Heterologous expression from the human D-Loop in organello

We report the expression of a linear reporter construct in isolated human mitochondria. The reporter construct contained the entire human D-Loop with adjacent tRNA (MTT) genes (mt.15956-647), the human ND1 gene with an in frame GFP gene and adjacent endogenous MTT genes and heterologous rat MTT genes. Natural competence of isolated human mitochondria of HepG2 cells was used to import reporter constructs. The import efficiency of various fluorescently labelled PCR-generated import substrates in the range of 250bp up to 3.5kb was assessed by quantitative PCR and evaluated by confocal microscopy. Heterologous expression of the imported construct was confirmed at RNA level by a circular RNA (cRNA)-RT-PCR assay for the expression of tRNAs and by in organello [α-(32)P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNA(Leu(UUR)) (rMT-TL1) was confirmed by co-/post-transcriptional trinucleotide (CCA) addition. Interestingly, the rat-specific MT-TL1 was correctly processed in isolated human mitochondria at the 3' end, but showed an aberrant 5' end processing. Correct 3' end processing of the heterologous expressed mitochondrial rat tRNA(Ser2) (MT-TS2) was detected. These findings demonstrate the feasibility of genetic manipulation of human mitochondria, providing a tool for characterisation of cis-acting elements of the human mitochondrial genome and for the study of human mitochondrial tRNA processing in organello.

Postnatal Notch1 activation induces T‑cell malignancy in conditional and inducible mouse models

The Notch1 signaling pathway is essential for hematopoietic development. However, the effects of postnatal activation of Notch1 signaling on hematopoietic system is not yet fully understood. We previously generated ZEG‑IC‑Notch1 transgenic mice that have a floxed β‑geo/stop signal between a CMV promoter and intracellular domain of Notch1 (IC‑Notch1). Constitutively active IC‑Notch1 is silent until the introduction of Cre recombinase. In this study, endothelial/hematopoietic specific expression of IC‑Notch1 in double transgenic ZEG‑IC‑Notch1/Tie2‑Cre embryos induced embryonic lethality at E9.5 with defects in vascular system but not in hematopoietic system. Inducible IC‑Notch1 expression in adult mice was achieved by using tetracycline regulated Cre system. The ZEG‑IC‑Notch1/Tie2‑tTA/tet‑O‑Cre triple transgenic mice survived embryonic development when maintained on tetracycline. Post‑natal withdrawal of tetracycline induced expression of IC‑Notch1 transgene in hematopoietic cells of adult mice. The triple transgenic mice displayed extensive T‑cell infiltration in multiple organs and T‑cell malignancy of lymph nodes. In addition, the protein levels of p53 and alternative reading frame (ARF) were decreased in lymphoma‑like neoplasms from the triple transgenic mice while their mRNA expression remained unchanged, suggesting that IC‑Notch1 might repress ARF‑p53 pathway by a post‑transcriptional mechanism. This study demonstrated that activation of constitutive Notch1 signaling after embryonic development alters adult hematopoiesis and induces T‑cell malignancy.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down-regulated in primary AML patient samples and healthy CD34(+) progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.

Ribosome-associated ncRNAs (rancRNAs) An emerging class of translation regulators

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. Most of the recently discovered regulatory ncRNAs acting on translation target the mRNA rather than the ribosome (e.g.: miRNAs, siRNAs, antisense RNAs). To address the question, whether ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes. Deep-sequencing analyses revealed thousands of putative rancRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and fine-tune the rate of protein biosynthesis (3,4). Many of the investigated rancRNAs appear to be processing products of larger functional RNAs, such as tRNAs (2,3), mRNAs (3), or snoRNAs (2). Post-transcriptional cleavage of RNA to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data disclose the ribosome as target for small regulatory RNAs. rancRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Ongoing work in our lab revealed first insight into rancRNA processing and mechanism of this emerging class of translation regulators.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [1].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-protein-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

The low abundance of U7 snRNA is partly determined by its Sm binding site.

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4- to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Sm-mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

H19 lncRNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis

Endometriosis affects approximately 15% of reproductive aged women and is associated with chronic pelvic pain and infertility. However, the molecular mechanisms by which endometriosis impacts fertility are poorly understood. The developmentally regulated, imprinted H19 long noncoding RNA (lncRNA) functions to reduce the bioavailability of microRNA let-7 by acting as a molecular sponge. Here we report that H19 expression is significantly decreased in the eutopic endometrium of women with endometriosis as compared to normal controls. We show that decreased H19 increases let-7 activity, which in turn inhibits Igf1r expression at the post-transcriptional level, thereby contributing to reduced proliferation of endometrial stromal cells. We propose that perturbation of this newly identified H19/Let-7/IGF1R regulatory pathway may contribute to impaired endometrial preparation and receptivity for pregnancy in women with endometriosis. Our finding represents the first example of a lncRNA-based mechanism in endometriosis and its associated infertility, thus holding potential in the development of novel therapeutics for women with endometriosis and infertility.

Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID)

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Analysis of Lim1 LIM domains in mouse development

Transcriptional regulation is fundamental for the precise development of all organisms. Through tight regulation, necessary genes are activated at proper spatial and temporal patterns, while unnecessary genes are repressed. A large family of regulator proteins that have been demonstrated to be involved in various developmental processes by activation and repression of target genes is the homeodomain family of proteins. To date, the function of many of these homeoproteins has been elucidated in diverse species. However, the molecular mechanism underlying the function of these proteins has not been fully understood. In this study, the molecular mechanism of the function of a LIM-homeoprotein, Lim1, was examined. In addition to the homeodomain, Lim1 contains two LIM domains that are highly conserved among species. This high conservation along with data from in vitro studies on Xenopus Lim1 suggests that the LIM domains might be important for the function of Lim1 as a transcriptional regulator. Here, the functional importance of the LIM domains of Lim1 was determined by using a novel gene-targeting strategy in mouse embryonic stem (ES) cells. A cre-loxP system was used in conjunction with the unique genomic organization of Lim1 to obtain four types of mutant ES cell lines that would allow for the in vivo analysis of the function of both the LIM domains of Lim1 together and also singularly. These four mutant Lim1 alleles either contained base-pair changes at the LIM encoding exons that alters zinc-binding amino acids of the LIM domains or contained only exogenous loxP sequences in the first intron of Lim1, which serves as the control allele. These mutations in the LIM domains would presumably abolish the zinc-finger tertiary structure of the domain and thus render the domain non-functional. Mice carrying mutations at both the LIM domains of Lim1, L1L2, die around E10 without anterior head structures anterior to rhombomere 3, identical in phenotype to the Lim1 null mutants in spite of the presence of mutant Lim1 RNA. This result demonstrates that the integrity of both the LIM domains are essential for the function of Lim1. This is further supported by the phenotype of mice carrying mutation at only the second LIM domain of Lim1, L2. The L2 mice although still carrying one intact Lim1 LIM domain, also die in utero. The L2 mice die at varying times, from around E8 to E10 with anterior defects in addition to other axial defects which have yet to be fully characterized. The results of this study so far demonstrates that the integrity of both LIM domains are required for the function of Lim1. ^

Changes in miRNA Expression in a Model of Microcephaly

miRNAs function to regulate gene expression through post-transcriptional mechanisms to potentially regulate multiple aspects of physiology and development. Whole transcriptome analysis has been conducted on the citron kinase mutant rat, a mutant that shows decreases in brain growth and development. The resulting differences in RNA between mutant and wild-type controls can be used to identify genetic pathways that may be regulated differentially in normal compared to abnormal neurogenesis. The goal of this thesis was to verify, with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), changes in miRNA expression in Cit-k mutants and wild types. In addition to confirming miRNA expression changes, bio-informatics software TargetScan 5.1 was used to identify potential mRNA targets of the differentially expressed miRNAs. The miRNAs that were confirmed to change include: rno-miR-466c, mmu-miR-493, mmu-miR-297a, hsa-miR-765, and hsa-miR-1270. The TargetScan analysis revealed 347 potential targets which have known roles in development. A subset of these potential targets include genes involved in the Wnt signaling pathway which is known to be an important regulator of stem cell development.

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Requirement of GCN5 histone acetyltransferase in mouse neural tube closure and skeletal patterning

Histone acetylation plays an essential role in many DNA-related processes such as transcriptional regulation via modulation of chromatin structure. Many histone acetytransferases have been discovered and studied in the past few years, but the roles of different histone acetyltransferases (HAT) during mammalian development are not well defined at present. Gcn5 histone acetyltransferase is highly expressed until E16.5 during development. Previous studies in our lab using a constitutive null allele demonstrated that Gcn5 knock out mice are embryonic lethal, precluding the study of Gcn5 functions at later developmental stages. The creation of a conditional Gcn5 null allele, Gcn5flox allele, bypasses the early lethality. Mice homozygous for this allele are viable and appear healthy. In contrast, mice homozygous for a Gcn5 Δex3-18 allele created by Cre-loxP mediated deletion display a phenotype identical to our original Gcn5 null mice. Strikingly, a Gcn5flox(neo) allele, which contain a neomycin cassette in the second intron of Gcn5 is only partially functional and gives rise to a hypomorphic phenotype. Initiation of cranial neural tube closure at forebrain/midbrain boundary fails, resulting in an exencephaly in some Gcn5flox(neo)/flox(neo) embryos. These defects were found at an even greater penetrance in Gcn5flox(neo)/Δ embryos and become completely penetrant in the 129Sv genetic background, suggesting that Gcn5 controls mouse neural tube closure in a dose dependent manner. Furthermore, both Gcn5flox(neo)/flox(neo) and Gcn5 flox(neo)/Δ embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by decreased expression levels and a shift in anterior expression boundary of Hoxc8 and Hoxc9. This study provides the first evidence that Gcn5 regulates Hox gene expression and is required for normal axial skeletal patterning in mice. ^