Translation elongation defects activate the integrated stress response (ISR), but whether and how ribosome stalls are cleared to enable mRNA release for ribonucleoprotein (RNP) granule assembly remain unclear. We show that blocking tRNA aminoacylation generates persistent uncollided ribosome stalls that inhibit stress granule and P-body assembly despite robust ISR activation. Collided ribosomes are rapidly cleared by ZNF598-dependent ribosome-associated quality control within 4 h, while uncollided stalls resist clearance and persist for >16 h. Puromycin releases persistent stalls and restores RNP granule formation. The block in stress granule assembly is generalizable across tRNA synthetase inhibitors and amino acid deprivation. Therefore, stress granules represent signal integrators reporting translation elongation status when initiation is suppressed. Our findings reveal that translation quality control pathways selectively clear collided ribosomes, establish that translation elongation stress uncouples RNP granule assembly from the ISR, and suggest that tolerating uncollided stalls may be adaptive for cotranslational processes essential for cellular function.

Human neurogenesis is disproportionately protracted, lasting >10 times longer than in mice, allowing neural progenitors to undergo more rounds of self-renewing cell divisions and generate larger neuronal populations. In the human spinal cord, expansion of the motor neuron lineage is achieved through a newly evolved progenitor domain called the ventral motor neuron progenitor (vpMN) that delays and expands motor neurogenesis. This behavior of vpMNs is controlled by transcription factor NKX2-2, which in vpMNs is coexpressed with classical motor neuron progenitor (pMN) marker OLIG2. In this study, we sought to determine the molecular basis of NKX2-2-mediated extension and expansion of motor neurogenesis. We found that, unlike in mice or chicks, NKX2-2 in the human spinal cord does not repress dorsoventral patterning genes like OLIG2 However, it retains its ability to repress NEUROG2, a proneural gene that promotes exit from the cell cycle and motor neurogenesis. Interestingly, we found that ectopic expression of Tinman mutant Nkx2-2 in mouse pMNs phenocopies human vpMNs, repressing Neurog2 but not Olig2, resulting in delayed motor neurogenesis. Thus, our studies reveal that the classical patterning function of NKX2-2 that depends on its Tinman repressive domain is dissociated from NKX2-2's ability to repress NEUROG2 to control the onset and duration of motor neurogenesis in human ventral motor neuron progenitors.

During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report that synthetic cell-cell signaling using designed ligand-receptor pairs can induce synchronized oscillations in PSM organoids. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; nonoscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock and provides a way to program temporal gene expression in organoids and artificial tissues.

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. DIO3 mutant organoids display precocious development of S cones, L/M cones, and rods; increased photoreceptor density; and subpopulations of photoreceptors that coexpress different opsin proteins. Our multiomics and chimeric organoid experiments show that cell-autonomous and non-cell-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared with a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass-like mechanism in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, triggering development of photoreceptor subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.

The exon junction complex (EJC) has roles in mRNA export and cytoplasmic quality control. However, the EJC is recruited to pre-mRNA by the spliceosome prior to the completion of splicing. When splicing is cotranscriptional, the EJC is deposited on nascent RNA early during synthesis, raising the question of whether the EJC regulates downstream RNA processing. Here we show, using long-read sequencing, that degron-mediated depletion of EJC component EIF4A3 leads to skipping of neighboring pairs of two or more exons on the same mRNA molecule. These data suggest that the entire "exon block" requires the EJC for inclusion. Introns flanking EJC-dependent exon blocks were longer and spliced after internal introns. In our working model, block exons are first spliced together to form a larger EJC-marked exon that promotes surrounding splicing events. Strikingly, analysis of 480 RNA binding protein knockdowns across two different human cell lines revealed block exons that are dependent on other splicing factors, indicating that coordinated splicing of adjacent exons is a general mechanism, of which the EJC is the dominant regulator. Cell type-specific coordinated splicing of adjacent exon pairs has been observed before. Here we identify the EJC as the main protein factor massively regulating this novel splicing mechanism in trans.

While homologous recombination (HR) is often considered to be an error-free DNA repair mechanism, the fidelity of this pathway depends on the cell's ability to engage the ideal template: the replicated sister chromatid. This is particularly challenging during repair of repetitive genome regions for which nonallelic sequences can errantly be used as templates. We developed a model to study spontaneous DNA damage and repair that occurs at repetitive protein-coding genes of the Schizosaccharomyces pombe flocculin family. We observed that genes encoding most members of this protein family constitutively reside at the nuclear periphery by virtue of their close proximity to binding sites for the CENP-B-like protein, Cbp1. Tethering via Cbp1 to the nuclear periphery enhances the stability of the flocculin genes against intragenic recombination and restrains intergenic recombination between homoeologous repeat-encoding sequences. The LINC complex component Kms1 also antagonizes both intragenic and intergenic recombination at the flocculin genes as well as microhomology-mediated end joining (MMEJ). Our observations suggest that S. pombe leverages nuclear compartmentalization to maintain the stability of repetitive genic regions at the nuclear periphery, while association of DSBs with Kms1-containing LINC complexes enforces stringency to avoid mutagenic end joining and use of the incorrect template during HR.

Nuclear receptors (NRs) are ligand-regulated transcription factors (TFs) that respond to hormonal, nutritional, and environmental signals. NRs as druggable targets are known for their therapeutic potential in treating a wide range of diseases, including metabolic disorders, inflammatory conditions, and various cancers. Due to their structure and ability to interact with DNA, ligands, and other proteins, NR transcriptional regulatory capacity is fine-tuned through dynamic interactions with a disparate array of coregulators, including coactivators and corepressors that form an intricate network that integrates multiple signaling and metabolic pathways. This review synthesizes insights into the functional interactions between NRs and the modulators of transcription, focusing on interactions between NRs and coregulators and how their model of interaction has evolved from a binary switch to a coregulator shift mechanism in regulating ligand-dependent transcription. These nuanced multifaceted interactions collectively direct dynamic gene expression by NRs across multiple tissues in physiology and diseases.

Chromatin architecture plays a key role in development and cancer, yet most studies lack mechanistic depth due to widespread epigenomic remodeling. To address this, we tracked chromatin structure dynamics during the progression of endocrine resistance in ER+ breast cancer using Hi-C, chromatin accessibility, epigenomic, and transcriptomic profiling. We uncovered a critical role for H3K9 methylation and the demethylase KDM4C association with SWI/SNF in driving proliferation of cells fated to become resistant through a nongenomic estrogen-mediated mechanism. These findings highlight the mechanistic contribution of chromatin regulation in therapy resistance and offer a blueprint for studying similar processes in cancer, development, and cell fate decisions.

The discovery of the p53 tumor suppressor protein raised fundamental questions about cell cycle regulation that have spanned several decades. TP53 mutations are found in most human cancers, most frequently as missense alterations in the DNA-binding domain (DBD). As a master regulator of both cell-intrinsic and cell-extrinsic functions, mutant p53 contributes to pro-oncogenic activities through gain-of-function (GOF) properties in addition to loss-of-function (LOF) and dominant-negative effects (DNEs). New technologies and improved fidelity of model systems are uncovering the functional consequences caused by p53 mutations at the molecular, cellular, and tissue levels. In a new era of precision medicine, with the context of recent success in targeting genetic mutations, ongoing and future understanding of fundamental mutant p53 biology is of paramount importance.