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Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome.Here, we used the newly developed hyperactiveTc Buster(Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function.Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

(1) Background: A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional alternative splicing contributes to the complexity of the proteome and is critical in understanding gene expression. mRNA-sequencing (RNA-seq) has been widely used to study the transcriptome and provides opportunity to detect alternative splicing events among different biological conditions. Despite the popularity of studying transcriptome variants with RNA-seq, few efficient and user-friendly bioinformatics tools have been developed for the genome-wide detection and visualization of alternative splicing events. (2) Results: We propose AS-Quant, (Alternative Splicing Quantitation), a robust program to identify alternative splicing events from RNA-seq data. We then extended AS-Quant to visualize the splicing events with short-read coverage plots along with complete gene annotation. The tool works in three major steps: (i) calculate the read coverage of the potential spliced exons and the corresponding gene, (ii) categorize the events into five different categories according to the annotation, and assess the significance of the events between two biological conditions, (iii) generate the short reads coverage plot for user specified splicing events. Our extensive experiments on simulated and real datasets demonstrate that AS-Quant outperforms the other three widely used baselines, SUPPA2, rMATS, and diffSplice for detecting alternative splicing events. Moreover, the significant alternative splicing events identified by AS-Quant between two biological contexts were validated by RT-PCR experiment. (3) Availability: AS-Quant is implemented in Python 3.0. Source code and a comprehensive user’s manual are freely available online.

Mammalian target of rapamycin (mTOR) pathway is crucial in cell proliferation. Many have associated its dysregulation with numerous human pathogenic processes. Previously, we reported 3’-untranslated region (UTR) length dynamics by alternative polyadenylation in the mTOR-activated transcriptome and its impact on the proteome. Here, we further explored the mTOR-activated transcriptome with regard to alternative splicing (AS) events and their functional relevance. We employed cellular models with genetic or pharmacological manipulation of mTOR activity and investigated the changes of transcriptome profile using RNA-Seq experiments and a custom-developed AS-Quant pipeline. Strikingly, we found that hyperactivation of mTOR in cells promotes transcriptome-wide exon skipping/exclusion. These AS events multifariously regulate the proteome: exon-skipping in the coding regions widely affects functional domains in the proteome; exon-skipping in the 5’-UTR can control translation efficiency. Moreover, a number of these exon-skipping events affects potential ubiquitination and phosphorylation sites and produces protein isoforms with varying stabilities. Furthermore, some of these exon-skipping events can switch transcripts from being coding to non-coding or vice versa, suggesting AS as a built-in post-transcriptional molecular switch for gene expression and/or functional regulation. Notably, we found that mTOR-activated widespread exon skipping is in part facilitated by splicing factor Srsf3. Our study reveals previously unappreciated mTOR-coordinated post-transcriptional pathways that regulate cellular proteome. These findings also highlight multifaceted mechanisms of AS in controlling functional proteome.

Background The eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3′-untranslated region (3′-UTR) of mRNA produces transcripts with shorter or longer 3′-UTR. Often, 3′-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3′-UTR APA is known to modulate translation and provides a mean to regulate gene expression at the post-transcriptional level. Current bioinformatics pipelines have limited capability in profiling 3′-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3′-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3′-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations. Methods APA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3′-UTR transcripts in the RNA-seq data. APA-Scan works in three major steps: (i) calculate the read coverage of the 3′-UTR regions of genes; (ii) identify the potential APA sites and evaluate the significance of the events among two biological conditions; (iii) graphical representation of user specific event with 3′-UTR annotation and read coverage on the 3′-UTR regions. APA-Scan is implemented in Python3. Source code and a comprehensive user’s manual are freely available at https://github.com/compbiolabucf/APA-Scan. Result APA-Scan was applied to both simulated and real RNA-seq datasets and compared with two widely used baselines DaPars and APAtrap. In simulation APA-Scan significantly improved the accuracy of 3′-UTR APA identification compared to the other baselines. The performance of APA-Scan was also validated by 3′-end-seq data and qPCR on mouse embryonic fibroblast cells. The experiments confirm that APA-Scan can detect unannotated 3′-UTR APA events and improve genome annotation. Conclusion APA-Scan is a comprehensive computational pipeline to detect transcriptome-wide 3′-UTR APA events. The pipeline integrates both RNA-seq and 3′-end-seq data information and can efficiently identify the significant events with a high-resolution short reads coverage plots.

A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional alternative splicing contributes to the complexity of proteome and are critical in understanding gene expression. mRNA-sequencing (RNA-seq) has been widely used to study the transcriptome and provides opportunity to detect alternative splicing events among different biological conditions. Despite the popularity of studying transcriptome variants with RNA-seq, few efficient and user-friendly bioinformatics tools have been developed for the genome-wide detection and visualization of alternative splicing events. We have developed AS-Quant (Alternative Splicing Quantitation), a robust program to identify alternative splicing events and visualize the short-read coverage with gene annotations. AS-Quant works in three steps: (i) calculate the read coverage of the potential splicing exons and the corresponding gene; (ii) categorize the splicing events into five different types based on annotation, and assess the significance of the events between two biological conditions; (iii) generate the short reads coverage plot with a complete gene annotation for user specified splicing events. To evaluate the performance, two significant alternative splicing events identified by AS-Quant between two biological contexts were validated by RT-PCR.ImplementationAS-Quant is implemented in Python. Source code and a comprehensive user’s manual are freely available at https://github.com/CompbioLabUCF/AS-Quant

Additional file 1 Figures S1-S6 and Table S1-S2.

3′-untranslated regions (UTRs) can vary through the use of alternative polyadenylation sites during pre-mRNA processing. Multiple publically available pipelines combining high profiling technologies and bioinformatics tools have been developed to catalog changes in 3′-UTR lengths. In our recent RNA-seq experiments using cells with hyper-activated mammalian target of rapamycin (mTOR), we found that cellular mTOR activation leads to transcriptome-wide alternative polyadenylation (APA), resulting in the activation of multiple cellular pathways. Here, we developed a novel bioinformatics algorithm, IntMAP, which integrates RNA-Seq and PolyA Site (PAS)-Seq data for a comprehensive characterization of APA events. By applying IntMAP to the datasets from cells with hyper-activated mTOR, we identified novel APA events that could otherwise not be identified by either profiling method alone. Several transcription factors including Cebpg (CCAAT/enhancer binding protein gamma) were among the newly discovered APA transcripts, indicating that diverse transcriptional networks may be regulated by mTOR-coordinated APA. The prevention of APA in Cebpg using the CRISPR/cas9-mediated genome editing tool showed that mTOR-driven 3′-UTR shortening in Cebpg is critical in protecting cells from endoplasmic reticulum (ER) stress. Taken together, we present IntMAP as a new bioinformatics algorithm for APA analysis by which we expand our understanding of the physiological role of mTOR-coordinated APA events to ER stress response. IntMAP toolbox is available at http://compbio.cs.umn.edu/IntMAP/.

