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79 Publications

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    Singer Lab
    06/01/14 | Heterogeneity in periodontitis prevalence in the Hispanic Community Health Study/Study of Latinos.
    Sanders AE, Campbell SM, Mauriello SM, Beck JD, Jimenez MC, Kaste LM, Singer RH, Beaver SM, Finlayson TL, Badner VM
    Annals of Epidemiology. 2014 Jun;24(6):455-62. doi: 10.1016/j.annepidem.2014.02.018

    PURPOSE: The aim of the study was to examine acculturation and established risk factors in explaining variation in periodontitis prevalence among Hispanic/Latino subgroups.

    METHODS: Participants were 12,730 dentate adults aged 18-74 years recruited into the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) from four U.S. field centers between 2008 and 2011. A standardized periodontal assessment measured probing pocket depth and gingival recession at six sites per tooth for up to 28 teeth. Periodontitis was defined according to the Centers for Disease Control and Prevention and American Academy of Periodontology case classifications developed for population surveillance. Covariates included acculturation indicators and established periodontitis risk factors. Survey estimation procedures took account of the complex sampling design. Adjusted multivariate binomial regression estimated prevalence ratios and 95% confidence limits (CLs).

    RESULTS: Unadjusted prevalence of moderate and severe periodontitis was 38.5% and ranged from 24.7% among Dominicans to 52.1% among Cubans. Adjusted prevalence ratios for subgroups relative to Dominicans were as follows: (1) 1.34 (95% CL, 1.13-1.58) among South Americans; (2) 1.37 (95% CL, 1.17-1.61) among Puerto Ricans; (3) 1.43 (95% CL, 1.25-1.64) among Mexicans; (4) 1.53 (95% CL, 1.32-1.76) among Cubans; and (5) 1.55 (95% CL, 1.35-1.78) among Central Americans.

    CONCLUSIONS: Heterogeneity in prevalence of moderate/severe periodontitis among Hispanic/Latino subpopulations was not explained by acculturation or periodontitis risk factors.

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    Singer Lab
    05/19/14 | Gene regulation: the HSP70 gene jumps when shocked.
    Vera M, Singer RH
    Current Biology. 2014 May 19;24(10):R396-8. doi: 10.1016/j.cub.2014.03.070

    Limited chromosome mobility has been observed in mammalian interphase nuclei. Live imaging shows unidirectional and actin-dependent movement of HSP70 loci towards speckles upon heat shock, resulting in enhanced transcription. This adds further impetus to understanding compartmentalization of function in the nucleus.

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    Singer Lab
    04/20/14 | Efficient Bayesian-based multiview deconvolution.
    Preibisch S, Amat F, Stamataki E, Sarov M, Singer RH, Myers E, Tomancak P
    Nature Methods. 2014 Apr 20;11:645-8. doi: 10.1038/nmeth.2929

    Light-sheet fluorescence microscopy is able to image large specimens with high resolution by capturing the samples from multiple angles. Multiview deconvolution can substantially improve the resolution and contrast of the images, but its application has been limited owing to the large size of the data sets. Here we present a Bayesian-based derivation of multiview deconvolution that drastically improves the convergence time, and we provide a fast implementation using graphics hardware.

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    Singer Lab
    03/01/14 | Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA-binding protein IMP1 facilitates its trafficking into motor neuron axons.
    Fallini C, Rouanet JP, Donlin-Asp PG, Guo P, Zhang H, Singer RH, Rossoll W, Bassell GJ
    Developmental Neurobiology. 2014 Mar;74(3):319-32. doi: 10.1002/dneu.22111

    Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the β-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the β-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1.

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    Singer Lab
    01/24/14 | Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability.
    Buxbaum AR, Wu B, Singer RH
    Science. 2014 Jan 24;343(6169):419-22. doi: 10.1126/science.1242939

    The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Local actin remodeling underlies some forms of plasticity and may be facilitated by local β-actin synthesis, but dynamic information is lacking. In this work, we use single-molecule in situ hybridization to demonstrate that dendritic β-actin messenger RNA (mRNA) and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. Ribosomes and single β-actin mRNA motility increase after stimulation, indicative of release from complexes. Hence, the single-molecule assays we developed allow for the quantification of activity-induced unmasking and availability for active translation. Further, our work demonstrates that β-actin mRNA and ribosomes are in a masked state that is alleviated by stimulation.

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    01/24/14 | Visualization of dynamics of single endogenous mRNA labeled in live mouse.
    Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH
    Science. 2014 Jan 24;343(6169):422-4. doi: 10.1126/science.1239200

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

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    Singer Lab
    01/09/14 | Background free imaging of single mRNAs in live cells using split fluorescent proteins.
    Wu B, Chen J, Singer RH
    Scientific Reports. 2014 Jan 9;4:3615. doi: 10.1038/srep03615

    We describe a technique for imaging single mRNAs in living cells based on fluorescent protein (FP) complementation. We employ the high affinity interaction between the bacterial phage MS2/PP7 coat proteins and their respective RNA binding motifs as an RNA scaffold to bring two halves of a split-FP together to image single reporter mRNAs without background fluorescence.

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    Singer Lab
    09/24/13 | Direct observation of frequency modulated transcription in single cells using light activation.
    Larson DR, Fritzsch C, Sun L, Meng X, Lawrence DS, Singer RH
    eLife. 2013 Sep 24;2:e00750. doi: 10.7554/eLife.00750

    Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI:http://dx.doi.org/10.7554/eLife.00750.001.

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    Singer Lab
    08/01/13 | Eukaryotic transcriptional dynamics: from single molecules to cell populations.
    Coulon A, Chow CC, Singer RH, Larson DR
    Nature Reviews Genetics. 2013 Aug;14(8):572-84. doi: 10.1038/nrg3484

    Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time- and energy-dependence at the molecular level.

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    Singer Lab
    07/12/13 | mRNA on the move: the road to its biological destiny.
    Eliscovich C, Buxbaum AR, Katz ZB, Singer RH
    The Journal of Biological Chemistry. 2013 Jul 12;288(28):20361-8. doi: 10.1074/jbc.R113.452094

    Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.

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