Splice Forms Research Articles

Deciphering the Role of CBF/DREB Transcription Factors and Dehydrins in Maintaining the Quality of Table Grapes cv. Autumn Royal Treated with High CO2 Levels and Stored at 0°C.

C-repeat/dehydration-responsive element binding factors (CBF/DREB) are transcription factors which play a role in improving plant cold stress resistance and recognize the DRE/CRT element in the promoter of a set of cold regulated genes. Dehydrins (DHNs) are proteins that accumulate in plants in response to cold stress, which present, in some cases, CBF/DREB recognition sequences in their promoters and are activated by members of this transcription factor family. The application of a 3-day gaseous treatment with 20 kPa CO2 at 0°C to table grapes cv. Autumn Royal maintained the quality of the bunches during postharvest storage at 0°C, reducing weight loss and rachis browning. In order to determine the role of CBF/DREB genes in the beneficial effect of the gaseous treatment by regulating DHNs, we have analyzed the gene expression pattern of three VviDREBA1s (VviDREBA1-1, VviDREBA1-6, and VviDREBA1-7) as well as three VviDHNs (VviDHN1a, VviDHN2, and VviDHN4), in both alternative splicing forms. Results showed that the differences in VviDREBA1s expression were tissue and atmosphere composition dependent, although the application of high levels of CO2 caused a greater increase of VviDREBA1-1 in the skin, VviDREBA1-6 in the pulp and VviDREBA1-7 in the skin and pulp. Likewise, the application of high levels of CO2 regulated the retention of introns in the transcripts of the dehydrins studied in the different tissues analyzed. The DHNs promoter analysis showed that VviDHN2 presented the cis-acting DRE and CRT elements, whereas VviDHN1a presented only the DRE motif. Our electrophoretic mobility shift assays (EMSA) showed that VviDREBA1-1 was the only transcription factor that had in vitro binding capacity to the CRT element of the VviDHN2 promoter region, indicating that the transcriptional regulation of VviDHN1a and VviDHN4 would be carried out by activating other independent routes of these transcription factors. Our results suggest that the application of high CO2 levels to maintain table grape quality during storage at 0°C, leads to an activation of CBF/DREBs transcription factors. Among these factors, VviDREBA1-1 seems to participate in the transcriptional activation of VviDHN2 via CRT binding, with the unspliced form of this DHN being activated by high CO2 levels in all the tissues analyzed.

IgH isotype-specific B cell receptor expression influences B cell fate

Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgμ > mIgγ1 > mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells-which essentially do not exist under normal conditions-to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

Novel landscape of HLA-G isoforms expressed in clear cell renal cell carcinoma patients.

Immune checkpoints are powerful inhibitory molecules that promote tumor survival. Their blockade is now recognized as providing effective therapeutic benefit against cancer. Human leukocyte antigen G (HLA‐G), a recently identified immune checkpoint, has been detected in many types of primary tumors and metastases, in malignant effusions as well as on tumor‐infiltrating cells, particularly in patients with clear cell renal cell carcinoma (ccRCC). Here, in order to define a possible anticancer therapy, we used a molecular approach based on an unbiased strategy that combines transcriptome determination and immunohistochemical labeling, to analyze in‐depth the HLA‐G isoforms expressed in these tumors. We found that the expression of HLA‐G is highly variable among tumors and distinct areas of the same tumor, testifying a marked inter‐ and intratumor heterogeneity. Moreover, our results generate an inventory of novel HLA‐G isoforms which includes spliced forms that have an extended 5′‐region and lack the transmembrane and alpha‐1 domains. So far, these isoforms could not be detected by any method available and their assessment may improve the procedure by which tumors are analyzed. Collectively, our approach provides the first extensive portrait of HLA‐G in ccRCC and reveals data that should prove suitable for the tailoring of future clinical applications.

