Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and ...diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.
The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating ...mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.
Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes ...were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.
(101955) Bennu is a dark, Earth orbit-crossing, asteroid thought to be assembled from the fragments of an ancient collision. Spatially-resolved visible and near-infrared spectra of Bennu provide ...details about its surface properties and composition. In addition to a hydrated phyllosilicate band, we detect a ubiquitous 3.4-micron absorption feature, which we attribute to a mix of organic and carbonate materials. The shape and depth of this absorption feature vary across Bennu’s surface, spanning the range seen among similar main-belt asteroids. Its
distribution does not correlate with the temperature, reflectance, spectral slope, or hydrated
minerals although those characteristics correlate with each other in some cases. The deepest 3.4-micron absorptions occur on individual boulders. The variations may be due to differences in abundance, recent exposure, or space weathering.
We use Thermal Emission Imaging System (THEMIS) data to identify and characterize the global distribution of distinct materials interpreted to contain chloride salts on the Martian surface. ...Previously mapped global geochemical and physical properties are used in concert with thermophysical and morphological observations to assess the materials' local and regional characteristics. The results of our survey have expanded the characterization of the materials from ∼200 to ∼640 distinct sites dispersed throughout low‐albedo Noachian‐ and Hesperian‐aged terrains. Our survey also shows that the materials are detected in locally thermophysically distinct terrains and display a range of morphologies. Topography indicates that the majority of the materials occur in local lows, although crosscutting relationships indicate that some sites are located in “geologic windows” implying that the materials may be older than the terrains in which they are situated. Once exposed, the materials appear to undergo erosion, which may be the reason we do not observe large laterally extensive materials at the surface. The materials are predominantly local in nature, yet their prevalence across the southern highlands suggests that they represent one or more globally ubiquitous processes. We consider a number of formation hypotheses but find that most observations are consistent with formation via ponding of surface runoff or groundwater upwelling, although efflorescence and hydrothermal activity may also be possible in some locales. The materials' inferred ages suggest that the conditions that enabled the deposition of the materials persisted for up to 1 billion years.
Alternative splicing (AS) is an important mRNA maturation step that allows increased variability and diversity of proteins in eukaryotes. AS is dysregulated in numerous diseases, and its implication ...in the carcinogenic process is well known. However, progress in understanding how oncogenic viruses modulate splicing, and how this modulation is involved in viral oncogenicity has been limited. Epstein-Barr virus (EBV) is involved in various cancers, and its EBNA1 oncoprotein is the only viral protein expressed in all EBV malignancies.
In the present study, the ability of EBNA1 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in 1238 cancer-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by EBNA1.
Upon EBNA1 expression, we detected modifications to the AS profiles of 89 genes involved in cancer. Moreover, we show that EBNA1 modulates the expression levels of various splicing factors such as hnRNPA1, FOX-2, and SF1. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that EBNA1 immunoprecipitates specific cellular mRNAs, but not the ones that are spliced differently in EBNA1-expressing cells.
The EBNA1 protein can modulate the AS profiles of numerous cellular genes. Interestingly, this modulation protein does not require the RNA binding activity of EBNA1. Overall, these findings underline the novel role of EBNA1 as a cellular splicing modulator.
Psoriasis is an immune-mediated inflammatory skin disease that affects millions worldwide. Studying immune cells involved in psoriasis pathogenesis is essential to identify effective and safe ...therapeutics for the disease. Using human psoriasis skin, activated macrophages were observed in both lesional and non-lesional skin, but were elevated in lesional skin. Activation of the IL-23/IL-17 pathway is integral to the development of psoriasis. To further characterize the monocyte/macrophage (Mon/Mac) population when the IL-23 pathway is activated, a murine model of intradermal injection of IL-23 was used. Flow cytometry revealed that Mon/Mac cells were the dominant immune population, particularly late in the model, highlighted by strong presence of Ly6C
MHC II
cells. The Mon/Mac cells were also shown to have high expression for TNFα but not IL-17A. Prophylactic dosing of a CSF-1R inhibitor to deplete Mon/Mac cells significantly reduced several inflammatory mediators from the skin tissue suggesting a pathogenic role for Mon/Mac. Treatment dosing of the inhibitor produced a less robust effect. Mon/Mac cells were also differentiated by levels of Ki67 and TNFα expression. These data point to an important contribution of Mon/Mac cells in IL-23 related skin inflammation and suggest that these cells are a significant player in the underlying pathophysiology of psoriasis.
Few studies have investigated the relative influence of individual susceptibility versus household exposure factors versus regional clustering of infection on soil transmitted helminth (STH) ...transmission. The present study examined reinfection dynamics and spatial clustering of Ascaris lumbricoides, Trichuris trichiura and hookworm in an extremely impoverished indigenous setting in rural Panamá over a 16 month period that included two treatment and reinfection cycles in preschool children.
Spatial cluster analyses were used to identify high prevalence clusters for each nematode. Multivariate models were then used (1) to identify factors that differentiated households within and outside the cluster, and (2) to examine the relative contribution of regional (presence in a high prevalence cluster), household (household density, asset-based household wealth, household crowding, maternal education) and individual (age, sex, pre-treatment eggs per gram (epg) feces, height-for-age, latrine use) factors on preschool child reinfection epgs for each STH. High prevalence spatial clusters were detected for Trichuris and hookworm but not for Ascaris. These clusters were characterized by low household density and low household wealth indices (HWI). Reinfection epg of both hookworm and Ascaris was positively associated with pre-treatment epg and was higher in stunted children. Additional individual (latrine use) as well as household variables (HWI, maternal education) entered the reinfection models for Ascaris but not for hookworm.
Even within the context of extreme poverty in this remote rural setting, the distinct transmission patterns for hookworm, Trichuris and Ascaris highlight the need for multi-pronged intervention strategies. In addition to poverty reduction, improved sanitation and attention to chronic malnutrition will be key to reducing Ascaris and hookworm transmission.
The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue ...maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.
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•AMPK blocks Shh-induced transcriptional activity•AMPK reduces GLI1 protein level and stability•AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074•GLI13A protein is resistant to AMPK and has higher stability and oncogenic ability
Li. et al. show that AMPK is linked to the Hh signaling pathway. Activation of AMPK phosphorylates GLI1, a Hedgehog transcriptional activator, and inhibits Hh activity. GLI1 phosphorylation decreases GLI1 protein stability and reduces cell growth, colony formation, and tumor growth in mice.
Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular ...genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data.
Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control.
This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.
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