Specific Subset Of mRNAs Research Articles

Abstract C082: Decoding the KRAS-dependent proteome through the ribosomal lens

Abstract Our study addresses the gap in understanding the KRAS-specific translation landscape and functional proteome in cancer. We demonstrate that mutant KRAS drives distinct mechanisms of translational control to fuel cancer growth. By leveraging ribosome footprinting and advanced sequencing techniques, we investigate the impact of sotorasib, a targeted inhibitor of G12C mutant KRAS activity, on translation. Notably, KRAS (G12C) inhibition profoundly alters the translation of ribosomal proteins and translation elongation factors. Additionally, we reveal that KRAS inhibition resulted in ribosome stalling on a specific subset of mRNAs. Acute treatment with sotorasib hampers the translation of crucial oncogenic proteins, including MYC, as well as proteins involved in metabolism. These translational changes are facilitated by specific RNA motifs and RNA binding proteins that respond to KRAS activity. In essence, our study delineates the KRAS (G12C)-specific regulation of translation and elucidates the mechanism underlying KRAS (G12C)-mediated control over the cancer proteome. Citation Format: Kamini Singh, Trang Uyen Nguyen, Eric Nels Pederson, Vinay Kumar, Xiaoling Yun, Zhengqing Ouyang. Decoding the KRAS-dependent proteome through the ribosomal lens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C082.

RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-β to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-β has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-β in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-β remain largely unknown. Using RNA interactome capture, we identified global TGF-β driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. Characterization of TGF-β-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-β at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-β drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. Together, we show that RBPs play a key role in the endothelial response to TGF-β stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-β profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Customization of the translational complex regulates mRNA-specific translation to control CNS regeneration

In the adult mammalian central nervous system (CNS), axons fail to regenerate spontaneously after injury because of a combination of extrinsic and intrinsic factors. Despite recent advances targeting the intrinsic regenerative properties of adult neurons, the molecular mechanisms underlying axon regeneration are not fully understood. Here, we uncover a regulatory mechanism that controls the expression of key proteins involved in regeneration at the translational level. Our results show that mRNA-specific translation is critical for promoting axon regeneration. Indeed, we demonstrate that specific ribosome-interacting proteins, such as the protein Huntingtin (HTT), selectively control the translation of a specific subset of mRNAs. Moreover, modulating the expression of these translationally regulated mRNAs is crucial for promoting axon regeneration. Altogether, our findings highlight that selective translation through the customization of the translational complex is a key mechanism of axon regeneration with major implications in the development of therapeutic strategies for CNS repair.

A Ribosomal Protein Homolog Governs Gene Expression and Virulence in a Bacterial Pathogen.

ABSTRACTThe molecular machine necessary for protein synthesis, the ribosome, is generally considered constitutively functioning and lacking any inherent regulatory capacity. Yet ribosomes are commonly heterogeneous in composition and the impact of ribosome heterogeneity on translation is not well understood. Here, we determined that changes in ribosome protein composition govern gene expression in the intracellular bacterial pathogen Francisella tularensis. F. tularensis encodes three distinct homologs for bS21, a ribosomal protein involved in translation initiation, and analysis of purified F. tularensis ribosomes revealed they are heterogeneous with respect to bS21. The loss of one homolog, bS21-2, resulted in significant changes to the cellular proteome unlinked to changes in the transcriptome. Among the reduced proteins were components of the type VI secretion system (T6SS), an essential virulence factor encoded by the Francisella Pathogenicity Island. Furthermore, loss of bS21-2 led to an intramacrophage growth defect. Although multiple bS21 homologs complemented the loss of bS21-2 with respect to T6SS protein abundance, bS21-2 was uniquely necessary for robust intramacrophage growth, suggesting bS21-2 modulates additional virulence gene(s) distinct from the T6SS. Our results indicate that ribosome composition in F. tularensis, either directly or indirectly, posttranscriptionally modulates gene expression and virulence. Our findings are consistent with a model in which bS21 homologs function as posttranscriptional regulators, allowing preferential translation of specific subsets of mRNAs, likely at the stage of translation initiation. This work also raises the possibility that bS21 in other organisms may function similarly and that ribosome heterogeneity may permit many bacteria to posttranscriptionally regulate gene expression.IMPORTANCE While bacterial ribosomes are commonly heterogeneous in composition (e.g., incorporating different homologs for a ribosomal protein), how heterogeneity impacts translation is unclear. We found that the intracellular human pathogen Francisella tularensis has heterogeneous ribosomes, incorporating one of three homologs for ribosomal protein bS21. Furthermore, one bS21 homolog posttranscriptionally governs the expression of the F. tularensis type VI secretion system, an essential virulence factor. This bS21 homolog is also uniquely important for robust intracellular growth. Our data support a model in which bS21 heterogeneity leads to modulation of translation, providing another source of posttranscriptional gene regulation. Regulation of translation by bS21, or other sources of ribosomal heterogeneity, may be a conserved mechanism to control gene expression across the bacterial phylogeny.

Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation

BackgroundVertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3′ UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date.ResultsBased on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2–4 are regulated by multiple proline-directed phosphorylations that control their liquid–liquid phase separation. CPEB2–4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs.ConclusionsAltogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Receptor-Ribosome Coupling: A Link Between Extrinsic Signals and mRNA Translation in Neuronal Compartments.

Axons receive extracellular signals that help to guide growth and synapse formation during development and to maintain neuronal function and survival during maturity. These signals relay information via cell surface receptors that can initiate local intracellular signaling at the site of binding, including local messenger RNA (mRNA) translation. Direct coupling of translational machinery to receptors provides an attractive way to activate this local mRNA translation and change the local proteome with high spatiotemporal resolution. Here, we first discuss the increasing evidence that different external stimuli trigger translation of specific subsets of mRNAs in axons via receptors and thus play a prominent role in various processes in both developing and mature neurons. We then discuss the receptor-mediated molecular mechanisms that regulate local mRNA translation with a focus on direct receptor-ribosome coupling. We advance the idea that receptor-ribosome coupling provides several advantages over other translational regulation mechanisms and is a common mechanism in cell communication.

Abstract 2444: Do all 4 NCL RNA Binding Domains play a role in mRNA target specificity? An in silico study to decipher NCL-mRNA interactions

Abstract RNA binding proteins (RBPs) are post-transcriptional regulators that associate with RNA molecules through one or more of their RNA-binding domains (RBDs). The dysregulation of RBPs, and the RNA metabolism pathways they intersect, are known drivers of tumorigenesis. Nucleolin (NCL) is a stress responsive RBP with four RBDs that directly impacts gene expression by altering rRNA processing, RNA localization, miRNA biogenesis, mRNA decay, and translation. NCL is known to interact with both the 3' and 5' untranslated regions (UTR) and coding regions of target mRNAs to either modulate their stability or translation via recognition of AU-rich or G-rich elements in the RNA. However, the consequences of NCL-mRNA interactions are target-specific as well as context and location dependent. While NCL interacts with both 3' and 5' UTR of TP53 mRNA and suppresses its translation, interactions of NCL with 5' UTR of an anti-apoptotic gene, BCL2, stabilizes this transcript. NCL-mediated enhanced biogenesis of tumorigenic mir-15a and mir-16, on the other hand, results in the translational repression of BCL2. Because NCL exerts its functions in such a complex manner, elucidation of its roles in tumorigenic pathways is paramount in the development of therapeutical applications targeting NCL-RNA interactions. Mechanisms driving NCL-mRNA interactions are currently unknown and this knowledge gap is further compounded by the incomplete nature of the current structural information for NCL and its mRNA targets. In this study, we aim to elucidate NCL RBD-mRNA interactions using computational tools. We investigated interactions of NCL with a specific subset of mRNAs that are implicated in breast cancer progression. We have previously generated and validated in silico models for all the four NCL-RBDs (1-4) in various combinations. Using several secondary and tertiary structure prediction programs along with structure refinement programs, we have generated 3D models of TP53, BCL2, CSF-1, and GADD45α mRNA UTR molecules. Our docking analyses suggests that RBD1-2 and RBD3-4 collaborate in most NCL-mRNA interactions using RNA binding motifs (RNP1 & RNP2) on NCL. We further identify additional contact points in the bridging linker regions between the RBDs that might be crucial for the structural stability of these complexes. This study provides details previously not reported in the literature and provides a comprehensive map of NCL RBD-mRNA interactions that have direct pathophysiological relevance in breast cancer. Altered levels of NCL expression are linked with poor prognosis in breast cancers and targeting NCL results in a better survival outcome. Understanding how NCL associates with its mRNA targets will reveal binding pockets and hence potential sites for drug interventions in addition to characterizing the complex nature of NCL-mRNA interactions/specificity in multiple cancer types. Citation Format: Avdar San, Anjana Saxena, Shaneen Singh. Do all 4 NCL RNA Binding Domains play a role in mRNA target specificity? An in silico study to decipher NCL-mRNA interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2444.

Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a "comPARE" (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.

Analysis of the TORC1 interactome reveals a spatially distinct function of TORC1 in mRNP complexes.

The target of rapamycin complex 1 (TORC1) is mainly localized to the vacuolar membrane and regulates eukaryotic cell growth in response to nutrient availability. To obtain deeper insights into the functional roles of TORC1, we performed a genome-wide analysis of the TORC1 interactome in yeast using the bimolecular fluorescence complementation (BiFC) assay. We found that while most of the BiFC signals are observed at the vacuolar membrane, a fraction of them are detected at cytoplasmic messenger ribonucleoprotein (mRNP) granules. Moreover, mRNA-binding proteins are enriched in the TORC1 interactome, suggesting a functional relationship between TORC1 and mRNA metabolism. We show that a portion of TORC1 is consistently associated with mRNP complexes and interacts with a specific subset of mRNAs. We also demonstrate that TORC1 directly targets a translational repressor Scd6 and that the activity of Scd6 is inhibited by TORC1-dependent phosphorylation. Collectively, our data suggest that TORC1 plays a novel role in posttranscriptional regulation by controlling the activity of Scd6.

5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

Ribosome heterogeneity in stem cells and development.

Translation control is critical to regulate protein expression. By directly adjusting protein levels, cells can quickly respond to dynamic transitions during stem cell differentiation and embryonic development. Ribosomes are multisubunit cellular assemblies that mediate translation. Previously seen as invariant machines with the same composition of components in all conditions, recent studies indicate that ribosomes are heterogeneous and that different ribosome types can preferentially translate specific subsets of mRNAs. Such heterogeneity and specialized translation functions are very important in stem cells and development, as they allow cells to quickly respond to stimuli through direct changes of protein abundance. In this review, we discuss ribosome heterogeneity that arises from multiple features of rRNAs, including rRNA variants and rRNA modifications, and ribosomal proteins, including their stoichiometry, compositions, paralogues, and posttranslational modifications. We also discuss alterations of ribosome-associated proteins (RAPs), with a particular focus on their consequent specialized translational control in stem cells and development.

Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis

Emerging evidence indicates that heterogeneity in ribosome composition can give rise to specialized functions. Until now, research mainly focused on differences in core ribosomal proteins and associated factors. The effect of posttranslational modifications has not been studied systematically. Analyzing ribosome heterogeneity is challenging because individual proteins can be part of different subcomplexes (40S, 60S, 80S, and polysomes). Here we develop polysome proteome profiling to obtain unbiased proteomic maps across ribosomal subcomplexes. Our method combines extensive fractionation by sucrose gradient centrifugation with quantitative mass spectrometry. The high resolution of the profiles allows us to assign proteins to specific subcomplexes. Phosphoproteomics on the fractions reveals that phosphorylation of serine 38 in RPL12/uL11, a known mitotic CDK1 substrate, is strongly depleted in polysomes. Follow-up experiments confirm that RPL12/uL11 phosphorylation regulates the translation of specific subsets of mRNAs during mitosis. Together, our results show that posttranslational modification of ribosomal proteins can regulate translation.

