Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.
Summary MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNP (microRNA-protein complex) that lacks the microRNP repressor, GW182. Their mechanism in these conditions of decreased mTOR signaling and therefore, reduced canonical (cap-and-poly(A)-tail-mediated) translation, remains undiscovered. Our data reveal that mTOR inhibition in human THP1 cells enables microRNA-mediated activation. Activation requires shortened/no poly(A)-tail targets; polyadenylated mRNAs are partially activated upon PAIP2 overexpression, which interferes with poly(A)-bound PABP, precluding PABP-enhanced microRNA-mediated inhibition and canonical translation. Consistently, inhibition of PARN deadenylase prevents activation. P97/DAP5, a homolog of canonical translation factor, eIF4G, which lacks PABP- and cap binding complex-interacting domains, is required for activation and thereby, for the oocyte immature state. P97 interacts with 3′-UTR-binding FXR1a-associated microRNPs and with PARN, which binds mRNA 5′ caps, forming a specialized complex to translate recruited mRNAs in these altered canonical translation conditions.
MicroRNAs can promote translation of specific mRNAs in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes. We report that microRNA-mediated upregulation of target mRNAs in oocytes is dependent on nuclear entry of the microRNA; cytoplasmically-injected microRNA repress target mRNAs. Components of the activation microRNP, AGO, FXR1 (FXR1-iso-a) and miR16 are present in the nucleus and cytoplasm. Importantly, microRNA target mRNAs for upregulation, Myt1, TNFα and a reporter bearing the TNFα AU-rich, microRNA target sequence, are associated with AGO in immature oocyte nuclei and AGO2 in G0 human nuclei, respectively. mRNAs that are repressed or lack target sites are not associated with nuclear AGO. Crosslinking-coupled immunopurification revealed greater association of AGO2 with FXR1 in the nucleus compared to cytoplasm. Consistently, overexpression of FXR1-iso-a rescues activation of cytoplasmically-injected RNAs and in low density, proliferating cells. These data indicate the importance of a compartmentalized AGO2-FXR1-iso-a complex for selective recruitment for microRNA-mediated upregulation.
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