Advances in cellular immunotherapy have led to multiple FDA approvals for autologous CAR-T cell therapies in acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas (NHL), and multiple myeloma (MM). While effective, autologous CAR-T therapies are limited by safety concerns, lack of scalability for patient derived starting material, and long vein-to-vein timelines. Allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefit without the safety and manufacturing challenges associated with CAR-T therapy. CAR-NK cell therapies are particularly attractive in AML as the inherent graft-versus-leukemia activity of NK cells can be effectively augmented by a CAR directed to an AML expressed antigen. CD70 expression is associated with several indications, including AML, NHL, and renal cell carcinoma (RCC), and it is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is absent in normal bone marrow hematopoietic stem cells. 1 While aberrant expression of CD70 is associated with several solid and hematological indications, its expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells. 2 Here we demonstrate that CD70 is not expressed in resting peripheral blood NK cells but is strongly upregulated in response to NK cell activation by engineered feeder cells. Introduction of CARs targeting CD70 into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. While CD70 is expressed in activated NK cells, knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair endogenous cytotoxicity against K562 target cells. Using the non-viral TcBuster™ Transposon System (Bio-Techne), we were able to deliver transposons containing a CD70 CAR and an IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. This single-step process resulted in >70% CAR integration/expression and >80% knockout of CD70. The resulting CD70 knockout CAR-NK cells were resistant to fratricide and expanded comparably to mock-engineered NK cells following feeder cell activation. The IL15 expression cassette enabled enhanced persistence of CAR-NK cells in vitro without exogenous cytokine support. In functional assays, CD70 KO NK cells engineered with the CD70 CAR and IL15 expression cassette mediated cytotoxicity against multiple CD70-positive tumor cell lines, expressed the degranulation marker CD107a (LAMP1), and expressed the cytokines IFNγ and TNFα. Overall, the results demonstrate the potential for targeting CD70 with CAR-NK cell therapy for the treatment of AML, RCC, and other CD70-positive malignancies while overcoming the risk posed by fratricide by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing. 1 Perna et al. 2017, Cancer Cell. 32:506-519. 2 McEarchern et al. 2008, Clin Cancer Res. 14(23):7763-7772. Disclosures Choi: Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Walsh: Obsidian Therapeutics: Ended employment in the past 24 months. Khamhoung: Rubius Therapeutics, Inc.: Ended employment in the past 24 months. Johnson: Celsius Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Franco: KSQ Therapeutics: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Swiech: Agenus: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months; Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Richardson: Novartis Pharma: Current equity holder in publicly-traded company; Obsidian Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months.

Background: Most eukaryotic genes produce different transcripts of multiple isoforms by inclusion or exclusion of particular exons. The isoforms of a gene often play diverse functional roles and thus, it is necessary to accurately measure isoform expressions as well as the genes'. While previous studies have demonstrated the strong agreement between mRNA-sequencing (RNA-seq) and array-based gene and/or isoform quantification platforms (Microarray gene expression and Exon-array), the more recently developed NanoString platform has not been systematically evaluated and compared, especially in large-scale studies across different cancer domains. Results: In this paper, we present a large-scale comparative study among RNA-seq, NanoString, array-based and RT-qPCR platforms using 46 cancer cell lines across different cancer types to understand and evaluate the calibers of the platforms for measuring gene and isoform expressions in cancer studies. We first performed NanoString experiments on 59 cancer cell lines with 403 custom-designed probes for measuring the expressions of 405 isoforms in 155 genes and additional RT-qPCR experiments for a subset of the measured isoforms in 13 cell lines, and then combined the data with the matched RNA-seq, Exon-array and Microarray data of 46 of the 59 cell lines for the comparative analysis. Conclusion: In the comparisons of the platforms for evaluating expressions at both isoform and gene levels, we found that (1) the degree of agreement across platforms on quantifying isoform expressions is lower than gene expressions; (2) NanoString and Exon-array are not consistent on isoform quantification even though both techniques are based on hybridization reactions; (3) RT-qPCR experiments are more consistent with RNA-seq quantification results on isoform-level compared to NanoString and Exon-array; (4) different RNA-seq isoform quantification algorithms showed inconsistent results, and two isoform quantification methods Net-RSTQ and eXpress are more consistent across the platforms in the comparison; (5) RNA-seq has the best overall consistent with the other platforms on gene expression quantification.

U2 auxiliary factor 1 (U2AF1) functions in 3′-splice site selection during pre-mRNA processing. Alternative usage of duplicated tandem exons in U2AF1 produces two isoforms, U2AF1a and U2AF1b, but their functional differences are unappreciated due to their homology. Through integrative approaches of genome editing, customized-transcriptome profiling and crosslinking-mediated interactome analyses, we discovered that the expression of U2AF1 isoforms is controlled by mTOR and they exhibit a distinctive molecular profile for the splice site and protein interactomes. Mechanistic dissection of mutually exclusive alternative splicing events revealed that U2AF1 isoforms’ inherent differential preferences of nucleotide sequences and their stoichiometry determine the 3′-splice site. Importantly, U2AF1a-driven transcriptomes feature alternative splicing events in the 5′-untranslated region (5′-UTR) that are favorable for translation. These findings unveil distinct roles of duplicated tandem exon-derived U2AF1 isoforms in the regulation of the transcriptome and suggest U2AF1a-driven 5′-UTR alternative splicing as a molecular mechanism of mTOR-regulated translational control.