MicroRNAs in prostate cancer: From function to biomarker discovery.

MicroRNAs (miRNAs) are a small functional non-coding RNAs that post-transcriptionally regulate gene expression through mRNA degradation or translational repression. miRNAs are key regulatory components of various cellular networks. Current evidence support that multiple mammalian genome-encoded miRNAs impact the cellular biology, including proliferation, apoptosis, differentiation, and tumorigenesis, by targeting specific subsets of mRNAs. This minireview is focused on the current themes underlying the interactions between miRNAs and their mRNA targets and pathways in prostate tumorigenesis and progression, and their potential clinical utility as biomarkers for prostate cancer. Impact statement The primary goal of this article was to review recent literature on miRNA biogenesis and further elaborate on the identity of newly discovered miRNAs and their potential functional significance in the complex biological network associated with prostate tumorigenesis and disease progression and as biomarkers for prostate cancer.

Trans‐acting translational regulatory RNA binding proteins

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans‐acting regulatory RNA‐binding proteins (RBPs) are necessary to provide mRNA‐specific translation, and these interact with 5′ and 3′ untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans‐acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans‐acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans‐acting ribosomal proteins in directing the translation of specific mRNAs.This article is categorized under: 1RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes2Translation > Translation Regulation3Translation > Translation Mechanisms

Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum.

Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum.

Mechanistic aspects of mammalian cell size control.

Size distribution in a group of differentiated cells often falls into a constant range. However, invitro and invivo studies have shown that cells can temporarily change their size in response to their surrounding environment and the stimuli they receive. Thus, there must be a mechanism that normally keeps cell size constant while allowing a shift to an alternative size when necessary. To investigate the molecular basis of mammalian cell size control, we conducted a genetic screen in a human T cell line to identify genes involved in cell size regulation. A prime candidate emerging from this screen increases cell size when it is overexpressed but reduces cell size when subjected to siRNA knockdown. Several lines of evidence indicate that the product of this gene, which we called "Largen", regulates mRNA translation in a manner associated with the upregulation of a specific subset of mRNAs, many of which affect mitochondrial function. In fact, cells overexpressing Largen increase both mitochondrial mass and activity, enhancing ATP production. These invitro observations have been replicated invivo using transgenic mouse models. With a focus on these findings, we discuss the possible contribution of mitochondria to the control of mammalian cell size.

IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories.

RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurological disease. Developing axons provide a model well suited to the study of RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing mouse brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal that it is required for normal axon pathfinding during vertebrate development.

Abstract PR07: Myc-dependent translation makes an impact: Tailor-made protein expression for cancer development