A novel gene, cilia flagella associated protein 44, encoding an enzyme cleaving FtsZ and tubulin contributes to the regulation of secretory pathway

We identified a novel gene, encoding the central region of the cilia and flagella associated protein 44 (Cfap44), that regulates trafficking of cellular components and morphology via the cleavage of cellular proteins (particularly β-tubulin). Although Cfap44 is registered in GenBank, the functions of both the central part and full-length protein are unknown except for a polymorphism associated with proprotein convertase 9 activity, the third gene of familiar hyper-cholesterolemia. In mice and humans, both unspliced and spliced RNAs were transcribed, and the spliced form was predominantly transcribed in the brain and embryonic tissues. In transfectants carrying this gene, various cellular processes such as cell division, transport of cellular components, and proteolytic processing of several proteins were found to be affected. The cleavage of β-tubulin was observed. A bacterial tubulin homolog, cell division protein FtsZ, was also cleaved in vivo and in vitro by the spliced form of Cfap44 product. Furthermore, the unspliced form showed proteolytic activity with low substrate specificity. Various biological activities of Cfap44 may be due to a direct effect of cleavage of β-tubulin inhibiting microtubule formation, or an indirect effect with the cross-recognition of the cleavage site between β-tubulin and other molecules.

Dietary Fat Quantity and Type Induce Transcriptome-Wide Effects on Alternative Splicing of Pre-mRNA in Rat Skeletal Muscle

Background: Fat-enriched diets produce metabolic changes in skeletal muscle, which in turn can mediate changes in gene regulation.Objective: We examined the high-fat-diet-induced changes in skeletal muscle gene expression by characterizing variations in pre-mRNA alternative splicing.Methods: Affymetrix Exon Array analysis was performed on the transcriptome of the gastrocnemius/plantaris complex of male obesity-prone Sprague-Dawley rats fed a 10% or 60% fat (lard) diet for 2 or 8 wk. The validation of exon array results was focused on troponin T (Tnnt3). Tnnt3 splice form analyses were extended in studies of rats fed 10% or 30% fat diets across 1- to 8-wk treatment periods and rats fed 10% or 45% fat diets with fat sources from lard or mono- or polyunsaturated fats for 2 wk. Nuclear magnetic resonance (NMR) was used to measure body composition.Results: Consumption of a 60% fat diet for 2 or 8 wk resulted in alternative splicing of 668 and 726 pre-mRNAs, respectively, compared with rats fed a 10% fat diet. Tnnt3 transcripts were alternatively spliced in rats fed a 60% fat diet for either 2 or 8 wk. The high-fat-diet-induced changes in Tnnt3 alternative splicing were observed in rats fed a 30% fat diet across 1- to 8-wk treatment periods. Moreover, this effect depended on fat type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard but not in response to diets enriched with mono- or polyunsaturated fatty acids. Fat mass (a proxy for obesity as measured by NMR) did not differ between groups in any study.Conclusions: Rat skeletal muscle responds to overconsumption of dietary fat by modifying gene expression through pre-mRNA alternative splicing. Variations in Tnnt3 alternative splicing occur independently of obesity and are dependent on dietary fat quantity and suggest a role for saturated fatty acids in the high-fat-diet-induced modifications in Tnnt3 alternative splicing.

Interneurons secrete prosaposin, a neurotrophic factor, to attenuate kainic acid-induced neurotoxicity

Prosaposin (PS) is a secretory neurotrophic factor, as well as a regulator of lysosomal enzymes. We previously reported the up-regulation of PS and the possibility of its axonal transport by GABAergic interneurons after exocitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, we performed double immunostaining with PS and three calcium binding protein markers: parvalbumin (PV), calbindin, and calretinin, for the subpopulation of GABAergic interneurons, and clarified that the increased PS around the hippocampal pyramidal neurons after KA injection existed mainly in the axons of PV positive interneurons. Electron microscopy revealed PS containing vesicles in the PV positive axon. Double immunostaining with PS and secretogranin or synapsin suggested that PS is secreted with secretogranin from synapses. Based on the results from in situ hybridization with two alternative splicing forms of PS mRNA, the increase of PS in the interneurons was due to the increase of PS+0 (mRNA without 9-base insertion) as in the choroid plexus, but not PS+9 (mRNA with 9-base insertion). These results were similar to those from the choroid plexus, which secretes an intact form PS+0 to the cerebrospinal fluid. Neurons, especially PV positive GABAergic interneurons, produce and secrete the intact form of PS around hippocampal pyramidal neurons to protect them against KA neurotoxicity.

