Toshimichi Yamada

Long Noncoding RNA NEAT1-Dependent SFPQ Relocation from Promoter Region to Paraspeckle Mediates IL8 Expression upon Immune Stimuli

Although thousands of long noncoding RNAs (lncRNAs) are localized in the nucleus, only a few dozen have been functionally characterized. Here we show that nuclear enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, is induced by influenza virus and herpes simplex virus infection as well as by Toll-like receptor3-p38 pathway-triggered poly I:C stimulation, resulting in excess formation of paraspeckles. We found that NEAT1 facilitates the expression of antiviral genes including cytokines such as interleukin-8 (IL8). We found that splicing factor proline/glutamine-rich (SFPQ), a NEAT1-binding paraspeckle protein, is a repressor of IL8 transcription, and that NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles, leading to transcriptional activation of IL8. Together, our data show that NEAT1 plays an important role in the innate immune response through the transcriptional regulation of antiviral genes by the stimulus-responsive cooperative action of NEAT1 and SFPQ.

Visualization of Nonengineered Single mRNAs in Living Cells Using Genetically Encoded Fluorescent Probes

Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.

Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio.

Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells.

Development of red-shifted mutants derived from luciferase of Brazilian click beetle Pyrearinus termitilluminans

Abstract. Luciferase, a bioluminescent protein, has been used as an analytical tool to visualize intracellular phenomena. Luciferase with red light emission is particularly useful for bioluminescence imaging because of its high transmittance in mammalian tissues. However, the luminescence intensity of existing luciferases with their emission over 600 nm is insufficient for imaging studies because of their weak intensities. We developed mutants of Emerald luciferase (Eluc) from Brazilian click beetle (Pyrearinus termitilluminans), which emits the strongest bioluminescence among beetle luciferases. We successively introduced four amino acid mutations into the luciferase based on a predicted structure of Eluc using homology modeling. Results showed that quadruple mutations R214K/H241K/S246H/H347A into the beetle luciferase emit luminescence with emission maximum at 626 nm, 88-nm red-shift from the wild-type luciferase. This mutant luciferase is anticipated for application in in vivo multicolor imaging in living samples.

Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres

Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.

Diminished nuclear RNA decay upon Salmonella infection upregulates antibacterial noncoding RNAs

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity‐related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

Techniques for Genome-Wide Expression Analysis of Noncoding RNA

Genome-wide expression analysis of RNAs was revolutionized by the development of RNA sequencing (RNA-seq). RNA-seq revealed that most genomic regions are transcribed pervasively, resulting in the production of a large number of noncoding RNAs (ncRNA), such as large intergenic ncRNAs (lincRNAs), microRNAs (miRNAs), cryptic unstable transcripts (CUTs), and enhancer RNAs (eRNAs). Subsequently, it was revealed that these ncRNAs are involved in epigenetic gene regulation. The instability and diversity of ncRNAs has meant that the precise determination of their gene structures and the expressions of ncRNA are a major challenge in field of transcriptomics. Here, we introduce several key steps in RNA-seq to detect ncRNAs, such as sample preparation optimized for ncRNAs and computational annotation of RNA-seq data to ncRNAs. We further review the methods used for functional studies of ncRNAs with developed applications based on RNA-seq.