Calsenilin is a calcium sensor protein that interacts with presenilin and increases calcium-triggered neuronal apoptosis, and γ-secretase activity. Notch is a cell surface receptor that regulates cell-fate decisions and synaptic plasticity in brain. The aim of the present study was to characterize the role of calsenilin as a regulator of the γ-secretase cleavage of Notch in ischemic stroke. Here, we determined the modulation of expression level and cellular distribution of calsenilin in neurons subjected to ischemic-like conditions. The levels of calsenilin and presenilin were increased in primary neurons after oxygen and glucose deprivation. Furthermore, calsenilin was found to enhance the γ-secretase cleavage of Notch and to contribute to cell death under ischemia-like conditions. The inhibition of γ-secretase activity and a presenilin deficiency were both found to protect against calsenilin-mediated ischemic neuronal death. The expression of calsenilin was found to be increased in brain following experimental ischemic stroke. These findings establish a specific molecular mechanism by which the induction of calsenilin enhances Notch activation in ischemic stroke, and identify calsenilin as an upstream of the γ-secretase cleavage of Notch.

Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses.

Huntington's disease (HD), an inherited neurodegenerative disorder, is caused by an expansion of cytosine-adenine-guanine repeats in the huntingtin gene. The aggregation of mutant huntingtin (mtHTT) and striatal cell loss are representative features to cause uncontrolled movement and cognitive defect in HD. However, underlying mechanism of mtHTT aggregation and cell toxicity remains still elusive. Here, to find new genes modulating mtHTT aggregation, we performed cell-based functional screening using the cDNA expression library and isolated IRE1 gene, one of endoplasmic reticulum (ER) stress sensors. Ectopic expression of IRE1 led to its self-activation and accumulated detergent-resistant mtHTT aggregates. Treatment of neuronal cells with ER stress insults, tunicamycin and thapsigargin, increased mtHTT aggregation via IRE1 activation. The kinase activity of IRE1, but not the endoribonuclease activity, was necessary to stimulate mtHTT aggregation and increased death of neuronal cells, including SH-SY5Y and STHdhQ111/111 huntingtin knock-in striatal cells. Interestingly, ER stress impaired autophagy flux via IRE1-TRAF2 pathway, thus enhancing cellular accumulation of mtHTT. Atg5 deficiency in M5-7 cells increased mtHTT aggregation but blocked ER stress-induced mtHTT aggregation. Further, ER stress markers including p-IRE1 and autophagy markers such as p62 were up-regulated exclusively in the striatal tissues of HD mouse models and in HD patients. Moreover, down-regulation of IRE1 expression rescues the rough-eye phenotype by mtHTT in a HD fly model. These results suggest that IRE1 plays an essential role in ER stress-mediated aggregation of mtHTT via the inhibition of autophagy flux and thus neuronal toxicity of mtHTT aggregates in HD.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent with cancer-selective apoptogenic activity. It evokes the canonical caspase-mediated cell death pathway through death-inducing signaling complex (DISC) formation. We identified that Peroxiredoxin 6 (Prx6) interacts with caspase-10 and caspase-8 via the death effector domain (DED). Prx6 suppresses TRAIL-mediated cell death in human cancer cells, but not that induced by intrinsic apoptosis inducers such as etoposide, staurosporine, or A23187. Among Prx1–6 members, only Prx6 binds to DED caspases and is most effective in suppressing TRAIL or DED caspase-induced cell death. The antiapoptotic activity of Prx6 against TRAIL is not likely associated with its peroxidase activity but is associated with its ability to bind to DED caspases. Increased expression of Prx6 enhances the binding of Prx6 to caspase-10 but reduces TRAIL-induced DISC formation and subsequently caspase activation. Interestingly, Prx6 is highly upregulated in metastatic gastric cancer cells, which are relatively resistant to TRAIL as compared with primary cancer cells. Downregulation of Prx6 sensitizes the metastatic cancer cells to TRAIL-induced cell death. Taken together, these results suggest that Prx6 modulates TRAIL signaling as a negative regulator of caspase-8 and caspase-10 in DISC formation of TRAIL-resistant metastatic cancer cells.

