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Neurodegenerative disorders such as Alzheimer&rsquo, s disease (AD) and Parkinson&rsquo, s disease (PD) are characterized by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and the unfolded protein response (UPR). Modulating the UPR is one of the major challenges to counteract the development of neurodegenerative disorders and other diseases with affected UPR. Here, we show that miR-34a-5p directly targets the IRE1&alpha, branch of the UPR, including the genes BIP, IRE1&alpha, and XBP1. Upon induction of ER stress in neuronal cells, miR-34a-5p overexpression impacts the resulting UPR via a significant reduction in IRE1&alpha, and XBP1s that in turn leads to decreased viability, increased cytotoxicity and caspase activity. The possibility to modify the UPR signaling pathway by a single miRNA that targets central genes of the IRE1&alpha, branch offers new perspectives for future therapeutic approaches against neurodegeneration.

SummarySince the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.

// Martin Hart 1 , Stefanie Rheinheimer 1 , Petra Leidinger 1 , Christina Backes 2 , Jennifer Menegatti 3 , Tobias Fehlmann 2 , Friedrich Grasser 3 , Andreas Keller 2 , Eckart Meese 1 1 Institute of Human Genetics, Saarland University, 66421 Homburg, Germany 2 Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrucken, Germany 3 Institute of Virology, Saarland University Medical School, 66421 Homburg, Germany Correspondence to: Martin Hart, email: martin.hart@uks.eu Keywords: network analysis, PKC family, miR-34a, cancer, immune system Received: March 08, 2016 Accepted: April 16, 2016 Published: April 29, 2016 ABSTRACT Circulating miRNAs have been associated with numerous human diseases. The lack of understanding the functional roles of blood-born miRNAs limits, however, largely their value as disease marker. In a systems biology analysis we identified miR-34a as strongly associated with pathogenesis. Genome-wide analysis of miRNAs in blood cell fractions highlighted miR-34a as most significantly up-regulated in CD3+ cells of lung cancer patients. By our in silico analysis members of the protein kinase C family (PKC) were indicated as miR-34a target genes. Using a luciferase assay, we confirmed binding of miR-34a-5p to target sequences within the 3’UTRs of five PKC family members. To verify the biological effect, we transfected HEK 293T and Jurkat cells with miR-34a-5p causing reduced endogenous protein levels of PKC isozymes. By combining bioinformatics approaches with experimental validation, we demonstrate that one of the most relevant disease associated miRNAs has the ability to control the expression of a gene family.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid tumor which is occasionally Epstein-Barr virus (EBV) positive and is further subtyped as activated B-cell DLBCL (ABC-DLBCL) and germinal center B-cell DLBCL (GCB-DLBCL), which has implications for prognosis and treatment. We performed Ago2 RNA immunoprecipitation followed by high-throughput RNA sequencing (Ago2-RIP-seq) to capture functionally active microRNAs (miRNAs) in EBV-negative ABC-DLBCL and GCB-DLBCL cell lines and their EBV-infected counterparts. In parallel, total miRNA profiles of these cells were determined to capture the cellular miRNA profile for comparison with the functionally active profile. Selected miRNAs with differential abundances were validated using real-time quantitative PCR (RT-qPCR) and Northern blotting. We found 6 miRNAs with differential abundances (2 upregulated and 4 downregulated miRNAs) between EBV-negative and -positive ABC-DLBCL cells and 12 miRNAs with differential abundances (3 upregulated and 9 downregulated miRNAs) between EBV-negative and -positive GCB-DLBCL cells. Eight and twelve miRNAs were confirmed using RT-qPCR in ABC-DLBCL and GCB-DLBCL cells, respectively. Selected miRNAs were analyzed in additional type I/II versus type III EBV latency DLBCL cell lines. Furthermore, upregulation of miR-221-3p and downregulation of let7c-5p in ABC-DLBCL cells and upregulation of miR-363-3p and downregulation of miR-423-5p in GCB-DLBCL cells were verified using RIP-Northern blotting. Our comprehensive sequence analysis of the DLBCL miRNA profiles identified sets of deregulated miRNAs by Ago2-RIP-seq. Our Ago2-IP-seq miRNA profile could be considered an important data set for the detection of deregulated functionally active miRNAs in DLBCLs and could possibly lead to the identification of miRNAs as biomarkers for the classification of DLBCLs or even as targets for personalized targeted treatment. IMPORTANCE Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive tumor of lymphoid origin which is occasionally Epstein-Barr virus (EBV) positive. MicroRNAs are found in most multicellular organisms and even in viruses such as EBV. They regulate the synthesis of proteins by binding to their cognate mRNA. MicroRNAs are tethered to their target mRNAs by “Argonaute” proteins. Here we compared the overall miRNA content of the Ago2 complex by differential loading to the overall content of miRNAs in two DLBCL cell lines and their EBV-converted counterparts. In all cell lines, the Ago2 load was different from the overall expression of miRNAs. In addition, the loading of the Ago2 complex was changed upon infection with EBV. This indicates that the virus not only changes the overall content of miRNAs but also influences the expression of proteins by affecting the Ago complexes.

Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca2+ influx through Ca2+ release-activated Ca2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions.

Background Protein kinase CK2 is induced early in adipogenesis whereas later on, this kinase seems to be dispensable. Here, we have analysed how CK2 might be involved in early steps of differentiation of 3T3-L1 cells. Methods 3T3-L1 cells were differentiated to adipocytes in the absence or presence of quinalizarin. The expression and localization of important transcription factors was analysed by Western blot and immunofluorescence. DNA binding capacity and transactivation was analysed with pull-down assays and with luciferase reporter experiments, respectively. mRNA was detected with qRT-PCR, miRNAs with Northern hybridization and qRT-PCR. Results We show that clonal expansion was considerably repressed upon inhibition of CK2 with quinalizarin. Moreover, to prevent adipogenesis CK2 inhibition had to take place before day 4 of differentiation. Neither the expression at the protein or at the RNA level nor the subcellular localization of the transcription factors C/EBPβ and C/EBPδ was affected by CK2 inhibition. There was, however, a drastic reduction in the mRNA and protein levels of C/EBPα and PPARγ2. Upon inhibition of CK2, we found a significant up-regulation of the level of the microRNAs miR-27a and miR-27b, which are known to target PPARγ mRNA. Conclusions Time course experiments revealed that CK2 seems to be required at early time points after the induction of differentiation. One important target of CK2 was identified as PPARγ, which is down-regulated after inhibition of CK2. General significance This is the first report about i) cellular targets of CK2 during adipogenesis and ii) a role of CK2 in microRNA regulation.

Small non-coding RNAs play a key role in many physiological and pathological processes. Since 2004, miRNA sequences have been catalogued in miRBase, which is currently in its 21st version. We investigated sequence and structural features of miRNAs annotated in the miRBase and compared them between different versions of this reference database. We have identified that the two most recent releases (v20 and v21) are influenced by next-generation sequencing based miRNA predictions and show significant deviation from miRNAs discovered prior to the high-throughput profiling period. From the analysis of miRBase, we derived a set of key characteristics to predict new miRNAs and applied the implemented algorithm to evaluate novel blood-borne miRNA candidates. We carried out 705 individual whole miRNA sequencings of blood cells and collected a total of 9.7 billion reads. Using miRDeep2 we initially predicted 1452 potentially novel miRNAs. After excluding false positives, 518 candidates remained. These novel candidates were ranked according to their distance to the features in the early miRBase versions allowing for an easier selection of a subset of putative miRNAs for validation. Selected candidates were successfully validated by qRT-PCR and northern blotting. In addition, we implemented a web-server for ranking potential miRNA candidates, which is available at: www.ccb.uni-saarland.de/novomirank.

The aim of this work was to establish the microRNA profile of SNK6 and SNT16, two Epstein-Barr virus (EBV)-infected cell lines derived from nasal NK/T-cell lymphoma (NKTL). The oncogenic EBV is strongly associated with the pathogenesis of nasal and extranodal NK/T-cell lymphoma and expresses 44 mature microRNAs and two noncoding EBV-encoded RNAs (EBERs). miRNAs are 19-25nt noncoding RNAs that affect host and viral gene expression post-transcriptionally. Deregulated miRNA patterns are frequently linked to a variety of human cancers including lymphomas. miRNA profiling of the two NK/T cell lines vs. primary cells revealed 10 and 4 up-regulated and 10 and 12 down-regulated miRNAs in SNK6 and SNT16 cells respectively. The results were validated by qRT-PCR for selected miRNAs. Target gene analyses confirmed cullin 5 (CUL5) and sphingosin-1-phosphate receptor 1 (S1PR1) as targets for the down-regulated hsa-miR-148a and viral ebv-miR-BART16 respectively. As recently demonstrated for the regulation of IL1-alpha by miR-142-3p, coexpression of the EBERs selectively exerted corepression of S1PR1 by BART16 but not of CUL5 by miR-148a, indicating selective corepression by the EBERs.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research.