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43 Publications

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    Singer Lab
    01/01/12 | Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ZBP1.
    Gu W, Katz Z, Wu B, Park HY, Li D, Lin S, Wells AL, Singer RH
    The Journal of Cell Science. 2012 Jan 1;125(Pt 1):81-91. doi: 10.1242/jcs.086132

    Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, β-actin, α-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.

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    Singer Lab
    04/15/15 | Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.
    Wu B, Miskolci V, Sato H, Tutucci E, Kenworthy CA, Donnelly SK, Yoon YJ, Cox D, Singer RH, Hodgson L
    Genes & Development. 2015 Apr 15;29(8):876-86. doi: 10.1101/gad.259358.115

    Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.

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    Singer Lab
    04/01/15 | Dynamic visualization of transcription and RNA subcellular localization in zebrafish.
    Campbell PD, Chao JA, Singer RH, Marlow FL
    Development. 2015 Apr 1;142(7):1368-74. doi: 10.1242/dev.118968

    Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.

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    Singer Lab
    04/01/15 | Reminiscences on my life with RNA: a self-indulgent perspective.
    Singer RH
    RNA. 2015 Apr;21(4):508-9. doi: 10.1261/rna.050922.115
    Singer Lab
    03/01/15 | Specific interaction of KIF11 with ZBP1 regulates the transport of β-actin mRNA and cell motility.
    Song T, Zheng Y, Wang Y, Katz Z, Liu X, Chen S, Singer RH, Gu W
    Journal of Cell Science. 2015 Mar 1;128(5):1001-10. doi: 10.1242/jcs.161679

    ZBP1-modulated localization of β-actin mRNA enables a cell to establish polarity and structural asymmetry. Although the mechanism of β-actin mRNA localization has been well established, the underlying mechanism of how a specific molecular motor contributes to the transport of the ZBP1 (also known as IGF2BP1) complex in non-neuronal cells remains elusive. In this study, we report the isolation and identification of KIF11, a microtubule motor, which physically interacts with ZBP1 and is a component of β-actin messenger ribonucleoprotein particles (mRNPs). We show that KIF11 colocalizes with the β-actin mRNA, and the ability of KIF11 to transport β-actin mRNA is dependent on ZBP1. We characterize the corresponding regions of ZBP1 and KIF11 that mediate the interaction of the two proteins in vitro and in vivo. Disruption of the in vivo interaction of KIF11 with ZBP1 delocalizes β-actin mRNA and affects cell migration. Our study reveals a molecular mechanism by which a particular microtubule motor mediates the transport of an mRNP through direct interaction with an mRNA-binding protein.

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    Singer Lab
    02/01/15 | In the right place at the right time: visualizing and understanding mRNA localization.
    Buxbaum AR, Haimovich G, Singer RH
    Nature Reviews Molecular Cell Biology. 2015 Feb;16(2):95-109. doi: 10.1038/nrm3918

    The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.

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    Singer Lab
    09/16/14 | The translation elongation factor eEF1A1 couples transcription to translation during heat shock response.
    Vera M, Pani B, Griffiths LA, Muchardt C, Abbott CM, Singer RH, Nudler E
    eLife. 2014 Sep 16;3:e03164. doi: 10.7554/eLife.03164

    Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3'UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.

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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
    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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