Chronic Periodontitis Genome-wide Association Study in the Hispanic Community Health Study / Study of Latinos.

Chronic periodontitis (CP) has a genetic component, particularly its severe forms. Evidence from genome-wide association studies (GWASs) has highlighted several potential novel loci. Here, the authors report the first GWAS of CP among a large community-based sample of Hispanics/Latinos. The authors interrogated a quantitative trait of CP (mean interproximal clinical attachment level determined by full-mouth periodontal examinations) among 10,935 adult participants (mean age: 45 y, range: 18 to 76 y) from the Hispanic Community Health Study / Study of Latinos. Genotyping was done with a custom Illumina Omni2.5M array, and imputation to approximately 20 million single-nucleotide polymorphisms was based on the 1000 Genomes Project phase 1 reference panel. Analyses were based on linear mixed models adjusting for sex, age, study design features, ancestry, and kinship and employed a conventional P < 5 × 10-8 statistical significance threshold. The authors identified a genome-wide significant association signal in the 1q42.2 locus ( TSNAX-DISC1 noncoding RNA, lead single-nucleotide polymorphism: rs149133391, minor allele [C] frequency = 0.01, P = 7.9 × 10-9) and 4 more loci with suggestive evidence of association ( P < 5 × 10-6): 1q22 (rs13373934), 5p15.33 (rs186066047), 6p22.3 (rs10456847), and 11p15.1 (rs75715012). We tested these loci for replication in independent samples of European-American ( n = 4,402) and African-American ( n = 908) participants of the Atherosclerosis Risk in Communities study. There was no replication among the European Americans; however, the TSNAX-DISC1 locus replicated in the African-American sample (rs149133391, minor allele frequency = 0.02, P = 9.1 × 10-3), while the 1q22 locus was directionally concordant and nominally significant (rs13373934, P = 4.0 × 10-2). This discovery GWAS of interproximal clinical attachment level-a measure of lifetime periodontal tissue destruction-was conducted in a large, community-based sample of Hispanic/Latinos. It identified a genome-wide significant locus that was independently replicated in an African-American population. Identifying this genetic marker offers direction for interrogation in subsequent genomic and experimental studies of CP.

A date with telomerase: pick you up at S phase.

Using the MS2 system for labeling mRNA, in this issue, Gallardo et al. (2011) find that telomere lengthening depends on a stable accumulation of multiple telomerase complexes in late S phase and that this process is temporally regulated by Rif1/2 proteins.

Nuclear physics: quantitative single-cell approaches to nuclear organization and gene expression.

The internal workings of the nucleus remain a mystery. A list of component parts exists, and in many cases their functional roles are known for events such as transcription, RNA processing, or nuclear export. Some of these components exhibit structural features in the nucleus, regions of concentration or bodies that have given rise to the concept of functional compartmentalization--that there are underlying organizational principles to be described. In contrast, a picture is emerging in which transcription appears to drive the assembly of the functional components required for gene expression, drawing from pools of excess factors. Unifying this seemingly dual nature requires a more rigorous approach, one in which components are tracked in time and space and correlated with onset of specific nuclear functions. In this chapter, we anticipate tools that will address these questions and provide the missing kinetics of nuclear function. These tools are based on analyzing the fluctuations inherent in the weak signals of endogenous nuclear processes and determining values for them. In this way, it will be possible eventually to provide a computational model describing the functional relationships of essential components.

A role for VICKZ proteins in the progression of colorectal carcinomas: regulating lamellipodia formation.

VICKZ proteins are a highly conserved family of RNA binding proteins, implicated in RNA regulatory processes such as intracellular RNA localization, RNA stability, and translational control. During embryogenesis, VICKZ proteins are required for neural crest migration and in adults, the proteins are overexpressed primarily in different cancers. We hypothesized that VICKZ proteins may play a role in cancer cell migration. In patients, VICKZ expression varies with tumour type, with over 60% of colon, lung, and ovarian tumours showing strong expression. In colorectal carcinomas (CRCs), expression is detected at early stages, and the frequency and intensity of staining increase with progression of the disease to lymph node metastases, of which 97% express the protein at high levels. Indeed, in stage II CRC, the level of VICKZ expression in the primary lesion correlates with the degree of lymph node metastasis. In culture, VICKZ proteins rapidly accumulate in processes at the leading edge of PMA-stimulated SW480 CRC cells, where they co-localize with beta-actin mRNA. Two distinct cocktails of shRNAs, each targeting all three VICKZ paralogues, cause a dramatic drop in lamellipodia and ruffle formation in stimulated cells. Thus, VICKZ proteins help to facilitate the dynamic cell surface morphology required for cell motility. We propose that these proteins play an important role in CRC metastasis by shuttling requisite RNAs to the lamellipodia of migrating cells.

Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes.

The coherence of mitochondrial biogenesis relies on spatiotemporally coordinated associations of 800-1000 proteins mostly encoded in the nuclear genome. We report the development of new quantitative analyses to assess the role of local protein translation in the construction of molecular complexes. We used real-time PCR to determine the cellular location of 112 mRNAs involved in seven mitochondrial complexes. Five typical cases were examined by an improved FISH protocol. The proteins produced in the vicinity of mitochondria (MLR proteins) were, almost exclusively, of prokaryotic origin and are key elements of the core construction of the molecular complexes; the accessory proteins were translated on free cytoplasmic polysomes. These two classes of proteins correspond, at least as far as intermembrane space (IMS) proteins are concerned, to two different import pathways. Import of MLR proteins involves both TOM and TIM23 complexes whereas non-MLR proteins only interact with the TOM complex. Site-specific translation loci, both outside and inside mitochondria, may coordinate the construction of molecular complexes composed of both nuclearly and mitochondrially encoded subunits.

RNP localization and transport in yeast.

The localization of mRNAs is used by various types of polarized cells to locally translate specific proteins, which restricts their distribution to a particular sub-region of the cytoplasm. This mechanism of protein sorting is involved in major biological processes such as asymmetric cell division, oogenesis, cellular motility, and synapse formation. With the finding of localized mRNAs in the yeast Saccharomyces cerevisiae, it is now possible to benefit from the powerful yeast laboratory tools to explore the molecular basis of RNA localization. Because mRNA transport and localization in yeast share many features with RNA localization in higher eukaryotes, including the formation of a large ribonucleoprotein (RNP) localization complex, the requirement of a polarized cytoskeleton and molecular motors, and the role of nuclear RNA-binding proteins in cytoplasmic localization, the yeast can be used as a paradigm for unraveling the molecular aspects of this process. This review summarizes the current knowledge on RNP transport and localization in yeast.

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility.

Neurotrophin regulation of actin-dependent changes in growth cone motility may depend on the signaling of beta-actin mRNA transport. Formation of an RNP complex between the beta-actin mRNA zipcode sequence and Zipcode Binding Protein 1 (ZBP1) was required for its localization to growth cones. Antisense oligonucleotides to the zipcode inhibited formation of this RNP complex in vitro and the neurotrophin-induced localization of beta-actin mRNA and ZBP1 granules. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bidirectional movements of granules in neurites that were inhibited by antisense treatment, as visualized by FRAP analysis. NT-3 stimulation of beta-actin protein localization was dependent on the 3'UTR and inhibited by antisense treatment. Growth cones exhibited impaired motility in the presense of antisense. These results suggest a novel mechanism to influence growth cone dynamics involving the regulated transport of mRNA.

A Rho-dependent signaling pathway operating through myosin localizes beta-actin mRNA in fibroblasts.

BACKGROUND: The sorting of mRNA is a determinant of cell asymmetry. The cellular signals that direct specific RNA sequences to a particular cellular compartment are unknown. In fibroblasts, beta-actin mRNA has been shown to be localized toward the leading edge, where it plays a role in cell motility and asymmetry. RESULTS: We demonstrate that a signaling pathway initiated by extracellular receptors acting through Rho GTPase and Rho-kinase regulates this spatial aspect of gene expression in fibroblasts by localizing beta-actin mRNA via actomyosin interactions. Consistent with the role of Rho as an activator of myosin, we found that inhibition of myosin ATPase, myosin light chain kinase (MLCK), and the knockout of myosin II-B in mouse embryonic fibroblasts all inhibited beta-actin mRNA from localizing in response to growth factors. CONCLUSIONS: We therefore conclude that the sorting of beta-actin mRNA in fibroblasts requires a Rho mediated pathway operating through a myosin II-B-dependent step and postulate that polarized actin bundles direct the mRNA to the leading edge of the cell.

