Simple and inexpensive ribosome profiling analysis of mRNA translation.
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2015-12
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The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.
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Reid, David W, Shirish Shenolikar and Christopher V Nicchitta (2015). Simple and inexpensive ribosome profiling analysis of mRNA translation. Methods (San Diego, Calif.), 91. pp. 69–74. 10.1016/j.ymeth.2015.07.003 Retrieved from https://hdl.handle.net/10161/17234.
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Shirish Shenolikar
Protein phosphorylation controls a wide range of physiological processes in mammalian tissues. Phosphorylation state of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases that are regulated by hormones, neurotransmitters, growth factors and other environmental cues. Our research attempts to understand the communication between protein kinases and phosphatases that dictates cellular protein phosphorylation and the cell's response to hormones. Over the last decade, our work has provided critical information about the role of protein phosphatase-1 (PP1) in controlling synaptic function, cell stress, gene expression and growth. We have generated a large repertoire of reagents to decipher PP1's role in signaling pathways in mammalian cells and tissues. Emerging evidence suggests that in many cells, PP1 activity is fine tuned by the protein, inhibitor-1 (I-1). A major focus of our research is to elucidate the role of I-1 in kinase-phosphatase cross-talk and impact of the altered I-1 gene expression seen in several human diseases. Our studies showed that recognition of cellular substrates by PP1 is also directed by its association with a variety of targeting subunits that are themselves also subject to physiological control. Thus, the overall focus of our research is to define the physiological mechanisms that regulate PP1 functions relevant to human health and disease.
Christopher Vincent Nicchitta
From the ER to Stress Granules: Defining Pathways of RNA Regulation:
Our laboratory investigates how cells control the location and timing of protein synthesis, with a focus on mRNA localization—the process by which mRNAs are targeted to specific sites within the cell to direct protein production. This spatial and temporal regulation is essential for cell signaling, division, and overall cellular dynamics.
We study mRNA localization to the endoplasmic reticulum (ER), where this process occurs on an unusually large scale. While the ER has long been recognized as the translation site for mRNAs encoding secretory and membrane proteins, our research has revealed that the ER functions far more broadly, supporting translation across the transcriptome. In particular, we have shown that newly exported mRNAs are preferentially translated on the ER, a process we hypothesize is coupled to RNA quality-control mechanisms during the pioneer rounds of translation.
Our recent work has also uncovered links between ER-directed mRNA localization and the pathways governing stress granule (SG) biogenesis. We are currently investigating how transcriptional status influences mRNA recruitment into SGs, the mechanisms that determine which mRNAs are selected, and the role of ER-associated sites in organizing SG assembly.
To address these questions, we combine biochemistry, cell biology, advanced imaging, genomics, and computational biology. Current research themes include:
- Cis-encoded signals and targeting mechanisms – defining mRNA sequence elements and cellular factors that direct ER localization. Beyond the canonical SRP pathway, our CRISPR/Cas studies have revealed additional, pathway-independent routes that recruit even cytosolic and nucleoplasmic mRNAs to the ER.
- RNA-binding proteins and stress responses – investigating how RNA-binding proteins mediate mRNA localization to the ER and regulate selective mRNA recruitment into SGs. Approaches include optical imaging, nucleoside analog pulse-labeling, cell fractionation, proteomics, ribosome footprinting, and RNA-seq methods (including 4SU-RNAseq).
Through these studies, our goal is to uncover fundamental principles of RNA regulation, quality control, and cellular organization.
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