Friedrich A. Grässer

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) induces the expression of the cellular microRNA miR-146a.

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g. the “vault” RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is up-regulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NF-κB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NF-κB-response elements. Conversely, a LMP1-mutant deficient in NF-κB-activation failed to activate the promoter. The “CAO”-LMP1 variant which has an increased potential to induce NF-κB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt’s lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt’s lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells up-regulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells.

Non-protein-coding RNAs (ncRNAs) fulfill a wide range of cellular functions from protein synthesis to regulation of gene expression. Identification of novel regulatory ncRNAs by experimental approaches commonly includes the generation of specialized cDNA libraries encoding small ncRNA species. However, such identification is severely hampered by the presence of constitutively expressed and highly abundant ‘house-keeping’ ncRNAs, such as ribosomal RNAs, small nuclear RNAs or transfer RNAs. We have developed a novel experimental strategy, designated as subtractive hybridization of ncRNA transcripts (SHORT) to specifically select and amplify novel regulatory ncRNAs, which are only expressed at certain stages or under specific growth conditions of cells. The method is based on the selective subtractive hybridization technique, formerly applied to the detection of differentially expressed mRNAs. As a model system, we applied SHORT to Epstein–Barr virus (EBV) infected human B cells. Thereby, we identified 21 novel as well as previously reported ncRNA species to be up-regulated during virus infection. Our method will serve as a powerful tool to identify novel functional ncRNAs acting as genetic switches in the regulation of fundamental cellular processes such as development, tissue differentiation or disease.

Downregulation of Sec23A protein by miRNA-375 in prostate carcinoma

Prostate carcinoma (CaP) is a leading cause of cancer-related death in men. We have previously determined the microRNA (miRNA) profile of primary CaP in comparison with nontumor prostate tissue. miRNAs are small, noncoding RNAs that inhibit protein synthesis on a posttranscriptional level by binding to the 3′-untranslated region (3′-UTR) of their target genes. In primary CaP tissue, we have previously found by miRNA sequencing that miR-375 and miR-200c were upregulated 9.1- and 4.5-fold, respectively. A computational analysis predicted the 3′-UTR of the SEC23A gene as a potential target for both miR-375 and miR-200c. Here, we show that the 3′-UTR of SEC23A mRNA is indeed a target for miR-375 and miR-200c and that both miRNAs downregulate Sec23A protein expression when ectopically expressed in human 293T cells. In primary samples of CaP, we found a direct correlation between reduction of SEC23A mRNA and overexpression of miR-375 but not of miR-200c. The reduced levels of Sec23A protein were inversely correlated to the increased amount of miR-375 in the LNCaP and DU145 CaP cell lines when compared with normal prostate fibroblasts. In primary CaP, we also detected decreased amounts of Sec23A protein when compared with corresponding normal prostate tissue. Ectopically overexpressed Sec23A in LNCaP and DU145 CaP cells significantly reduced the growth properties, indicating that Sec23A might play a role in the induction or growth of prostate carcinoma. Sec23A overexpression reduced cell growth but did not induce apoptosis, whereas inhibition of Sec23A stimulated cell proliferation. Mol Cancer Res; 9(6); 791–800. ©2011 AACR.

Epstein-Barr Virus-Induced Expression of a Novel Human Vault RNA

Non-protein-coding RNAs (ncRNAs) have recently emerged on the scene of genomic research as prominent players in the regulation of gene expression. Many functionally characterized ncRNAs have been shown to be differentially expressed in various organisms during specific environmental or developmental conditions, thus establishing regulatory networks crucial for shaping cellular life. Here, we show that the expression of vault RNAs (vtRNAs) is specifically up-regulated in human lymphocytes upon infection by gamma-herpesviruses, such as the Epstein-Barr virus and Kaposis sarcoma virus. vtRNAs are ncRNAs that are integral to the vault complex, a gigantic (13 MDa) hollow ribonucleoprotein particle with a thus far elusive biological role. Stimulation of vtRNA expression by the Epstein-Barr virus was evident for all three canonical vtRNAs (hvg1-hvg3) and also for a novel ncRNA candidate, initially termed CBL-3. This ncRNA shares clear primary- and secondary-structure similarities with the three known vtRNAs. Importantly, CBL-3 co-sediments with intact vault particles in density gradients of various human cell lines, thus strongly indicating this ncRNA as a novel, fourth vault-complex-associated RNA.

The NP9 protein encoded by the human endogenous retrovirus HERV-K(HML-2) negatively regulates gene activation of the Epstein-Barr virus nuclear antigen 2 (EBNA2).

