipated, SMN-dependent and neuron-specific liabilities warrant caution on the long-term safety of treating individuals with SMA with AAV9-SMN and the risks of uncontrolled protein expression by gene therapy.
We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click ...chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.
The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise ...these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.
Coal and gas outbursts are among the most severe disasters threatening the safety of coal mines around the world. They are dynamic phenomena characterized by large quantities of coal and gas ejected ...from working faces within a short time. Numerous researchers have conducted studies on outburst prediction, and a variety of indices have been developed to this end. However, these indices are usually empirical or based on local experience, and the accurate prediction of outbursts is not feasible due to the complicated mechanisms of outbursts. This study conducts outburst experiments using large-scale multifunctional equipment developed in the laboratory to develop a more robust outburst prediction method. In this study, the coal temperature during the outburst process was monitored using temperature sensors. The results show that the coal temperature decreased rapidly as the outburst progressed. Meanwhile, the coal temperature in locations far from the outburst mouth increased. The coal broken in the stress concentration state is the main factor causing the abnormal temperature rise. The discovery of these phenomena lays a theoretical foundation and provides an experimental basis for an effective outburst prediction method. An outburst prediction method based on monitoring temperature was proposed, and has a simpler and faster operation process and is not easily disturbed by coal mining activities. What is more, the critical values of coal temperature rises or temperature gradients can be flexibly adjusted according to the actual situations of different coal mines to predict outbursts more effectively and accurately.
Alternative splicing (AS) is one crucial step of gene expression that must be tightly regulated during neurodevelopment. However, the precise timing of developmental splicing switches and the ...underlying regulatory mechanisms are poorly understood. Here we systematically analyze the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis reveals early-switch and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl, and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the "immature" splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.
In the process of green and smart mine construction under the context of carbon neutrality, China's coal safety situation has been continuously improved in recent years. In order to recognize the ...development of coal production in China and prepare for future monitoring and prevention of safety incidents, this study mainly elaborated on the basic situation of coal resources and national mining accidents over the past five years (2017–2021), from four dimensions (accident level, type, region, and time), and then proposed the preventive measures based on accident statistical laws. The results show that the storage of coal resources has obvious geographic characteristics, mainly concentrated in the Midwest, with coal resources in Shanxi and Shaanxi accounting for about 49.4%. The proportion of coal consumption has dropped from 70.2% to 56% between 2011 and 2021, but still accounts for more than half of the all. Meanwhile, the accident-prone areas are positively correlated with the amount of coal production. Among different levels of coal mine accidents, general accidents had the highest number of accidents and deaths, with 692 accidents and 783 deaths, accounting for 87.6% and 54.64% respectively. The frequency of roof, gas, and transportation accidents is relatively high, and the number of single fatalities caused by gas accidents is the largest, about 4.18. In terms of geographical distribution of accidents, the safety situation in Shanxi Province is the most severe. From the time distribution of coal mine accidents, the accidents mainly occurred in July and August, and rarely occurred in February and December. Finally, the "4 + 4" safety management model is proposed, combining the statistical results with coal production in China. Based on the existing health and safety management systems, the managements are divided into four sub-categories, and more specific measures are suggested.
Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 ...and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3′ untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to misregulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Misregulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM, resulting in the persistence of neonatal polyadenylation patterns. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels.
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•Microsatellite CUG and CCUG expansion RNAs sequester MBNL proteins•MBNL proteins regulate alternative polyadenylation•MBNL loss induces a reversion to fetal polyadenylation patterns•Abnormal polyadenylation occurs in the RNA-mediated disease myotonic dystrophy
In the microsatellite expansion disorder myotonic dystrophy (DM), previous studies have implicated RNA splicing dysregulation as the major molecular signature associated with this disease. In this issue of Molecular Cell, Batra et al. demonstrate that alternative polyadenylation is also profoundly affected in DM.
Estimating the intensity of outbursts of coal and gas is important as the intensity and frequency of outbursts of coal and gas tend to increase in deep mining. Fully understanding the major factors ...contributing to coal and gas outbursts is significant in the evaluation of the intensity of the outburst. In this paper, we discuss the correlation between these major factors and the intensity of the outburst using Analysis of Variance（ANOVA） and Contingency Table Analysis（CTA）. Regression analysis is used to evaluate the impact of these major factors on the intensity of outbursts based on physical experiments. Based on the evaluation, two simple models in terms of multiple linear and nonlinear regression were constructed for the prediction of the intensity of the outburst. The results show that the gas pressure and initial moisture in the coal mass could be the most significant factors compared to the weakest factor-porosity. The P values from Fisher＇s exact test in CTA are： moisture（0.019）, geostress（0.290）, porosity（0.650）, and gas pressure（0.031）. P values from ANOVA are moisture（0.094）, geostress（0.077）, porosity（0.420）, and gas pressure（0.051）. Furthermore, the multiple nonlinear regression model（RMSE： 3.870） is more accurate than the linear regression model（RMSE： 4.091）.
China's first Mars mission Tianwen-1 successfully touched down the surface of the southern Utopia Planitia on 15th May 2021. In this study we investigate the geological characteristics of the target ...landing site (Site 1) and a backup landing site (Site 2) on Utopia Planitia for Tianwen-1 Mission. Site 1 is located in a topographic low and generally flat area to the southern Utopia basin, while Site 2 is located in the Utopia-Elysium transition zone. Several geological features indicative of the presence of a large reservoir of volatiles are identified and mapped in detail for both landing sites, including pitted cones, giant polygons, rampart and pancake craters. The volcanism-related morphologies, e.g., lava flows, lahars, and dikes, are also mapped for Site 2. The compositional analysis reveals little exposures of hydrous materials due to dust cover and/or lacking of data coverage over the regions. Crater counting analysis showed that the Site 1 has an absolute model age of ~3.68 Ga and ~3.45 Ga when including and excluding ghost craters, respectively. The absolute model age of Site 2 is ~3.34 Ga. Finally, the evolutionary history of the Utopia Planitia is discussed, and the related scientific hypotheses to be tested for the in situ exploration are suggested. Our study provides a framework for the Tianwen-1 in situ exploration, which will impose important constraints on the nature and evolution history of the northern lowlands.
•The detailed geological survey of the landing site of China's first Mars mission Tianwen-1 is conducted.•Various landforms within the landing site have been mapped and interpreted as the morphological indicators of water/ice.•The evolutionary history of the Utopia Planitia is discussed and the related scientific hypotheses to be tested are outlined.
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