On the Possibility of an Early Evolutionary Origin for the Spliced Leader Trans-Splicing.

Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

MicroRNA-9 regulates fetal alcohol-induced changes in D2 receptor to promote prolactin production.

Fetal alcohol exposure (FAE) is known to increase prolactin (PRL) secretion from the pituitary lactotropes. In this study, we determined whether microRNAs (miRs) are involved in FAE-induced alteration in PRL release. We employed a rat animal model of FAE involving feeding pregnant Fisher 344 rats with a liquid diet containing 6.7% alcohol between gestational days 7-21 (AF). Both cyclic and estradiol-implanted FAE females showed increased levels of plasma PRL and pituitary Prl mRNA but reduced levels of pituitary dopamine D2 receptor (D2r) and its short spliced form (D2s). FAE increased the expression levels of miR-9 and miR-326 and did not produce any significant changes in miR-153 or miR-200a levels in the pituitary. Effects of FAE on miR-9 and miR-326 were associated with reduced levels of D2r and D2s, increased levels of Prl in the pituitary, and in plasma. These effects of FAE on D2r, D2s and Prl were enhanced following estradiol treatment. In PRL-producing MMQ cells, ethanol increased miR-9 but not miR-326, reduced levels of D2r and D2s and increased levels of Prl Treatment of MMQ cells with an anti-miR-9 oligo reduced ethanol effects on miR-9, D2r, D2s and Prl miR-9 mimic oligos reduced the luciferase activity of reporter vector containing D2r 3'UTR, but failed to reduce the mutant luciferase activity. These data suggest that FAE programs the pituitary to produce increased amounts of miR-9 expression that represses the D2r gene and its spliced variant D2s by targeting its 3'UTR leading to an increase in PRL production and secretion.

Abstract 1292: Germline genetic, molecular and environmental factors modulate APOBEC mutagenesis in human tumors

Abstract Background: Activity of APOBEC3 enzymes has been identified as a main source of somatic mutations of a specific type (C to T or G substitutions in the TCA or TCT motifs). This mutation pattern, known as the APOBEC signature, has been described in 16 of 30 tumor types explored by TCGA, with bladder cancer being one of top cancers enriched with APOBEC-signature mutations. Recently, we demonstrated that a risk allele of a bladder cancer GWAS SNP rs1014971 was significantly associated with increased APOBEC3B (A3B) mRNA expression and APOBEC mutagenesis in bladder tumors. Here, we aimed to further explore factors that can modulate APOBEC mutagenesis in human tumors. Methods: Isoform-specific expression analysis of all APOBEC3 genes (A3A, A3B, A3C, A3D, A3G, A3F, and A3H) was performed in TCGA datasets for seven tumor types enriched for APOBEC mutations (> 25% of the samples) - bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical and endocervical cancers (CESC), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and ovarian serous cystadenocarcinoma (OV). Expression and APOBEC mutagenesis pattern were analyzed by multivariate linear regression in relation to rs1014971 genotypes and adjusting for age, sex, race, DNA CpG methylation, copy number alterations (CNA), and human papilloma virus (HPV) infection status (where relevant). Survival analysis was performed using multivariate Cox regression models that included age, sex, tumor stage, rs1014971, and load of APOBEC mutations or mRNA expression of APOBEC3 splicing forms. Results: Analysis of APOBEC mutagenesis identified significant interplay between SNP rs1014971, expression of specific APOBEC3 splicing isoforms, and environmental factors (such as HPV infection). For example, SNP was important in BLCA and sub-types of HNSC, while HPV infection status was the main factor associated with APOBEC mutagenesis in CESC and HNSC. Importantly, variable contribution of mutagenic and non-mutagenic isoforms of A3A and A3B, which are generated by alternative splicing, emerged as significant factors contributing to APOBEC mutagenesis across human tumors. Survival of the cancer patients was also differentially influenced by these factors. Conclusions: These findings suggest that APOBEC mutagenesis pattern is tissue-specifically affected by germline genetic, molecular and environmental factors. Deeper understanding of these factors and their interplay may pave the way for preventive and treatment measures of human cancers towards precision medicine. Citation Format: A. Rouf Banday, Ariunaa Bayanjargal, Krizia-Ivana Udquim, Olusegun O Onabajo, Ludmila Prokunina-Olsson. Germline genetic, molecular and environmental factors modulate APOBEC mutagenesis in human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1292. doi:10.1158/1538-7445.AM2017-1292

Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches

Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

IgH isotype-specific B cell receptor dosage regulates B cell fate

Abstract Immunoglobulin heavy chain (IgH) isotypes (e.g. IgM, IgG and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCR) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is unclear. We examined IgH isotype-specific BCR function by engineering polyclonal Ighγ1/γ1 and Ighɛ/ɛ mice, which initially produce IgG1 or IgE from their respective native genomic configurations. Transcripts producing IgM, IgG1, and IgE are produced in an alternative splice form bias hierarchy, where mIgμ>mIgγ1>mIgɛ—influencing respective BCR densities and fitness for populating the periphery—a pattern replicated in activated and memory B cells from wild-type mice. Restrained B cell development from Ighγ1/γ1 and Ighɛ/ɛ mice was proportional to sIg/mIg ratios, and was rescued by enforced expression of the respective mIgs. In addition, enhanced BCR signaling by way of PTEN deletion in IgE B cells generated functional IgE memory responses from IgE memory B cells themselves, which are normally absent under normal conditions, indicating that weak signal strength normally restrains IgE responses. Thus, IgH isotype-specific mIg dosage regulates B cell fate by influencing quantitative baseline BCR signal strength.

The protein subunit of telomerase displays patterns of dynamic evolution and conservation across different metazoan taxa

BackgroundMost animals employ telomerase, which consists of a catalytic subunit known as the telomerase reverse transcriptase (TERT) and an RNA template, to maintain telomere ends. Given the importance of TERT and telomere biology in core metazoan life history traits, like ageing and the control of somatic cell proliferation, we hypothesised that TERT would have patterns of sequence and regulatory evolution reflecting the diverse life histories across the Animal Kingdom.ResultsWe performed a complete investigation of the evolutionary history of TERT across animals. We show that although TERT is almost ubiquitous across Metazoa, it has undergone substantial sequence evolution within canonical motifs. Beyond the known canonical motifs, we also identify and compare regions that are highly variable between lineages, but show conservation within phyla. Recent data have highlighted the importance of alternative splice forms of TERT in non-canonical functions and although animals may share some conserved introns, we find that the selection of exons for alternative splicing appears to be highly variable, and regulation by alternative splicing appears to be a very dynamic feature of TERT evolution. We show that even within a closely related group of triclad flatworms, where alternative splicing of TERT was previously correlated with reproductive strategy, we observe highly diverse splicing patterns.ConclusionsOur work establishes that the evolutionary history and structural evolution of TERT involves previously unappreciated levels of change and the emergence of lineage specific motifs. The sequence conservation we describe within phyla suggests that these new motifs likely serve essential biological functions of TERT, which along with changes in splicing, underpin diverse functions of TERT important for animal life histories.

A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation

Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life1, yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS2-4. This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.

Last rolls of the yoyo: Assessing the human canonical protein count.

