metabolic-labeling-and-profiling-of-transfer-rnas-using-macroarrays

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

MUSC

Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.

SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels.

Epithelial-mesenchymal transition (EMT) is a spatially- and temporally-regulated process involved in physiological and pathological transformations, such as embryonic development and tumor progression. While the role of TGF-β as an EMT-inducer has been extensively documented, the molecular mechanisms regulating this transition and their implications in tumor metastasis are still subjects of intensive debates and investigations. TGF-β regulates EMT through both transcriptional and post-transcriptional mechanisms, and recent advances underline the critical roles of non-coding RNAs in these processes. Although microRNAs and lncRNAs have been clearly identified as effectors of TGF-β-mediated EMT, the contributions of other atypical non-coding RNA species, such as piRNAs, snRNAs, snoRNAs, circRNAs, and even housekeeping tRNAs, have only been suggested and remain largely elusive. This review discusses the current literature including the most recent reports emphasizing the regulatory functions of non-coding RNA in TGF-β-mediated EMT, provides original experimental evidence, and advocates in general for a broader approach in the quest of new regulatory RNAs.

Pleiotropic Roles of Non-Coding RNAs in TGF-β-Mediated Epithelial-Mesenchymal Transition and Their Functions in Tumor Progression.

In order to better understand the process of breast cancer metastasis, we have generated a mammary epithelial progression series of increasingly aggressive cell lines that metastasize to lung. Here we demonstrate that upregulation of an endoplasmic reticulum (ER) to Golgi trafficking gene signature in metastatic cells enhances transport kinetics, which promotes malignant progression. We observe increased ER-Golgi trafficking, an altered secretome and sensitivity to the retrograde transport inhibitor brefeldin A (BFA) in cells that metastasize to lung. CREB3 was identified as a transcriptional regulator of upregulated ER-Golgi trafficking genes ARF4, COPB1, and USO1, and silencing of these genes attenuated the metastatic phenotype in vitro and lung colonization in vivo. Furthermore, high trafficking gene expression significantly correlated with increased risk of distant metastasis and reduced relapse-free and overall survival in breast cancer patients, suggesting that modulation of ER-Golgi trafficking plays an important role in metastatic progression.

Simon Grelet

Department of Biochemistry and Molecular Biology

MUSC

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Simon Grelet

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology

MUSC

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Department of Biochemistry and Molecular Biology