Visualization of newly synthesized neuronal RNA in vitro and in vivo using click-chemistry

Akbalık, Güney; Langebeck-Jensen, Kasper; Tushev, Georgi; Sambandan, Sivakumar; Rinne, Jennifer; Epstein, Irina; Cajigas, Iván J.; Vlatkovic, Irena; Schuman, Erin M.

doi:10.1080/15476286.2016.1251541

Cited by 39 publications

(54 citation statements)

References 43 publications

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“…Neuropil Localized Transcripts Are Long Lived Differences in mRNA half-life might also play an important role in both the localization and the function of neuropil mRNAs. To determine the half-lives of neuronal transcript 3 0 UTR isoforms, we blocked transcription (e.g., Akbalik et al, 2017) and then collected cultured hippocampal neuron samples at successive time points (0 hr, 2 hr, 4 hr, 9 hr, and 16 hr) ( Figure 7A). We performed 3 0 end sequencing on these samples and, after normalization (see STAR Methods and Figures S7A and S7B), fit an exponential decay function to the time course data of each 3 0 UTR isoform measured.…”

Section: Properties and Elements Within Neuronal 3 0 Utrsmentioning

confidence: 99%

Alternative 3′ UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

Tushev

Glock

Heumüller

et al. 2018

Neuron

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Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain.

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Section: Properties and Elements Within Neuronal 3 0 Utrsmentioning

confidence: 99%

Alternative 3′ UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

Tushev

Glock

Heumüller

et al. 2018

Neuron

Self Cite

325

336

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“…Nascent guanylin mRNA was labeled with ethynyl-uridine and biotinazide click-chemistry, followed by affinity purification and quantification by qRT-PCR analysis (Figure 5a). 45,46 In HT29 human colorectal cancer cells, induction of transgenic wild type APC (Figure 5b (Figure 5j). These observations demonstrate that dysregulated APC-β-catenin-TCF signaling mediates guanylin hormone loss as part of its canonical re-programming of nuclear transcription.…”

Section: Apc-β-catenin-tcf Signaling Regulates Guanylin Nuclear Transmentioning

confidence: 99%

APC-β-catenin-TCF signaling silences the intestinal guanylin-GUCY2C tumor suppressor axis

Blomain

Rappaport

Pattison

et al. 2020

Cancer Biology & Therapy

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Sporadic colorectal cancer initiates with mutations in APC or its degradation target β-catenin, producing TCF-dependent nuclear transcription driving tumorigenesis. The intestinal epithelial receptor, GUCY2C, with its canonical paracrine hormone guanylin, regulates homeostatic signaling along the crypt-surface axis opposing tumorigenesis. Here, we reveal that expression of the guanylin hormone, but not the GUCY2C receptor, is lost at the earliest stages of transformation in APC-dependent tumors in humans and mice. Hormone loss, which silences GUCY2C signaling, reflects transcriptional repression mediated by mutant APC-β-catenin-TCF programs in the nucleus. These studies support a pathophysiological model of intestinal tumorigenesis in which mutant APC-β-catenin-TCF transcriptional regulation eliminates guanylin expression at tumor initiation, silencing GUCY2C signaling which, in turn, dysregulates intestinal homeostatic mechanisms contributing to tumor progression. They expand the mechanistic paradigm for colorectal cancer from a disease of irreversible mutations in APC and β-catenin to one of guanylin hormone loss whose replacement, and reconstitution of GUCY2C signaling, could prevent tumorigenesis ARTICLE HISTORY

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“…Other uses of click-chemistry have been to develop better labelling of RNA in vitro and in vivo to better profile RNA populations in distinct cellular populations. 12–13 Furthermore, click chemistry has also been shown to be an effective tool to analyze the transport of stem cells to different locations 14–15 and has been used to understand the molecular dynamics and potential heterogeneity in drug activity in vivo 16 .…”

Section: Emerging Targets In Gene Therapy and Use Of Synthetic Biomentioning

confidence: 99%

“Clicking” Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions

et al. 2018

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The use of nucleic acid, DNA and RNA, based strategies to disrupt gene expression as a therapeutic is quickly emerging. Indeed, synthetic oligonucleotides represent a major component of emerging gene therapeutics. However, the efficiency and specificity of intracellular uptake for non-modified oligonucleotides is rather poor. Utilizing RNA based oligonucleotides as therapeutics is even more challenging to deliver, due to extremely fast enzymatic degradation of the RNAs. Like unmodified oligonucleotides, RNAs also get rapidly degraded in vivo and demonstrate large off-target binding events when they can reach and enter the desired target cells. One approach that holds much promise is the utilization of “click chemistry” to conjugate receptor or cell specific targeting molecules directly to the effector oligonucleotides. We discuss here the applications of the breakthrough technology of CuAAC “click-chemistry” and the immense potential in utilizing “click chemistry” in the development of new age targeted oligonucleotide therapeutics.

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Visualization of newly synthesized neuronal RNA in vitro and in vivo using click-chemistry

Cited by 39 publications

References 43 publications

Alternative 3′ UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

Alternative 3′ UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

APC-β-catenin-TCF signaling silences the intestinal guanylin-GUCY2C tumor suppressor axis

“Clicking” Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions

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