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The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5 -expressing intestinal stem cells (ISCs). However, several Lgr5 -negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5 -negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity. Intestinal stem cells are responsible for replenishing cells within the high-turnover intestinal epithelium. Here they show that ribosome dynamics affect intestinal stem cell identity through a mechanism that is triggered by changes in nutrient availability.

Increased protein synthesis supports the rapid cell proliferation associated with cancer. The Rpl24 Bst mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here, we show that Rpl24 Bst also suppresses tumorigenesis and proliferation in a model of colorectal cancer (CRC) with two common patient mutations, Apc and Kras . In contrast to previous reports, Rpl24 Bst mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in Rpl24 Bst mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the Rpl24 Bst mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in CRC, as well as in other cancers where the Rpl24 Bst mutation has a tumour suppressive effect in mouse models.

Increased protein synthesis supports the rapid cell proliferation associated with cancer. The mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here, we show that also suppresses tumorigenesis and proliferation in a model of colorectal cancer (CRC) with two common patient mutations, and . In contrast to previous reports, mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in CRC, as well as in other cancers where the mutation has a tumour suppressive effect in mouse models.

mRNA transcripts have limited potential for protein synthesis, defined by their open reading frames1. However, recent advances have revealed a far more complex reality, in which the proteome exceeds the perceived limits of the transcriptome2–6 exposing a significant gap in our understanding. Our prior studies have demonstrated that mRNA can undergo further processing, yielding truncated, uncapped mRNAs with translation potential2,7. Yet, the biological importance of this process remains mostly unclear. Here, we demonstrate that cleavage within the coding sequence of JAK1 mRNA produces an uncapped variant downstream to the cleavage site, at the expense of the full-length transcript. This results in the independent translation of the JH1 kinase domain, a process we term ‘RNA dicing’. Notably, canonical and diced JAK1 variants have distinct impacts on cell proliferation and tumorigenesis, operating independently, localizing to distinct cellular compartments. In addition, the activation of JAK1 signaling through IFNγ induction promotes the dicing of JAK1, thereby altering the balance of isoforms present. Base editor screens reveal that stop-codon nonsense mutations, which are typically considered loss-of-function, have differing impacts depending on their position relative to the dicing site. In agreement with this, JAK1 nonsense frameshift (JAK1fs) mutations in endometrial tumors inhibit the tumor-suppressive functions of canonical JAK1, while amplifying the oncogenic potential of the diced JH1 kinase domain. Lastly, we demonstrate that Momelotinib8, a JAK1-specific inhibitor, is more effective in cancer cells carrying a “loss-of-function” JAK1fs mutation, highlighting the significant impact of RNA dicing biology as a potent tool for patient stratification. Our findings characterize RNA dicing as a fundamental regulatory machinery that diversifies the potential products of a single mRNA molecule, and allows for significant variation in biological function.

Inflammatory cytokines are pivotal to immune responses. Upon cytokine exposure, cells enter an “alert-state” that enhances their visibility to the immune system. Here, we identified an “alert-state” subpopulation of ribosomes (ASRs) defined by the presence of the P-stalk. We show that ASRs are formed in response to cytokines linked to tumor immunity, and are involved in the preferential translation of mRNAs vital for the cytokine response. Mechanistically, ASRs are required for the efficient translation of transmembrane domains of receptor molecules involved in cytokine-mediated processes. Importantly, loss of the ASR prevents CD8+ T cell recognition and killing, and inhibitory cytokines like TGFβ hinder ASR formation, suggesting that the ASR is a central regulatory hub upon which multiple signals converge. Thus, the ASR is an essential mediator of the cellular rewiring that occurs following cytokine exposure, via the translational regulation of this process.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

Experimental data exists for only a vanishingly small fraction of sequenced microbial genes. This community page discusses the progress made by the COMBREX project to address this important issue using both computational and experimental resources.

[...]that DNA sequencing has become orders of magnitude faster and less expensive, focus has shifted to sequencing entire genomes. Since biochemistry and genetics have not, by and large, enjoyed the same improvement of scale, public sequence repositories now predominantly contain putative protein sequences for which there is no direct experimental evidence of function. COMBREX (COMputational BRidges to EXperiments, http://combrex.bu.edu) is an NIH-funded enterprise that has brought computational and experimental biologists together, with the goal of greatly improving our overall understanding of microbial protein function [1],[2]. Since its inception, it has made significant progress toward the following goals: identifying the minority of proteins that have already been experimentally characterized, serving as a public repository of novel protein function predictions made by diverse methods, producing a clear chain of evidence from experiment to prediction, identifying (\"recommending\") those functional predictions whose verification will contribute most to our overall understanding of protein function, and actually funding the experiments to test function. [...]the project issues small monetary awards (COMBREX grants) to biologists to fund the experimental testing of such predictions.