We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq), for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular ...RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus) enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.
RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA ...targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.
Pulmonary neuroendocrine (NE) cells are neurosensory cells sparsely distributed throughout the bronchial epithelium, many in innervated clusters of 20–30 cells. Following lung injury, NE cells ...proliferate and generate other cell types to promote epithelial repair. Here, we show that only rare NE cells, typically 2–4 per cluster, function as stem cells. These fully differentiated cells display features of classical stem cells. Most proliferate (self-renew) following injury, and some migrate into the injured area. A week later, individual cells, often just one per cluster, lose NE identity (deprogram), transit amplify, and reprogram to other fates, creating large clonal repair patches. Small cell lung cancer (SCLC) tumor suppressors regulate the stem cells: Rb and p53 suppress self-renewal, whereas Notch marks the stem cells and initiates deprogramming and transit amplification. We propose that NE stem cells give rise to SCLC, and transformation results from constitutive activation of stem cell renewal and inhibition of deprogramming.
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•Rare, fully differentiated neuroendocrine cells are stem cells activated by injury•Injury induces stem cell renewal, dispersal, transit amplification, and reprogramming•Tumor suppressors Rb, p53, and Notch control specific steps in stem cell program•Small-cell lung cancer arises by genetic activation of stem cell renewal and dispersal
A small fraction of differentiated neuroendocrine cells possesses the ability to act as reserve stem cells during lung injury.
Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of ...single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.
Epicardial cells on the heart's surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and ...caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease.
In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, ...lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.
Characterization of the molecular attributes and spatial arrangements of cells and features within complex human tissues provides a critical basis for understanding processes involved in development ...and disease. Moreover, the ability to automate steps in the analysis and interpretation of histological images that currently require manual inspection by pathologists could revolutionize medical diagnostics. Toward this end, we developed a new imaging approach called multidimensional microscopic molecular profiling (MMMP) that can measure several independent molecular properties in situ at subcellular resolution for the same tissue specimen. MMMP involves repeated cycles of antibody or histochemical staining, imaging, and signal removal, which ultimately can generate information analogous to a multidimensional flow cytometry analysis on intact tissue sections. We performed a MMMP analysis on a tissue microarray containing a diverse set of 102 human tissues using a panel of 15 informative antibody and 5 histochemical stains plus DAPI. Large-scale unsupervised analysis of MMMP data, and visualization of the resulting classifications, identified molecular profiles that were associated with functional tissue features. We then directly annotated H&E images from this MMMP series such that canonical histological features of interest (e.g. blood vessels, epithelium, red blood cells) were individually labeled. By integrating image annotation data, we identified molecular signatures that were associated with specific histological annotations and we developed statistical models for automatically classifying these features. The classification accuracy for automated histology labeling was objectively evaluated using a cross-validation strategy, and significant accuracy (with a median per-pixel rate of 77% per feature from 15 annotated samples) for de novo feature prediction was obtained. These results suggest that high-dimensional profiling may advance the development of computer-based systems for automatically parsing relevant histological and cellular features from molecular imaging data of arbitrary human tissue samples, and can provide a framework and resource to spur the optimization of these technologies.
Post-transcriptional regulation of gene expression, including mRNA localization, translation and decay, is ubiquitous yet still largely unexplored. How is the post-transcriptional regulatory program ...of each mRNA encoded in its sequence? Hundreds of specific RNA-binding proteins (RBPs) appear to play roles in mediating the post-transcriptional regulatory program, akin to the roles of specific DNA-binding proteins in transcription. As a step toward decoding the regulatory programs encoded in each mRNA, we focused on specific mRNA-protein interactions. We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements. Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3. Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.
The unfolded protein response (UPR) is a conserved mechanism that mitigates accumulation of unfolded proteins in the ER. The yeast UPR is subject to intricate post-transcriptional regulation, ...involving recruitment of the RNA encoding the Hac1 transcription factor to the ER and its unconventional splicing. To investigate the mechanisms underlying regulation of the UPR, we screened the yeast proteome for proteins that specifically interact with HAC1 RNA. Protein microarray experiments revealed that HAC1 interacts specifically with small ras GTPases of the Ypt family. We characterized the interaction of HAC1 RNA with one of these proteins, the yeast Rab1 homolog Ypt1. We found that Ypt1 protein specifically associated in vivo with unspliced HAC1 RNA. This association was disrupted by conditions that impaired protein folding in the ER and induced the UPR. Also, the Ypt1-HAC1 interaction depended on IRE1 and ADA5, the two genes critical for UPR activation. Decreasing expression of the Ypt1 protein resulted in a reduced rate of HAC1 RNA decay, leading to significantly increased levels of both unspliced and spliced HAC1 RNA, and delayed attenuation of the UPR, when ER stress was relieved. Our findings establish that Ypt1 contributes to regulation of UPR signaling dynamics by promoting the decay of HAC1 RNA, suggesting a potential regulatory mechanism for linking vesicle trafficking to the UPR and ER homeostasis.
How best to manage forest patches, mitigate the consequences of forest fragmentation, and enable landscape permeability are key questions facing conservation scientists and managers. Many temperate ...forests have become increasingly fragmented, resulting in reduced interior forest habitat, increased edge habitats, and reduced connectivity. Using a citizen science landscape-scale mark-release-recapture study on 87 macro-moth species, we investigated how both life-history traits and landscape characteristics predicted macro-moth responses to forest fragmentation. Wingspan, wing shape, adult feeding, and larval feeding guild predicted macro-moth mobility, although the predictive power of wingspan and wing shape depended on the species' affinity to the forest. Solitary trees and small fragments functioned as "stepping stones," especially when their landscape connectivity was increased, by being positioned within hedgerows or within a favorable matrix. Mobile forest specialists were most affected by forest fragmentation: despite their high intrinsic dispersal capability, these species were confined mostly to the largest of the forest patches due to their strong affinity for the forest habitat, and were also heavily dependent on forest connectivity in order to cross the agricultural matrix. Forest fragments need to be larger than five hectares and to have interior forest more than 100 m from the edge in order to sustain populations of forest specialists. Our study provides new insights into the movement patterns of a functionally important insect group, with implications for the landscape-scale management of forest patches within agricultural landscapes.
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