Abstract Our research delineates how the “cancer translatome” can reprogram gene expression at the post-transcriptional level, by controlling the translation of specific subsets of mRNAs during distinct steps of cancer progression. Here, we have genetically engineered a novel mouse model in which two of the most commonly perturbed oncogenes, MYC and KRAS, are stochastically activated in a small number of hepatocytes resulting in a dramatic increase in metastatic tumors, recapitulating the process of liver tumor development in human cancers. Therefore, we have used this model to detail the evolution of gene expression from primary tumor initiation to metastasis at both the transcriptional and translational levels. We used a genome-wide ribosome profiling approach to assess changes in gene expression at the earliest possible time point after oncogenic activation to metastasis formation in order to develop the first functional comprehensive network of widespread changes in translational regulation that occur during the course of tumor development. We characterized networks of translational regulation unique to each individual oncogene, as well as networks that are synergistically regulated upon activation of both oncogenes. For example, we have identified common networks that are translationally induced upon activation of either MYC or KRAS (e.g. protein synthesis, energy metabolism). We discovered that each oncogene also regulates the translation of unique mRNA networks. MYC induces the translation of genes involved in cell proliferation, mRNA splicing, and lipid metabolism, while KRAS specifically induces the translation of stress response genes, especially the pathway that responds to hypoxia. In addition to the additive effects of regulating their cognate mRNA networks, the combination of MYC and KRAS activation in the same cells can also induce a synergistic effect on the translation of new networks including genes involved in integrin signaling, which may partially explain the increased metastasis observed in this mouse model. Additionally, we uncovered independent regulatory mechanisms that may allow for temporal regulation of discrete oncogenic processes, either at the level of transcription or translation, required at different times during the course of tumor development. Understanding the mechanisms and specific targets of translational regulation downstream of MYC and KRAS activation will provide a novel platform from which to develop therapeutic strategies targeting cancers possessing two of the most common and currently “undruggable” oncogenic lesions. Citation Format: Craig Stumpf, Tom Cunningham, Davide Ruggero. Myc-dependent translation makes an impact: Tailor-made protein expression for cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR07.

Eukaryotic translation elongation factor-1 alpha is associated with a specific subset of mRNAs in Trypanosoma cruzi.

BackgroundRegulation of gene expression in trypanosomatids is mainly posttranscriptional. Tight regulation of mRNA stability and access to polysomes allows Trypanosoma cruzi to adapt to different environmental conditions during its life cycle. Posttranscriptional regulation requires association between mRNAs and specific proteins to form mRNP complexes. Proteins that lack a canonical RNA-binding domain, such as eukaryotic elongation factor-1α (EF-1α), may also associate with mRNPs. EF-1α is conserved in many organisms, and it plays roles in many cellular processes other than translation, including RNA transport, the cell cycle, and apoptosis.ResultsIn a previous study, EF-1α was found associated with mRNP-forming mRNAs in polysome-free fractions both in epimastigotes growing under normal conditions and in nutritionally stressed parasites. This finding suggested the possibility that EF-1α has a non-canonical function. Thus, we investigated the dynamics of EF-1α in association with T. cruzi epimastigote mRNAs under normal and stressed nutritional conditions. EF-1α is expressed throughout the parasite life cycle, but it shows a slight decrease in protein levels in the metacyclic trypomastigote form. The protein is cytoplasmically localized with a granular pattern in all forms analyzed. Following puromycin treatment, EF-1α migrated with the heaviest gradient fractions in a sucrose polysome profile, indicating that its association with large protein complexes was independent of the translation machinery. We next characterized the EF-1α-associated mRNAs in unstressed and stressed epimastigotes. We observed that specific subsets of mRNAs were associated with EF-1α-mRNPs in unstressed or stressed epimastigotes. Some mRNAs were identified in both physiological conditions, whereas others were condition-specific. Gene ontology analysis identified enrichment of gene sets involved in single-organism metabolic processes, amino acid metabolic processes, ATP and metal ion binding, glycolysis, glutamine metabolic processes, and cobalt and iron ion binding.ConclusionThese results indicate that in T. cruzi, as in other eukaryotes, EF-1α may play a non-canonical cellular role. We observed the enrichment of functionally related transcripts bound to EF-1α in normal growth conditions as well as in nutritionally stressed cell indicating a potential role of EF-1α mRNP in stress response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0436-2) contains supplementary material, which is available to authorized users.

Distinct inhibitory effects on mTOR signaling by ethanol and INK128 in diffuse large B-cell lymphoma.

BackgroundThe mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH’s modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL).ResultsTreatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis.ConclusionGiven that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128’s complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0091-0) contains supplementary material, which is available to authorized users.