In 2004, when the protein estimate from the finished human genome was only 24,000, the surprise was compounded as reviewed estimates fell to 19,000 by 2014. However, variability in the total canonical protein counts (i.e. excluding alternative splice forms) of open reading frames (ORFs) in different annotation portals persists. This work assesses these differences and possible causes. A 16-year analysis of Ensembl and UniProtKB/Swiss-Prot shows convergence to a protein number of ~20,000. The former had shown some yo-yoing, but both have now plateaued. Nine major annotation portals, reviewed at the beginning of 2017, gave a spread of counts from 21,819 down to 18,891. The 4-way cross-reference concordance (within UniProt) between Ensembl, Swiss-Prot, Entrez Gene and the Human Gene Nomenclature Committee (HGNC) drops to 18,690, indicating methodological differences in protein definitions and experimental existence support between sources. The Swiss-Prot and neXtProt evidence criteria include mass spectrometry peptide verification and also cross-references for antibody detection from the Human Protein Atlas. Notwithstanding, hundreds of Swiss-Prot entries are classified as non-coding biotypes by HGNC. The only inference that protein numbers might still rise comes from numerous reports of small ORF (smORF) discovery. However, while there have been recent cases of protein verifications from previous miss-annotation of non-coding RNA, very few have passed the Swiss-Prot curation and genome annotation thresholds. The post-genomic era has seen both advances in data generation and improvements in the human reference assembly. Notwithstanding, current numbers, while persistently discordant, show that the earlier yo-yoing has largely ceased. Given the importance to biology and biomedicine of defining the canonical human proteome, the task will need more collaborative inter-source curation combined with broader and deeper experimental confirmation in vivo and in vitro of proteins predicted in silico. The eventual closure could be well be below ~19,000.

In silico analysis of the sequence features responsible for alternatively spliced introns in the model green alga Chlamydomonas reinhardtii.

Alternatively spliced introns are the ones that are usually spliced but can be occasionally retained in a transcript isoform. They are the most frequently used alternative splice form in plants (~50% of alternative splicing events). Chlamydomonas reinhardtii, a unicellular alga, is a good model to understand alternative splicing (AS) in plants from an evolutionary perspective as it diverged from land plants a billion years ago. Using over 7million cDNA sequences from both pyrosequencing and Sanger sequencing, we found that a much higher percentage of genes (~20% of multi-exon genes) undergo AS than previously reported (3-5%). We found a full component of SR and SR-like proteins possibly involved in AS. The most prevalent type of AS event (40%) was retention of introns, most of which were supported by multiple cDNA evidence (72%) while only 20% of them have coding capacity. By comparing retained and constitutive introns, we identified sequence features potentially responsible for the retention of introns, in the framework of an "intron definition" model for splicing. We find that retained introns tend to have a weaker 5' splice site, more Gs in their poly-pyrimidine tract and a lesser conservation of nucleotide 'C' at position -3 of the 3' splice site. In addition, the sequence motifs found in the potential branch-point region differed between retained and constitutive introns. Furthermore, the enrichment of G-triplets and C-triplets among the first and last 50 nt of the introns significantly differ between constitutive and retained introns. These could serve as intronic splicing enhancers. All the alternative splice forms can be accessed at http://bioinfolab.miamioh.edu/cgi-bin/PASA_r20140417/cgi-bin/status_report.cgi?db=Chre_AS .

Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis.

Cool ambient temperatures are major cues determining flowering time in spring. The mechanisms promoting or delaying flowering in response to ambient temperature changes are only beginning to be understood. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) regulates flowering in the ambient temperature range and FLM is transcribed and alternatively spliced in a temperature-dependent manner. We identify polymorphic promoter and intronic sequences required for FLM expression and splicing. In transgenic experiments covering 69% of the available sequence variation in two distinct sites, we show that variation in the abundance of the FLM-ß splice form strictly correlate (R2 = 0.94) with flowering time over an extended vegetative period. The FLM polymorphisms lead to changes in FLM expression (PRO2+) but may also affect FLM intron 1 splicing (INT6+). This information could serve to buffer the anticipated negative effects on agricultural systems and flowering that may occur during climate change.

