Transcription of Muscle Actin Genes by a Nuclear Form of Mitochondrial RNA Polymerase

Actins are the major constituent of the cytoskeleton. In this report we present several lines of evidence that muscle actin genes are transcribed by nuclear isoform of mitochondrial RNA polymerase (spRNAP-IV) whereas the non-muscle actin genes are transcribed by the conventional RNA polymerase II (PolII). We show that mRNA level of muscle actin genes are resistant to PolII inhibitors α-amanitin and triptolide as well as insensitive to knockdown of PolII but not to knockdown of spRNAP-IV, in contrast to non-muscle actin genes in several cell lines. Similar results are obtained from nuclear run-on experiments. Reporter assay using muscle actin or PolII gene promoters also demonstrate the differential sensitivity to PolII inhibitors. Finally, chromatin-immunoprecipitation experiment was used to demonstrate that spRNAP-IV is associated with promoter of muscle actin genes but not with that of non-muscle gene and knockdown of spRNAP-IV depleted this polymerase from muscle actin genes. In summary, these experiments indicate that the two types of actin genes are transcribed by different transcription machinery. We also found that POLRMT gene is transcribed by spRNAP-IV, and actin genes are sensitive to oligomycin, suggesting a transcription coupling between mitochondria and nucleus

A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-κB

The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors

B3GALNT2 mutations associated with non-syndromic autosomal recessive intellectual disability reveal a lack of genotype-phenotype associations in the muscular dystrophy-dystroglycanopathies.

BACKGROUND: The phenotypic severity of congenital muscular dystrophy-dystroglycanopathy (MDDG) syndromes associated with aberrant glycosylation of α-dystroglycan ranges from the severe Walker-Warburg syndrome or muscle-eye-brain disease to mild, late-onset, isolated limb-girdle muscular dystrophy without neural involvement. However, muscular dystrophy is invariably found across the spectrum of MDDG patients. METHODS: Using linkage mapping and whole-exome sequencing in two families with an unexplained neurodevelopmental disorder, we have identified homozygous and compound heterozygous mutations in B3GALNT2. RESULTS: The first family comprises two brothers of Dutch non-consanguineous parents presenting with mild ID and behavioral problems. Immunohistochemical analysis of muscle biopsy revealed no significant aberrations, in line with the absence of a muscular phenotype in the affected siblings. The second family includes five affected individuals from an Iranian consanguineous kindred with mild-to-moderate intellectual disability (ID) and epilepsy without any notable neuroimaging, muscle, or eye abnormalities. Complementation assays of the compound heterozygous mutations identified in the two brothers had a comparable effect on the O-glycosylation of α-dystroglycan as previously reported mutations that are associated with severe muscular phenotypes. CONCLUSIONS: In conclusion, we show that mutations in B3GALNT2 can give rise to a novel MDDG syndrome presentation, characterized by ID associated variably with seizure, but without any apparent muscular involvement. Importantly, B3GALNT2 activity does not fully correlate with the severity of the phenotype as assessed by the complementation assay

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity

A dual function TAR Decoy serves as an anti-HIV siRNA delivery vehicle

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed

A Novel RNAi Lethality Rescue Screen to Identify Regulators of Adipogenesis

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an atractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting

Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

Single-stranded (ss) circular oligodeoxynucleotides were previously found to undergo rolling circle transcription (RCT) by phage and bacterial RNA polymerases (RNAPs) into tandemly repetitive RNA multimers. Here, we redesign them to encode minimal primary miRNA mimics, with the long term aim of intracellular transcription followed by RNA processing and maturation via endogenous pathways. We describe an improved method for circularizing ss synthetic DNA for RCT by using a recently described thermostable RNA ligase, which does not require a splint oligonucleotide to juxtapose the ligating ends. In vitro transcription of four templates demonstrates that the secondary structure inherent in miRNA-encoding vectors does not impair their RCT by RNAPs previously shown to carry out RCT. A typical primary-miRNA rolling circle transcript was accurately processed by a human Drosha immunoprecipitate, indicating that if human RNAPs prove to be capable of RCT, the resulting transcripts should enter the endogenous miRNA processing pathway in human cells. Circular oligonucleotides are therefore candidate vectors for small RNA delivery in human cells, which express RNAPs related to those tested here

Expression of Multiple Artificial MicroRNAs from a Chicken miRNA126-Based Lentiviral Vector

Background: The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector. Methods/Principal Findings: We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector. Conclusions/Significance: This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.National Institutes of Health (U.S.) (Grant R01AI056267)Cobb-Vantress, inc

Functional Characterization of Human Cancer-Derived TRKB Mutations

Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKBT695I and TRKBD751N). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKBL138F). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKBP507L) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKBT695I and TRKBD751N, displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKBL138F and TRKBP507L) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations