The RNA-binding protein CSDE1 is a key regulator of mRNA stability and translation in a broad spectrum of biological processes. We have previously shown that CSDE1 functions as an oncoprotein promoting invasion and metastasis in melanoma, whereas it behaves as a tumor suppressor promoting cellular senescence in squamous cell carcinoma. The reasons underlying these context-specific behaviors are unknown. To identify melanoma-specific vulnerabilities, we have compared CSDE1 protein isoforms and post-translational modifications in melanoma cells, keratinocytes, and melanocytic cells of different tumorigenic potential. By combining long-read Nanopore sequencing with two-dimensional gel electrophoresis and transcriptome analysis, we identify one major isoform expressed in melanoma cells and patient samples. This isoform is phosphorylated early during cellular transformation, correlating with changes in its subcellular localization. We provide extensive interactome analysis of mammalian CSDE1, showing increased interactions with ribosomes in melanoma cells compared to healthy melanocytes. Importantly, interactions of CSDE1 with the ribosome are promoted by CSDE1 phosphorylation. Our data uncover a specific feature of melanoma cells that could be harnessed for therapeutic intervention.

Identification of components of a noncanonical cytoplasmic polyadenylation machinery in Drosophila expands the diversity of RNA-binding proteins involved in poly(A) tail length control. Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative noncanonical, CPEB-independent complex in Drosophila , of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein cofactors in cytoplasmic polyadenylation. Using RIP-Seq analysis, we identify hundreds of potential Dicer-2 target transcripts, ∼60% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Complex analyses indicated that both factors form an RNA-independent complex with Dicer-2 and mediate interactions of Dicer-2 with Wispy. Functional poly(A)-test analyses showed that Twenty-four and Ataxin-2 are required for cytoplasmic polyadenylation of a subset of Dicer-2 targets. Our results reveal components of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis.

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.

Cold-inducible RNA binding protein (CIRBP) is a stress-responsive protein that promotes cancer development and inflammation. Critical to most CIRBP functions is its capacity to bind and posttranscriptionally modulate mRNA. However, a transcriptome-wide analysis of CIRBP mRNA targets in cancer has not yet been performed. Here, we use an ex vivo breast cancer model to identify CIRBP targets and mechanisms. We find that CIRBP transcript levels correlate with breast cancer subtype and are an indicator of luminal A/B prognosis. Accordingly, overexpression of CIRBP in nontumoral MCF-10A cells promotes cell growth and clonogenicity, while depletion of CIRBP from luminal A MCF-7 cells has opposite effects. We use RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) to identify a set of 204 high confident CIRBP targets in MCF-7 cells. About 10% of these showed complementary changes after CIRBP manipulation in MCF-10A and MCF-7 cells, and were highly interconnected with known breast cancer genes. To test the potential of CIRBP-mediated regulation of these targets in breast cancer development, we focused on Cystatin C (CST3), one of the most highly interconnected genes, encoding a protein that displays tumor suppressive capacities. CST3 depletion restored the effects of CIRBP depletion in MCF-7 cells, indicating that CIRBP functions, at least in part, by down-regulating CST3 levels. Our data provide a resource of CIRBP targets in breast cancer, and identify CST3 as a novel downstream mediator of CIRBP function.

Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. Here we identify Hrp48 as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. Our results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3′ UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.

Basic research in Drosophila melanogaster has benefited from a plethora of powerful genetics tools. Detailed biochemical analysis, however, has often been difficult due to the lack of in vitro systems that faithfully recapitulate the observations made in vivo. In the field of posttranscriptional regulation, the recent establishment of robust in vitro systems from embryo and ovary material has fueled the mechanistic understanding of a variety of processes. Here we describe protocols to obtain and use extracts from Drosophila embryos that are competent for cytoplasmic polyadenylation and translation of exogenously added transcripts.

Cytoplasmic polyadenylation is the process by which dormant, translationally inactive mRNAs become activated via the elongation of their poly(A) tails in the cytoplasm. This process is regulated by the conserved cytoplasmic polyadenylation element binding (CPEB) protein family. Recent studies have advanced our understanding of the molecular code that dictates the timing of CPEB-mediated poly(A) tail elongation and the extent of translational activation. In addition, evidence for CPEB-independent mechanisms has accumulated, and the breath of biological circumstances in which cytoplasmic polyadenylation plays a role has expanded. These observations underscore the versatility of CPEB as a translational regulator, and highlight the diversity of cytoplasmic polyadenylation mechanisms.

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation that requires two sequences in the 3' untranslated region (UTR) of vertebrate substrates: the polyadenylation hexanucleotide, and the cytoplasmic polyadenylation element (CPE). Using a cell-free Drosophila system, we show that these signals are not relevant for Toll polyadenylation but, instead, a "polyadenylation region" (PR) is necessary. Competition experiments indicate that PR-mediated polyadenylation is required for viability and is mechanistically distinct from the CPE/hexanucleotide-mediated process. These data indicate that Toll mRNA is polyadenylated by a noncanonical mechanism, and suggest that a novel machinery functions for cytoplasmic polyadenylation during Drosophila embryogenesis.

