Single Molecule Imaging of RNA In Situ

Batish, Mona; Raj, Arjun; Tyagi, Sanjay

doi:10.1007/978-1-61779-005-8_1

Cited by 70 publications

(74 citation statements)

References 6 publications

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“…1C). The FISH probes were 26 singly labeled oligonucleotides which should permit detection of RNA at a single-molecule sensitivity (52,53). Using a particle-counting algorithm (54), we found that cells had 50 to 500 discrete RNA signals per cell.…”

Section: Studying Hbv Subcellular Localization During Infectionmentioning

confidence: 99%

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal

Unchwaniwala

Sherer

Loeb

2016

J Virol

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To understand subcellular sites of hepatitis B virus (HBV) replication, we visualized core (Cp), polymerase (Pol), and pregenomic RNA (pgRNA) in infected cells. Interestingly, we found that the majority of Pol localized to the mitochondria in cells undergoing viral replication. The mitochondrial localization of Pol was independent of both the cell type and other viral components, indicating that Pol contains an intrinsic mitochondrial targeting signal (MTS). Neither Cp nor pgRNA localized to the mitochondria during active replication, suggesting a role other than DNA synthesis for Pol at the mitochondria. The Pol of duck hepatitis B virus (DHBV) also localized to the mitochondria. This result indicates that localization of Pol to mitochondria is likely a feature of all hepadnaviruses. To map the MTS within HBV Pol, we generated a series of Pol-green fluorescent protein (Pol-GFP) fusions and found that a stretch spanning amino acids (aa) 141 to 160 of Pol was sufficient to target GFP to the mitochondria. Surprisingly, deleting aa 141 to 160 in full-length Pol did not fully ablate Pol's mitochondrial localization, suggesting that additional sequences are involved in mitochondrial targeting. Only by deleting the N-terminal 160 amino acids in fulllength Pol was mitochondrial localization ablated. Crucial residues for pgRNA packaging are contained within aa 141 to 160, indicating a multifunctional role of this region of Pol in the viral life cycle. Our studies show an unexpected Pol trafficking behavior that is uncoupled from its role in viral DNA synthesis. IMPORTANCEChronic infection by HBV is a serious health concern. Existing therapies for chronically infected individuals are not curative, underscoring the need for a better understanding of the viral life cycle to develop better antiviral therapies. To date, the most thoroughly studied function of Pol is to package the pgRNA and reverse transcribe it to double-stranded DNA within capsids. This study provides evidence for mitochondrial localization of Pol and defines the MTS. Recent findings have implicated a nonreverse transcription role for Pol in evading host innate immune responses. Mitochondria play an important role in controlling cellular metabolism, apoptosis, and innate immunity. Pol may alter one or more of these host mitochondrial functions to gain a replicative advantage and persist in chronically infected individuals.

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Section: Studying Hbv Subcellular Localization During Infectionmentioning

confidence: 99%

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal

Unchwaniwala

Sherer

Loeb

2016

J Virol

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“…As a complementary approach, we assayed for wg transcript using a highly sensitive single-molecule fluorescence in situ hybridization (FISH) (Batish et al, 2011). We observed puncta throughout the anterior half of the germarium, and the signal was substantially diminished by pretreatment of the fixed tissue with RNase ( Fig.…”

Section: Escort Cells Are the Predominant Source Of Wg For The Fsc LImentioning

confidence: 99%

A dynamic population of stromal cells contributes to the follicle stem cell niche in theDrosophilaovary

2013

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“…Microscopy is undoubtedly the first device for single-cell analysis, since cells were first discovered under a microscope. Along the way, other methods include micromanipulator, histological staining, in situ hybridization (ISH), patch-clamp, mass cytometry, and flow cytometry were developed and people turn to these methods gradually (Batish et al 2011;Bendall et al 2011;Gaynor et al 1996;Krutzik et al 2008;Krutzik and Nolan 2006;Spruce et al 1989;Evans and Yeung 1989).…”

Section: The Development Of Microfluidic Devices For Single Cell Sequmentioning

confidence: 99%

The Application of Single-Cell Sequencing in Dynamic Transcriptomes

Chen¹,

Dong²,

Wu³

2015

Single Cell Sequencing and Systems Immunology

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There has been long-standing interest in developing procedures for profiling single-cell gene expression dynamics. Single-cell transcriptome profiling is useful for studying heterogeneity and mosaicism in tissues and understanding cellular behaviors and compositions. Recent advancements in RNA sequencing technologies dramatically improved sensitivity and specificity of gene expression quantifications, leading to rapid progress in single-cell transcriptome profiling methods. In this chapter, we provide a timely review on basic principles of singlecell RNA sequencing methods and discuss the technical challenges and their applications in research and medicine.

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Single Molecule Imaging of RNA In Situ

Cited by 70 publications

References 6 publications

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal

A dynamic population of stromal cells contributes to the follicle stem cell niche in theDrosophilaovary

The Application of Single-Cell Sequencing in Dynamic Transcriptomes

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