Epstein‐Barr virus (EBV) is a human tumour virus that efficiently growth‐transforms primary human B‐lymphocytes in vitro. The viral nuclear antigen 2 (EBNA2) is essential for immortalisation of B‐cells and stimulates viral and cellular gene expression through interaction with DNA‐bound transcription factors. Like its cellular homologue Notch, it associates with the DNA‐bound repressor RBPJκ (CSL/CBF1) thereby converting RBPJκ into the active state. For instance, both EBNA2 and Notch activate the cellular HES1 promoter. In EBV‐transformed lymphocytes, the RNA of the NP9 protein encoded by human endogenous retrovirus HERV‐K(HML‐2) Type 1 is strongly up‐regulated. The NP9 protein is detectable both in EBV‐positive Raji cells, a Burkitts lymphoma cell line, and in IB4, an EBV‐transformed human lymphoblastoid cell line. NP9 binds to LNX that forms a complex with the Notch regulator Numb. Therefore, the function of NP9 vis‐à‐vis Notch and EBNA2 was analysed. Here, we show that NP9 binds to EBNA2 and negatively affects the EBNA2‐mediated activation of the viral C‐ and LMP2A promoters. In contrast, NP9 did neither interfere in the activation of the HES1 promoter by Notch nor the induction of the viral LMP1 promoter by EBNA2. In an electrophoretic mobility shift analysis, NP9 reduced the binding of EBNA2 to DNA‐bound RBPJκ by about 50%. The down‐regulation of EBNA2‐activity by NP9 might represent a cellular defence mechanism against viral infection or could, alternatively, represent an adaptation of the virus to prevent excessive viral protein production that might otherwise be harmful for the infected cell.

A new strategy for the development of monoclonal antibodies for the determination of human procalcitonin in serum samples

Procalcitonin (PCT)—a diagnostic serum parameter for bacterial infection and sepsis—is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC50) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7xa0μgxa0L−1 (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R2: assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.

The LARK/RBM4a protein is highly expressed in cerebellum as compared to cerebrum

The RNA binding motif protein 4 genes RBM4a and RBM4b are located on human chromosome 11q13.2 and encode highly similar proteins of 363 and 359 amino acids, respectively. They contain two RNA recognition motifs (RRMs) and a retroviral-type Zn-finger. RBM4a binds RNA, is involved in alternative splicing and is also a part of the microRNA-processing RISC complex. In particular, RBM4a is involved in exon 10 inclusion of the tau protein. The function of RBM4b is unknown. With new monoclonal antibodies we show that RBM4a is detectable in virtually all tissues and cell lines tested while RBM4b was only found in kidney and liver. Both RBM4a and RBM4b are nuclear phosphoproteins with half-lives of 2.5h and 4.5h, respectively. To our knowledge, this is the first description of RBM4b protein in human tissue. In human brain, expression of RBM4a was strongly up-regulated in cerebellum as compared to forebrain.

Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2.

Humans and primates are long-lived animals with long reproductive phases. One factor that appears to contribute to longevity and fertility in humans, as well as to cancer-free survival, is the transcription factor and tumor suppressor p53, controlled by its main negative regulator MDM2. However, p53 and MDM2 homologs are found throughout the metazoan kingdom from Trichoplacidae to Hominidae. Therefore the question arises, if p53/MDM2 contributes to the shaping of primate features, then through which mechanisms. Previous findings have indicated that the appearances of novel p53-regulated genes and wild-type p53 variants during primate evolution are important in this context. Here, we report on another mechanism of potential relevance. Human endogenous retrovirus K subgroup HML-2 (HERV-K(HML-2)) type 1 proviral sequences were formed in the genomes of the predecessors of contemporary Hominoidea and can be identified in the genomes of Nomascus leucogenys (gibbon) up to Homo sapiens. We previously reported on an alternative splicing event in HERV-K(HML-2) type 1 proviruses that can give rise to nuclear protein of 9 kDa (Np9). We document here the evolution of Np9-coding capacity in human, chimpanzee and gorilla, and show that the C-terminal half of Np9 binds directly to MDM2, through a domain of MDM2 that is known to be contacted by various cellular proteins in response to stress. Np9 can inhibit the MDM2 ubiquitin ligase activity toward p53 in the cell nucleus, and can support the transactivation of genes by p53. Our findings point to the possibility that endogenous retrovirus protein Np9 contributes to the regulation of the p53-MDM2 pathway specifically in humans, chimpanzees and gorillas.

Abstract 4036: The microRNA profile of prostate carcinoma obtained by deep sequencing reveals potential miRNA targets such as myosin VI

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify and characterize the underlying molecular mechanisms, we performed microRNA (miRNA) profiling of primary prostate cancers and non-cancer prostate tissue. Furthermore, using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs. Material and Methods: We performed comparative miRNA expression profiling in ten prostate cancer specimens and ten non-cancer prostate tissue samples using deep sequencing of cDNA libraries. The deregulation of selected miRNAs was validated in established prostate cancer cell lines by Northern blotting as well as in 26 paired samples of prostate cancer tissue and adjacent normal tissue by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulative capabilities of selected miRNAs on potential target genes were analyzed in vitro using reporter gene constructs and transfection of miRNA expression vectors Results: Using deep sequencing we found that 16 miRNAs were up-regulated more than 1.5-fold and 17 miRNAs were down regulated more than 1.5-fold. The most pronounced deregulation was noted for miRNAs miR-375 and miR-200c which showed an up-regulation of more than 4.5-fold and miR-143 and miR-145 which were down regulated 4-fold each. The differential expression of these miRNAs could be confirmed by Northern blot analysis of established prostate cancer cell lines. By analyzing 26 primary prostate cancer samples by qRT-PCR we could detect a significant deregulation of the miRNAs miR-375, miR-143 and miR-145 (p Conclusion: Our findings indicate that specific miRNAs are reproducibly found to be deregulated in different cohorts of prostate cancer specimens using independent experimental methods. This is the first report to show that miRNAs miR-143 and miR-145 are capable of negatively regulating the protein expression of Myosin VI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4036.