The inhibition of male-specific lethal 2 (msl-2) mRNA translation by the RNA-binding protein sex-lethal (SXL) is an essential regulatory step for X-chromosome dosage compensation in Drosophila melanogaster. The mammalian upstream of N-ras (UNR) protein has been implicated in the regulation of mRNA stability and internal ribosome entry site (IRES)-dependent mRNA translation. Here we have identified the Drosophila homolog of mammalian UNR as a cofactor required for SXL-mediated repression of msl-2 translation. UNR interacts with SXL, a female-specific protein. Although UNR is present in both male and female flies, binding of SXL to uridine-rich sequences in the 3' untranslated region (UTR) of msl-2 mRNA recruits UNR to adjacent regulatory sequences, thereby conferring a sex-specific function to UNR. These data identify a novel regulator of dosage compensation in Drosophila that acts coordinately with SXL in translational control.

This study examined the role of ceramide generated by exogenous sphingomyelinases (SMases) on transcription nuclear factor-kappa B (NF-kappa B) activation and apoptosis in human colon epithelial HT-29 cells. Exogenous neutral (N) and acidic (A) SMase activated NF-kappa B with different kinetics, accounting for the diverse pattern of DNA binding of NF-kappa B complexes activated by tumor necrosis factor-alpha (TNF). NSMase activated predominantly RelA/p52 and RelA/p50 dimers within 30 min, while ASMase activated the p50/p50 homodimer by 20 h. The predominant activation of RelA-containing kappa B complexes by TNF or NSMase paralleled the induction of interleukin-8. HT-29 cells were sensitive to ASMase and TNF but resistant to NSMase. However, the apoptotic potential of NSMase was masked by NF-kappa B, as its prior inactivation sensitized HT-29 cells to NSMase. Thus, the generation of ceramide by exogenous SMases participates differentially in inflammation and apoptosis.

BACKGROUNDCyclosporine (CsA) nephrotoxicity can be identified by functional changes and chronic renal damage. CsA-associated renal fibrosis has been related to the overproduction of transforming growth factor (TGF)-beta1, a fibrogenic cytokine. Mycophenolate mofetil (MMF) may allow CsA dose reduction without increasing the risk of rejection.METHODSWe studied the impact of CsA dose reduction in association with MMF on renal function and TGF-beta1, production in 16 long-term renal allograft recipients with suspected CsA nephrotoxicity. Two grams/day of MMF were introduced, and CsA dose was reduced to reach whole-blood levels between 40 and 60 ng/ml within 1 month. CsA dose and levels, renal function parameters, and platelet-poor plasma TGF-beta1 levels were evaluated before and 6 months thereafter.RESULTSMMF allowed a decrease in both the mean dose of CsA (3.8+/-1.35 vs. 2.2+/-0.73 mg/kg/day; P<0.01) and CsA levels (148+/-36 vs. 53+/-19 ng/ml; P<0.001). The reduction of CsA was associated with a decrement of serum creatinine levels (210+/-46 vs. 172+/-41 micromol/L; P<0.001) and an increase in both the glomerular filtration rate (32.9+/-12 vs. 39.1+/-14 ml/min/1.73 m2; P<0.02) and renal plasma flow (195+/-79 to 218.6+/-74.02 ml/min/1.73 m2; P<0.02). There was a reduction in plasma TGF-beta1 levels (4.6+/-4.2 vs. 2.0+/-1.4 ng/ml; P=0.003) and CsA levels correlated with TGF-beta1 (r=0.536, P=0.002). No rejection episodes occurred, and an improvement in both systolic (149+/-13 vs. 137+/-12 mmHg; P<0.01) and diastolic blood pressure (89+/-14 vs. 83+/-10 mmHg; P<0.04) were observed.CONCLUSIONSThese short-term results show that MMF introduction allows a CsA dose reduction, which improves renal function, reduces TGF-beta1 production, and improves the control of hypertension, without increasing the incidence of acute rejection.

Incubation of HT-29 M6 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces cell scattering, loss of cellular contacts and inactivation of E-cadherin. We have investigated the involvement of different protein kinase C (PK-C) isoforms in these processes using specific activators. Thymeleatoxin, a derivative of mezerein that activates conventional PK-Cs (cPK-Cs) but not novel PK-Cs (nPK-Cs), promoted effects that were similar to those of PMA, i.e. at concentrations of 200 nM it induced scattering of HT-29 M6 colonies, loss of homotypic contacts and dissociation of E-cadherin from the cytoskeleton. Among the isoforms activated by this compound, only cPK-C alpha was detected in HT-29 M6 cells by Western blot. The specificity of this compound with respect to the rest of the PK-C isoforms present in these cells was determined; thymeleatoxin induced, as did PMA, the translocation of cPK-C alpha from the cytosol to the membrane and the cytoskeleton, and its partial down-regulation, On the other hand, thymeleatoxin did not modify the cellular levels or localization of nPK-C epsilon or atypical PK-C zeta. 'In vitro' assays also showed that thymeleatoxin did not activate nPK-C epsilon at the concentrations added to the cell cultures. These results indicate that thymeleatoxin is selective for cPK-C alpha over nPK-C epsilon and show a role for the former enzyme in the regulation of cell-cell contacts and the inactivation of E-cadherin in HT-29 M6 cells.