Hallmarks of Alzheimer’s Disease in Stem-Cell-Derived Human Neurons Transplanted into Mouse Brain

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, invitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.

IRE1\u03b1-XBP1 pathway promotes melanoma progression by regulating IL-6/STAT3 signaling

BackgroundThe IRE1α-XBP1 pathway is the most conserved branch of the unfolded protein response pathways, which are activated during endoplasmic reticulum (ER) stress caused by the accumulation of unfolded/misfolded proteins in the ER lumen. The IRE1α-XBP1 pathway plays a critical role in various cancers. However, the role of this pathway in melanoma cell growth remains unclear.MethodsSixty-one pairs of melanoma specimens and corresponding normal tissues from patients were stained with XBP1. Then, XBP1 splicing levels were detected in human tissues and cell lines at the mRNA level. IL-6 expression levels were determined in both melanocytes (HEMn-MP) and melanoma cells (Mel-RMu) overexpressing the spliced form of XBP1 (XBP1s). IL-6 expression was also examined in 4μ8C-treated HEMn-MP and Mel-RMu cells overexpressing IRE1α. Next, we analyzed potential XBP1s binding sites within the IL-6 promoter and conducted ChIP experiments. IL-6/STAT3 signaling was detected by western blotting. Melanoma cell proliferation was examined by CCK8 and BrdU assays.ResultsThe mRNA and protein expression levels of XBP1s were significantly elevated in human melanoma tissues and cell lines compared with normal tissues or melanocytes, thus indicating the activation of the IRE1α-XBP1 branch in melanoma. Ectopic expression of IRE1α or XBP1s robustly enhanced IL-6 expression in HEMn-MP and Mel-RMu cells. Moreover, the inhibition of the RNase activity of IRE1α also abolished the effect of IRE1α in promoting IL-6 expression. Mechanistically, XBP1 binds the IL-6 promoter and activates its expression. Furthermore, secreted IL-6 functions in an autocrine/paracrine manner, activates the intracellular JAK/STAT3 pathway and promotes the proliferation of melanoma cells.ConclusionOur results reveal that the IRE1α-XBP1 pathway regulates Mel-RMu cell proliferation and progression by activating IL-6/STAT3 signaling.

Herpes Simplex Virus 1 UL41 Protein Suppresses the IRE1/XBP1 Signal Pathway of the Unfolded Protein Response via Its RNase Activity.

During viral infection, viruses hijack the host translation apparatus to produce large amounts of viral proteins, which leads to ER stress. To restore ER homeostasis, cells initiate the UPR to alleviate the effects of ER stress. The IRE1/XBP1 pathway is the most conserved UPR branch, and it activates ER-associated protein degradation (ERAD) to reduce the ER load. The IRE1/XBP1 branch is repressed during HSV-1 infection, but little is known about the underlying molecular mechanism. Our results show for the first time that UL41 suppresses the IRE1/XBP1 signal pathway by reducing the accumulation of XBP1 mRNA, and characterization of the underlying molecular mechanism provides new insight into the modulation of UPR by HSV-1.

BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

Breast cancer amplified sequence 2 (BCAS2) is involved in multiple biological processes, including pre-mRNA splicing. However, the physiological roles of BCAS2 are still largely unclear. Here we report that BCAS2 is specifically enriched in spermatogonia of mouse testes. Conditional disruption of Bcas2 in male germ cells impairs spermatogenesis and leads to male mouse infertility. Although the spermatogonia appear grossly normal, spermatocytes in meiosis prophase I and meiosis events (recombination and synapsis) are rarely observed in the BCAS2-depleted testis. In BCAS2 null testis, 245 genes are altered in alternative splicing forms; at least three spermatogenesis-related genes (Dazl, Ehmt2 and Hmga1) can be verified. In addition, disruption of Bcas2 results in a significant decrease of the full-length form and an increase of the short form (lacking exon 8) of DAZL protein. Altogether, our results suggest that BCAS2 regulates alternative splicing in spermatogonia and the transition to meiosis initiation, and male fertility.