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Background Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. Results As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1 , are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. Conclusions Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility. Graphical abstract

Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr–Igf1r–Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications. A combination of single-cell approaches, lineage tracing and metabolomics is used to characterize the changes to intestinal stem cell function in the small intestine that underlie intestinal maladaptation in mice fed an obesogenic diet.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 ( IL2 ) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest. Truong et al. report a system to monitor RNA expression by modifying an intron within a gene of interest. This additional engineered transcript then hijacks nuclear export machinery for subsequent translation of a reporter gene.

Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.

Generating stem cell-derived glucagon-producing α (SC-α cells) and insulin-producing β cells (SC-β cells) allows to engineer an in vitro biomimetic of the islet of Langerhans, the micro-organ controlling blood glucose, however, there is still a major knowledge gap in the mode and mechanism by which human SC-α and β cells are specified. Mouse studies postulated that Aristaless Related homeobox (Arx) and Paired box 4 (Pax4) transcription factors cross-inhibit each other in endocrine progenitors to promote α or β cell fate allocation, respectively. To test this model in human, we generated an ARXCFP/CFP; PAX4mCherry/mCherry double knock-in reporter induced pluripotent stem cell (iPSC) line to combine time-resolved cell lineage labeling with high-resolution single cell multiomic analysis. Strikingly, lineage labelling and tracing, proteomic and gene regulatory network (GRN) analysis and potency assays revealed a human specific mode and regulatory logic of α versus β cell fate allocation. Importantly, pharmacological perturbation using drugs previously proposed to trigger α-to-β cell transdifferentiation or identified via our GRN analysis led to enhanced endocrine induction and directed α vs β cell fate commitment. Thus, shedding light on basic mechanisms of endocrine induction and fate segregation not only paves the way to engineer islets from pluripotent stem cells, but also has broader implications for cell-replacement therapy, disease modelling and drug screening.