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Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean expression levels. This amplified expression noise originates from shorter-duration, higher-intensity transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we refer to as "discordant transcription through repair" ("DiThR," which is pronounced "dither"), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.

Mitochondria are dynamic organelles that must precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial surface, the importance of this localization is unclear. As yeast switch to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. Our data point to this change in mitochondrial volume fraction increasing the localization of certain nuclear-encoded mRNAs to the surface of the mitochondria. We show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to mitochondria. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle-specific gene expression through mRNA localization.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19. [ABSTRACT FROM AUTHOR]

Molecular noise is a natural phenomenon that is inherent to all biological systems.sup.1,2. How stochastic processes give rise to the robust outcomes that support tissue homeostasis remains unclear. Here we use single-molecule RNA fluorescent in situ hybridization (smFISH) on mouse stem cells derived from haematopoietic tissue to measure the transcription dynamics of three key genes that encode transcription factors: PU.1 (also known as Spi1), Gata1 and Gata2. We find that infrequent, stochastic bursts of transcription result in the co-expression of these antagonistic transcription factors in the majority of haematopoietic stem and progenitor cells. Moreover, by pairing smFISH with time-lapse microscopy and the analysis of pedigrees, we find that although individual stem-cell clones produce descendants that are in transcriptionally related states--akin to a transcriptional priming phenomenon--the underlying transition dynamics between states are best captured by stochastic and reversible models. As such, a stochastic process can produce cellular behaviours that may be incorrectly inferred to have arisen from deterministic dynamics. We propose a model whereby the intrinsic stochasticity of gene expression facilitates, rather than impedes, the concomitant maintenance of transcriptional plasticity and stem cell robustness. Single-molecule fluorescence in situ hybridization and live-cell imaging are used to study the contribution of transcriptional noise to stem cell heterogeneity, revealing that stochastic transcription dynamics are conducive to concomitant stem-cell maintenance and tissue homeostasis., Author(s): Justin C. Wheat [sup.1] [sup.2] , Yehonatan Sella [sup.3] , Michael Willcockson [sup.1] , Arthur I. Skoultchi [sup.1] , Aviv Bergman [sup.3] [sup.4] [sup.5] [sup.6] , Robert H. Singer [...]

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.