Earlier reports found that calsenilin is a transcriptional repressor or a subunit of plasma membrane channel, and indicated that calsenilin was present in the nucleus or plasma membrane. Immunohistochemical and subcellular fractionation analysis, however, revealed that calsenilin/DREAM/KChlP3 was distributed throughout the cytoplasm of SK-N-BE2(C), Jurkat, and HeLa cells. In addition, the expression of calsenilin suppressed the ATP-induced increase in intracellular Ca 2+ concentrations. By increase in intracellular calcium concentration, calsenilin was translocated into the nucleus.

Calsenilin/DREAM/KChlPS, a neuronal Ca 2+ -binding protein, has multifunctions in nucleus and cytosol. Here, we identified CLN3 as a calsenilin-binding partner whose mutation or deletion is observed in Batten disease. In vitro binding and immunoprecipitation assays show that calsenilin interacts with the C-terminal region of CLN3 and the increase of Ca 2+ concentration in vitro and in cells causes significant dissociation of calsenilin from CLN3. Ectopic expression of CLN3 or its deletion mutant containing only the C-terminus (153-438) and capable of binding to calsenilin suppresses thapsigargin or A23187-induced death of neuronal cells. In contrast, CLN3 deletion mutant containing the N-terminus (1 -153) or (1-263), which is frequently found in Batten disease, induces the perturbation of Ca 2+ transient and fails to inhibit the cell death. In addition, the expression of calsenilin is increased in the brain tissues of CLN3 knock-out mice and SH-SY5Y/CL/V3 knock-down cells. Down-regulation of CLN3 expression sensitizes SH-SY5Y cells to thapsigargin or A23187. However, additional decrease of calsenilin expression rescues the sensitivity of SH-SY5Y/CL/V3 knock-down cells to Ca 2+ -mediated cell death. These results suggest that the vulnerability of CLN3 knock-out or CLN3 deletion (1-153)-expressing neuronal cells to Ca 2+ -induced cell death may be mediated by calsenilin.

Mammalian target of rapamycin (mTOR) enhances translation from a subset of messenger RNAs containing distinct 5'-untranslated region (UTR) sequence features. Here we identify 3'-UTR shortening of mRNAs as an additional molecular signature of mTOR activation and show that 3'-UTR shortening enhances the translation of specific mRNAs. Using genetic or chemical modulations of mTOR activity in cells or mouse tissues, we show that cellular mTOR activity is crucial for 3'-UTR shortening. Although long 3'-UTR-containing transcripts minimally contribute to translation, 3-'UTR-shortened transcripts efficiently form polysomes in the mTOR-activated cells, leading to increased protein production. Strikingly, selected E2 and E3 components of ubiquitin ligase complexes are enriched by this mechanism, resulting in elevated levels of protein ubiquitination on mTOR activation. Together, these findings identify a previously uncharacterized role for mTOR in the selective regulation of protein synthesis by modulating 3'-UTR length of mRNAs.

High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/., Author Summary New sequencing technologies for transcriptome-wide profiling of RNAs have greatly promoted the interest in isoform-based functional characterizations of a cellular system. Elucidation of gene expressions at the isoform resolution could lead to new molecular mechanisms such as gene-regulations and alternative splicings, and potentially better molecular signals for phenotype predictions. However, it could be overly optimistic to derive the proportion of the isoforms of a gene solely based on short read alignments. Inherently, systematical sampling biases from RNA library preparation and ambiguity of read origins in overlapping isoforms pose a problem in reliability. The work in this paper exams the possibility of using protein domain-domain interactions as prior knowledge in isoform transcript quantification. We first made the observation that protein domain-domain interactions positively correlate with isoform co-expressions in TCGA data and then designed a probabilistic EM approach to integrate domain-domain interactions with short read alignments for estimation of isoform proportions. Validated by qRT-PCR experiments on three cell lines, simulations and classifications of TCGA patient samples in several cancer types, Net-RSTQ is proven a useful tool for isoform-based analysis in functional genomes and systems biology.