The physiological significance of beta -actin mRNA localization in determining cell polarity and directional motility.

beta-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3' untranslated region, the "zip code." Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3' untranslated region leads to delocalization of beta-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after beta-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of beta-actin protein. These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Terminal minihelix, a novel RNA motif that directs polymerase III transcripts to the cell cytoplasm. Terminal minihelix and RNA export.

Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport. We have characterized the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic RNA transcribed by RNA polymerase III. Using a large panel of VA1 mutants in both transfected COS cells and injected Xenopus oocytes, we showed that the terminal stem of VA1 is necessary and sufficient for its export. Surprisingly, we found that the nucleotide sequence within the terminal stem is not important. Rather, the salient features of this motif are its length and its relative position within the RNA. Such stems thus define a novel and degenerate cytoplasmic localization motif that we termed the minihelix. This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway.

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Determination of transgenic loci by expression FISH.

DNA targeting by homologous recombination in mouse embryonic stem (ES) cells has become a widely used method for manipulating the mouse genome and for studying the role of specific genes in mammalian development. For certain studies, it is necessary to target two or more DNA sequences residing on a particular chromosome. In these situations, it would be important to distinguish whether two sequential gene targeting events in the ES cells have occurred in cis or in trans. We report here a new application of fluorescence in situ hybridization to RNA molecules present at sites of transcription that allows the identification of cis and trans gene targeting events in ES cells. The method is based on detection of transcripts from commonly used selectable marker genes inserted during homologous recombination. Transcripts are detected in interphase nuclei, making the preparation of mitotic cells unnecessary and obviating the necessity for the more technically demanding DNA detection of genes. The method is applicable to any chromosomal locus, and compared with other methods (e.g., genetic linkage testing in chimeric mice), it will greatly shorten the time required for distinguishing cis and trans gene targeting events in ES cells. The method also may be useful for detecting changes in ploidy of individual chromosomes and loss of heterozygosity of genes in single cells in culture and also in animals, for example, during processes such as tumorigenesis.

A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles.

We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.

She2p is a novel RNA-binding protein that recruits the Myo4p-She3p complex to ASH1 mRNA.

In Saccharomyces cerevisiae, Ash1p is a specific repressor of transcription that localizes exclusively to daughter cell nuclei through the asymmetric localization of ASH1 mRNA. This localization requires four cis-acting localization elements located in the ASH1 mRNA, five trans-acting factors, one of which is a myosin, and the actin cytoskeleton. The RNA-binding proteins that interact with these cis-elements remained to be identified. Starting with the 3' most localization element of ASH1 mRNA in the three-hybrid assay, element E3, we isolated a clone corresponding to the C-terminus of She3p. We also found that She3p and She2p interact, and this interaction is essential for the binding of She3p with element E3 in vivo. Moreover, She2p was observed to bind the E3 RNA directly in vitro and each of the ASH1 cis-acting localization elements requires She2p for their localization function. By tethering a She3p-MS2 fusion protein to a reporter RNA containing MS2 binding sites, we observed that She2p is dispensable for She3p-MS2-dependent RNA localization.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing.

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

Ultrastructural distribution of poly (A)+ RNA during Trypanosoma cruzi-cardiomyocyte interaction in vitro: a quantitative analysis of the total mRNA content by in situ hybridization.

Ultrastructural in situ hybridization was used to visualize the spatial distribution of poly (A)+ RNA and quantitate its relative amount within different cellular compartments of cardiomyocytes after T. cruzi infection. The amount of poly (A)+ RNA remained about the same up to 24 h post-infection. In contrast, its content was reduced 65% after 72 h of interaction, showing a marked decrease in the cell cytoplasm. This decline in poly (A)+ RNA level in host cell cytoplasm was concomitant with intracellular proliferation of T. cruzi amastigotes. Thus, T. cruzi may affect host cell cytoplasmic mRNA stability, associated with the parasite's intracellular multiplication.

Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells.

We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.