Live-cell imaging of budding yeast telomerase RNA and TERRA.

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs.

MS2 Labeling of Endogenous Beta-Actin mRNA Does Not Result in Stabilization of Degradation Intermediates.

The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of ß-actin mRNA extracted from the Actb-MBS knock-in and MBS×MCP hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of ß-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.

Visualization of Single mRNAs in Live Neurons.

Transcription and post-transcriptional regulations are critical in gene expression. To study the spatiotemporal regulation of RNA inside a cell, techniques for high-resolution imaging of RNA have been developed. In this chapter, we describe RNA fluorescent labeling methods using MS2 and PP7 systems to detect single RNA molecules in live neurons. We use hippocampal neurons cultured from knock-in mouse models in which ß-actin or Arc mRNAs are tagged with MS2 or PP7 stem-loops. Adeno-associated virus (AAV) or lentiviral vectors are used to express MS2 or PP7 capsid proteins fused with GFP in those neurons. Then, GFP-labeled RNAs in live neurons can be detected by epifluorescence microscopy, and their moving pathways can be analyzed using single-particle tracking software. For these processes, we introduce protocols for neuron culture, transfection, imaging, and particle tracking methods.

Dynamic imaging demonstrates that pulsed electromagnetic fields (PEMF) suppress IL-6 transcription in bovine nucleus pulposus cells.

Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Pulsed electromagnetic fields (PEMF) are noninvasive biophysical stimulus that has been used extensively in the orthopaedic field for many years. However, the specific cellular responses and mechanisms involved are still unclear. The objective of this study was to assess the time-dependent PEMF effects on pro-inflammatory factor IL-6 expression in disc nucleus pulposus cells using a novel green fluorescence protein (GFP) reporter system. An MS2-tagged GFP reporter system driven by IL-6 promoter was constructed to visualize PEMF treatment effect on IL-6 transcription in single living cells. IL-6-MS2 reporter-labeled cells were treated with IL-1α to mimic the in situ inflammatory environment of degenerative disc while simultaneously exposed to PEMF continuously for 4 h. Time-lapse imaging was recorded using a confocal microscope to track dynamic IL-6 transcription activity that was demonstrated by GFP. Finally, real-time RT-PCR was performed to confirm the imaging data. Live cell imaging demonstrated that pro-inflammatory factor IL-1α significantly promoted IL-6 transcription over time as compared with DMEM basal medium condition. Imaging and PCR data demonstrated that the inductive effect of IL-1α on IL-6 expression could be significantly inhibited by PEMF treatment in a time-dependent manner (early as 2 h of stimulus initiation). Our data suggest that PEMF may have a role in the clinical management of patients with chronic low back pain. Furthermore, this study shows that the MS2-tagged GFP reporter system is a useful tool for visualizing the dynamic events of mechanobiology in musculoskeletal research. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:778-787, 2018.

Visualizing mRNA Dynamics in Live Neurons and Brain Tissues.

Localization of mRNA plays a crucial role in a variety of neuronal processes including synaptogenesis, axonal guidance, and long-term plasticity. Recent advances in fluorescence imaging and RNA labeling techniques allow us to visualize how individual mRNA molecules are dynamically regulated inside live neurons and brain tissues. Here, we describe key methods in imaging mRNA dynamics, including preparation of neuron culture and brain slices from transgenic mice expressing GFP-labeled mRNA, high-resolution detection of single molecules, live tissue imaging, and analysis of mRNA transport.

Imaging Single-mRNA Localization and Translation in Live Neurons.

Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and regeneration. To reveal the underlying mechanisms of local translation, it is crucial to understand mRNA transport, localization and translation in live neurons. Among various techniques for mRNA analysis, fluorescence microscopy has been widely used as the most direct method to study localization of mRNA. Live-cell imaging of single RNA molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein (mRNP) complexes in neurons. Here, we review recent advances in the study of mRNA localization and translation in live neurons using novel techniques for single-RNA imaging.

Imaging Single mRNA Dynamics in Live Neurons and Brains.

RNA is a key player in the process of gene expression. Whereas fluorescence in situ hybridization allows single mRNA imaging in fixed cells, the MS2-GFP labeling technique enables the observation of mRNA dynamics in living cells. Recently, two genetically engineered mouse models have been developed for the application of the MS2-GFP system in live animals. First, the Actb-MBS mouse was generated by knocking in 24 repeats of the MS2 stem-loop sequence in the 3' untranslated region of the ß-actin gene. Second, the MCP mouse was made to express the NLS-HA-MCP-GFP transgene in all cell types. By crossing Actb-MBS and MCP mice, a double homozygous mouse line, MCP×MBS, was established to visualize endogenous ß-actin mRNA labeled with multiple green fluorescent proteins. By imaging hippocampal neurons or brain slices from MCP×MBS mice, the dynamics of mRNA, such as transcription, transport, and localization, can be studied at single mRNA resolution. In this chapter, we explain the basics of MCP×MBS mice and describe methods for utilizing these animals.