A Specialized Mechanism of Translation Mediated by FXR1a-Associated MicroRNP in Cellular Quiescence.

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 microRNA-protein complex (microRNP) 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.

Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages.

Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Met(i)) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met(i) upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Meti for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.

Regulation of RNA methylation by therapy treatment, promotes tumor survival.

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5'UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.

Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells.

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.

m5C modification of mRNA serves a DNA damage code to promote homologous recombination.

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.

Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis.

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.

Analysis of MicroRNA-Mediated Translation Activation of In Vitro Transcribed Reporters in Quiescent Cells.

Quiescence (G0) is defined as an assortment of cell cycle arrested states that exhibit distinct properties. Leukemias harbor a subpopulation of G0 cells that can be enriched by growth factor deprivation or serum starvation. Target site reporters with shortened poly(A) tails show translation activation by microRNAs, via a noncanonical mechanism, when introduced into the nucleus of G0 cells. This is because recruitment by the activation causing FXR1a-microRNA-protein complex (FXR1a-microRNP) is nuclear and requires shortened poly(A) tails to avoid repressive factors and canonical translation. When introduced into the cytoplasm, target mRNAs and microRNAs are directed toward repression rather than translation activation. Leukemic cell lines are difficult to transfect but can be routinely nucleofected-where in vitro transcribed mRNA reporters and microRNAs are introduced into the nucleus of G0 leukemic cells. Nucleofection of a microRNA target reporter and either cognate, targeting microRNA, or control microRNA, into the nucleus of G0 cells, enables analysis of translation activation by microRNAs in G0. We discuss a modified protocol that we developed for transfection of mRNAs along with microRNAs to test translation regulation by microRNAs in G0 leukemic cells.

The large-scale deployment of fish aggregation devices alters environmentally-based migratory behavior of skipjack tuna in the Western Pacific Ocean.

Fish aggregation devices (FADs) have been used extensively in the tuna purse seine fishery since the 1980s. This long-term modification of natural habitat has generated discussions as to whether FADs impact movement patterns of tuna species. We examined this question using data collected from the skipjack tuna (Katsuwonus pelamis) fishery. We used the longitudinal gravitational center of catch (G) to examine temporal variability in skipjack movement in the Western and Central Pacific Ocean, and related this to El Niño Southern Oscillation (ENSO) events. We found that in most cases G for free-swimming school sets changed with the onset of ENSO events, while G for floating-object-associated school sets remained relatively constant. This suggests that skipjack exhibit distinguishable behavioral strategies in response to ENSO events: they either react by moving long distances or they associate with floating objects. There has been no previous attempt to evaluate the interaction between FADs and the environmentally-determined movement of skipjack; this study shows evidence of an interaction, which should be considered when managing skipjack populations.