Harnessing synthetic biology to engineer organoids and tissues.

The development of an organism depends on intrinsic genetic programs of progenitor cells and their spatiotemporally complex extrinsic environment. Ex vivo generation of organoids from progenitor cells provides a platform for recapitulating and exploring development. Current approaches rely largely on soluble morphogens or engineered biomaterials to manipulate the physical environment, but the emerging field of synthetic biology provides a powerful toolbox to genetically manipulate cell communication, adhesion, and even cell fate. Applying these modular tools to organoids should lead to a deeper understanding of developmental principles, improved organoid models, and an enhanced capability to design tissues for regenerative purposes.

Repression of PUM1-mediated mRNA decay activates translesion synthesis after DNA damage.

Biological roles of Pumilio1 (PUM1) in ubiquitous cells remain unclear. Here we identify 48 degrading target mRNAs by combined analysis of transcriptome-wide mRNA stabilities and the binding of mRNAs. Further analysis revealed that cells respond to DNA damage by inhibiting PUM1-mediated mRNA decay to activate translesion synthesis (46/50).

Metabolic labeling of RNA using multiple ribonucleoside analogs enables the simultaneous evaluation of RNA synthesis and degradation rates.

Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this, simultaneous measurements of RNA synthesis and degradation are required. Here, we report the development of "Dyrec-seq," which uses 4-thiouridine and 5-bromouridine to simultaneously quantify RNA synthesis and degradation rates. Dyrec-seq enabled the quantification of RNA synthesis and degradation rates of 4702 genes in HeLa cells. Functional enrichment analysis showed that the RNA synthesis and degradation rates of genes are actually determined by the genes' biological functions. A comparison of theoretical and experimental analyses revealed that the amount of RNA is determined by the ratio of RNA synthesis to degradation rates, whereas the rapidity of responses to external stimuli is determined only by the degradation rate. This study emphasizes that not only RNA synthesis but also RNA degradation is important in shaping gene expression patterns.

Systematic Analysis of Targets of Pumilio-Mediated mRNA Decay Reveals that PUM1 Repression by DNA Damage Activates Translesion Synthesis.

RNA-binding proteins (RBPs) play a pivotal role in gene expression by modulating the stability of transcripts. However, the identification of degradation target mRNAs of RBPs remains difficult. By the combined analysis of transcriptome-wide mRNA stabilities and the binding of mRNAs to human Pumilio 1 (PUM1), we identify 48 mRNAs that both bind to PUM1 and exhibit PUM1-dependent degradation. Analysis of changes in the abundance of PUM1 and its degradation target mRNAs in RNA-seq data indicate that DNA-damaging agents negatively regulate PUM1-mediated mRNA decay. Cells exposed to cisplatin have reduced PUM1 abundance and increased PCNA and UBE2A mRNAs encoding proteins involved in DNA damage tolerance by translesion synthesis (TLS). Cells overexpressing PUM1 exhibit impaired DNA synthesis and TLS and increased sensitivity to the cytotoxic effect of cisplatin. Thus, our method identifies target mRNAs of PUM1-mediated decay and reveals that cells respond to DNA damage by inhibiting PUM1-mediated mRNA decay to activate TLS.

Contributions of regulated transcription and mRNA decay to the dynamics of gene expression.

Organisms have acquired sophisticated regulatory networks that control gene expression in response to cellular perturbations. Understanding of the mechanisms underlying the coordinated changes in gene expression in response to external and internal stimuli is a fundamental issue in biology. Recent advances in high-throughput technologies have enabled the measurement of diverse biological information, including gene expression levels, kinetics of gene expression, and interactions among gene expression regulatory molecules. By coupling these technologies with quantitative modeling, we can now uncover the biological roles and mechanisms of gene regulation at the system level. This review consists of two parts. First, we focus on the methods using uridine analogs that measure synthesis and decay rates of RNAs, which demonstrate how cells dynamically change the regulation of gene expression in response to both internal and external cues. Second, we discuss the underlying mechanisms of these changes in kinetics, including the functions of transcription factors and RNA-binding proteins. Overall, this review will help to clarify a system-level view of gene expression programs in cells. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Turnover and Surveillance > Regulation of RNA Stability RNA Methods > RNA Analyses in vitro and In Silico.

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.

5'-Bromouridine IP Chase (BRIC)-Seq to Determine RNA Half-Lives.

Analysis of RNA stability at genome-wide level is an advanced method in RNA biology that examines the half-life of each transcript. In particular, a pulse-labeling method using uridine analogs enables the determination of half-life of each transcript under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5'-bromouridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq.

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.

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

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.

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.

Fluorescent probes for imaging endogenous ß-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 ß-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 ß-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 ß-actin mRNA localization and dynamics in living cells.

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, ß-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 ß-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 ß-actin mRNA and that distinct spatial and temporal movement of the individual ß-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.

Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.

Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.