Molecular Cell最新文献

The cytoplasm of eukaryotic cells is crowded with macromolecules. In principle, this crowding could have either a positive or a negative effect on the rates of biochemical reactions. Here, we review two commonly invoked theories to account for these possible effects then survey recent experimental work in cells and extracts that measures the effects. The evidence so far suggests that the effective second-order rate constants (a measure of the speed of a reaction for a given concentration of reactants) for reactions in vivo generally go down when crowding increases due to the slowing of diffusion. If the evidence presented so far proves to be general, it would have important implications for how we view the trade-offs that determine the biochemical dynamics of the cytoplasm.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, driven by oncogenic KRAS mutations and dysregulated oncogenes, including SRSF1, MYC, and Aurora kinase A (AURKA). Although KRAS-targeted therapies are in development, resistance mechanisms underscore the need to identify alternative vulnerabilities. Here, we uncover an SRSF1-AURKA-MYC oncogenic circuit, wherein SRSF1 regulates AURKA 5' UTR alternative splicing, enhancing AURKA protein expression; AURKA positively regulates SRSF1 and MYC post-translationally, independently of its kinase activity; and MYC transcriptionally upregulates both SRSF1 and AURKA. Elevated SRSF1 in tumor cells promotes inclusion of an Alu-derived exon in the AURKA 5' UTR, resulting in splicing-dependent mRNA accumulation and exon-junction-complex deposition. Modulating 5' UTR splicing with splice-switching antisense oligonucleotides (ASOs) collapses the oncogenic circuit, reducing PDAC cell viability and triggering apoptosis. Our findings identify AURKA alternative splicing as a critical regulatory node and highlight a potential therapeutic strategy that simultaneously targets SRSF1, AURKA, and MYC oncogenes.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs that prevent FUS fibrillization by promoting liquid phases and distinct short RNAs that prevent and reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. We define a short RNA that dissolves cytoplasmic FUS aggregates, restores nuclear FUS, and mitigates FUS toxicity in optogenetic models and ALS patient-derived motor neurons. Another short RNA dissolves cytoplasmic TDP-43 aggregates, restores nuclear TDP-43, and mitigates TDP-43 toxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have utility for ALS/FTD and related disorders.

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin protein levels in patient-derived cells. We further show that ZNF207's zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (snRNP) components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-seq to uncover key regulators and domains of alternative splicing.

Mitochondria and the endoplasmic reticulum (ER) contain large areas that are in close proximity. Yet the mechanism of how these inter-organellar adhesions are formed remains elusive. Tight functional connections, termed "membrane contact sites," assemble at these areas and are essential for exchanging metabolites and lipids between the organelles. Recently, the ER-resident protein PDZ domain-containing protein 8 (PDZD8) was identified as a tether between the ER and mitochondria or late endosomes/lysosomes. Here, we show that PDZD8 can undergo phase separation via its intrinsically disordered region (IDR). Endogenously labeled PDZD8 forms condensates on membranes both in vitro and in mammalian cells. Electron microscopy analyses indicate that the expression of full-length PDZD8 rescues the decrease in inter-organelle contacts in PDZD8 knockout cells but not PDZD8 lacking its IDR. Together, this study identifies that PDZD8 condensates at the lipid interfaces act as an adhesive framework that stitches together the neighboring organelles and supports the structural and functional integrity of inter-organelle communication.

In this issue of Molecular Cell, Ishiguro et al.¹ describe new RNA modifications near the active site of the E. coli ribosome that appear only under anaerobic conditions. These modifications enhance ribosome activity and increase anaerobic growth rates.

Oxidative phosphorylation (OXPHOS) fulfills energy metabolism and biosynthesis through the tricarboxylic acid (TCA) cycle and an intact electron transport chain (ETC). Mitochondrial glutamine import (MGI) replenishes the TCA cycle through glutaminolysis, but its broader roles in cancer remain unclear. Here, we show that MGI sustains OXPHOS independently of glutaminolysis by maintaining ETC integrity. Exogenous glutamate availability abrogates cellular dependence on glutaminolysis but not SLC1A5var-mediated MGI. Blocking MGI elicits severe mitochondrial defects, reducing mitochondrial glucose oxidation and increasing glutamine reductive carboxylation. MGI, but not glutaminolysis, is essential for mitochondrial translation by enabling biogenesis of Gln-mt-tRNA^Gln, the most limiting mitochondrial aminoacyl-tRNA in cancer cells. Finally, deleting SLC1A5 in mice and targeting SLC1A5var in xenograft tumors inhibit Gln-mt-tRNA^Gln biogenesis and mitochondrial translation and blunt tumor growth. Our findings uncover a previously unrecognized role of MGI in safeguarding ETC integrity independently of glutaminolysis and inform a therapeutic option by targeting MGI to abrogate OXPHOS for cancer treatment.

In this issue of Molecular Cell, Zhu et al.¹ show that mitochondria of cancer cells rely on the import of glutamine not only to fuel metabolite synthesis via the tricarboxylic acid cycle but also to charge mt-tRNA^Gln to allow mitochondrial protein synthesis and respiration.