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid β1–42 (Aβ42)-induced autophagy activation through its interaction with BECN1. Treatment with Aβ42 enhanced autophagy flux in neuronal cells. Aβ42-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aβ42. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aβ42, defining a novel role of PRNP in the regulation of autophagy.

Accumulation of expanded polyglutamine proteins is considered to be a major pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel regulator of cellular accumulation of expanded polyglutamine track protein using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the formation of ubiquitin-positive and detergent-resistant aggregates of mutant huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum of Huntington disease patients and is induced in cultured striatal neurons by endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs endocytosis, which in turn enhances mtHTT aggregation. On the contrary, down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and endocytosis inhibition. Moreover, stereotactic injection into the striatum and intraperitoneal injection of tunicamycin significantly increase mtHTT aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice, respectively. Taken together, these results suggest that exposure to ER stress increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation via the endocytosis pathway.

The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse α-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe2GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe2GlyDH, shedding new insights into the protection of heart from hypoxic damages.

Apoptosis or programmed cell death is genetically controlled and plays a central role in normal development and tissue homeostasis, including development of the nervous system and regulation of the immune system (8, 25, 35). Dysregulated apoptosis has been implicated in the pathogenesis of cancer and autoimmune, neurodegenerative, and cardiovascular diseases (28). The cell death machinery that is conserved throughout evolution is composed of activators, inhibitors, and effectors (14). The effector arm of the cell death pathway consists of a family of cysteinyl aspartate-specific proteases called caspases (2). Data suggest that apoptotic cell death can be brought about by the loss of Ca2+ homeostatic control, but it can also be finely tuned positively or negatively by more subtle changes in Ca2+ distribution within intracellular compartments (29). While protein kinases such as AKT and ERK have been reported to modulate caspase activity through phosphorylation (1, 7), there have been few regulatory molecules directly linking cytotoxic Ca2+ signaling to caspase activity. Based on sequence similarities, three prominent interaction motifs involved in apoptosis are recognized. The death domain superfamily consists of death domain (DD), death effector domain (DED), and caspase recruitment domain (CARD) families (16, 26). In recent years, a number of CARD-containing proteins have been identified and participate in various signaling pathways during apoptosis and NF-κB activation. ARC is a CARD protein that selectively interacts with the initiator caspase 8 and significantly attenuates death receptor-induced apoptosis (22). Recently, ARC was also found capable of blocking caspase-independent events such as hypoxia-induced cytochrome c release and hydrogen peroxide (H2O2)-induced necrotic cell death (10, 27). We also described the protective role of ARC during hypoxia of hippocampal neurons (17). In addition, ARC is known to be phosphorylated by protein kinase CK2, modulating the subcellular localization of ARC (23). While increasing evidence suggests an inhibitory role for ARC in the diverse cell death processes, the precise mechanism by which ARC interferes with caspase-dependent and caspase-independent cell death has not been defined yet. In the present study, we postulate that ARC is a Ca2+-binding CARD protein that modulates activation of caspase 8.

Mutant presenilins cause early-onset of familial Alzheimer's disease and render cells vulnerable to apoptosis. Calsenilin/DREAM/KChIP3 is a multifunctional calcium-binding protein that interacts with presenilin and mediates calcium-mediated apoptosis. In the present study, we report that the calsenilin-mediated apoptosis is regulated by presenilin. The expression of calsenilin was highly up-regulated in neuronal cells undergoing Abeta42-triggered cell death. The incidence of calsenilin-mediated apoptosis was diminished in presenilin-1(-/-) mouse embryonic fibroblast cells or neuronal cells stably expressing a loss-of-function presenilin-1 mutant. On the contrary, an array of familial Alzheimer's disease-associated presenilin mutants (gain-of-function) increased calsenilin-induced cell death. Moreover, gamma-secretase inhibitors, including compound E and DAPT, decreased the calsenilin-induced cell death. These results suggest that the pro-apoptotic activity of calsenilin coordinates with presenilin/gamma-secretase activity to play a crucial role in the neuronal death of Alzheimer's disease.