Hsp83 loss suppresses proteasomal activity resulting in an upregulation of caspase-dependent compensatory autophagy

The 2 main degradative pathways that contribute to proteostasis are the ubiquitin-proteasome system and autophagy but how they are molecularly coordinated is not well understood. Here, we demonstrate an essential role for an effector caspase in the activation of compensatory autophagy when proteasomal activity is compromised. Functional loss of Hsp83, the Drosophila ortholog of human HSP90 (heat shock protein 90), resulted in reduced proteasomal activity and elevated levels of the effector caspase Dcp-1. Surprisingly, genetic analyses showed that the caspase was not required for cell death in this context, but instead was essential for the ensuing compensatory autophagy, female fertility, and organism viability. The zymogen pro-Dcp-1 was found to interact with Hsp83 and undergo proteasomal regulation in an Hsp83-dependent manner. Our work not only reveals unappreciated roles for Hsp83 in proteasomal activity and regulation of Dcp-1, but identifies an effector caspase as a key regulatory factor for sustaining adaptation to cell stress in vivo

Activation of an Endogenous Retrovirus-Associated Long Non-Coding RNA in Human Adenocarcinoma

Background Long non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology. Methods We used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA). Results We identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or ‘EVADR’, by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives. Conclusions Our results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics

The Oncogenic Roles of DICER1 RNase IIIb Domain Mutations in Ovarian Sertoli-Leydig Cell Tumors

AbstractDICER1, an endoribonuclease required for microRNA (miRNA) biogenesis, is essential for embryogenesis and the development of many organs including ovaries. We have recently identified somatic hotspot mutations in RNase IIIb domain of DICER1 in half of ovarian Sertoli-Leydig cell tumors, a rare class of sex-cord stromal cell tumors in young women. These hotspot mutations lost IIIb cleavage activity of DICER1 in vitro and failed to produce 5p-derived miRNAs in mouse Dicer1-null ES cells. However, the oncogenic potential of these hotspot DICER1 mutations has not been studied. Here, we further revealed that the global expression of 5p-derived miRNAs was dramatically reduced in ovarian Sertoli-Leydig cell tumors carrying DICER1 hotspot mutations compared with those without DICER1 hotspot mutation. The miRNA production defect was associated with the deregulation of genes controlling cell proliferation and the cell fate. Using an immortalized human granulosa cell line, SVOG3e, we determined that the D1709N-DICER1 hotspot mutation failed to produce 5p-derived miRNAs, deregulated the expression of several genes that control gonadal differentiation and cell proliferation, and promoted cell growth. Re-expression of let-7 significantly inhibited the growth of D1709N-DICER1 SVOG3e cells, accompanied by the suppression of key regulators of cell cycle control and ovarian gonad differentiation. Taken together, our data revealed that DICER1 hotspot mutations cause systemic loss of 5p-miRNAs that can both drive pseudodifferentiation of testicular elements and cause oncogenic transformation in the ovary

Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II

CrkRS (Cdc2-related kinase, Arg/Ser), or cyclin-dependent kinase 12 (CKD12), is a serine/threonine kinase believed to coordinate transcription and RNA splicing. While CDK12/CrkRS complexes were known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II), the cyclin regulating this activity was not known. Using immunoprecipitation and mass spectrometry, we identified a 65-kDa isoform of cyclin K (cyclin K1) in endogenous CDK12/CrkRS protein complexes. We show that cyclin K1 complexes isolated from mammalian cells contain CDK12/CrkRS but do not contain CDK9, a presumed partner of cyclin K. Analysis of extensive RNA-Seq data shows that the 65-kDa cyclin K1 isoform is the predominantly expressed form across numerous tissue types. We also demonstrate that CDK12/CrkRS is dependent on cyclin K1 for its kinase activity and that small interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 has similar effects on the expression of a luciferase reporter gene. Our data suggest that cyclin K1 is the primary cyclin partner for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

Background: CIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts. Results: Using mammalian cells lacking functional CIC, we found that CIC deficiency was associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability. Conclusions: Our study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC's increasingly apparent role as a "pan-cancer" tumour suppressor

Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.&nbsp

Large-scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein–protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations