Ribosomal Protein Translation Research Articles

MatK impacts differential chloroplast translation by limiting spliced tRNA-K(UUU) abundance.

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we used RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatK's preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for splice site selection, and we discovered alternative 5'-splice sites in three MatK target introns. The resulting alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Ribosome profiling analysis of the overexpressor revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

Exposure to 1-nitropyrene after weaning induces anxiety-like behavior partially by inhibiting steroid hormone synthesis in prefrontal cortex

1-Nitropyrene (1-NP) is a neurodevelopmental toxicant. This study was to evaluate the impact of exposure to 1-NP after weaning on anxiety-like behavior. Five-week-old mice were administered with 1-NP (0.1 or 1 mg/kg) daily for 4 weeks. Anxiety-like behaviour was measured using elevated-plus maze (EPM) and open field test (OFT). In EPM test, time spending in open arm and times entering open arm were reduced in 1-NP-treated mice. In OFT test, time spent in the center region and times entering the center region were diminished in 1-NP-treated mice. Prefrontal dendritic length and number of dendrite branches were decreased in 1-NP-treated mice. Prefrontal PSD95, an excitatory postsynaptic membrane protein, and gephyrin, an inhibitory postsynaptic membrane protein, were downregulated in 1-NP-treated mice. Further analysis showed that peripheral steroid hormones, including serum testosterone (T) and estradiol (E2), testicular T, and ovarian E2, were decreased in 1-NP-treated mice. Interestingly, T and E2 were diminished in 1-NP-treated prefrontal cortex. Prefrontal T and E2 synthases were diminished in 1-NP-treated mice. Mechanistically, GCN2-eIF2α, a critical pathway that regulates ribosomal protein translation, was activated in 1-NP-treated prefrontal cortex. These results indicate that exposure to 1-NP after weaning induces anxiety-like behaviour partially by inhibiting steroid hormone synthesis in prefrontal cortex.

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.

A Potential Link between hnRNP K and Cebpα: Implications for Ribosomal Dysfunction in Acute Myeloid Leukemia (AML)

Deletion of the long arm of chromosome 9, del(9q), is a recurrent genetic abnormality involving the haploinsufficient loss of the RNA binding protein hnRNPK and is often associated with poor treatment responses in AML. In addition to reduced hnRNP K expression, other critical proteins, not localized to this locus, are also aberrantly expressed in del9q AML. One protein of critical interest is CEBPα, a well-established myeloid differentiation transcription factor. Recently, CEBPα was discovered to have a novel role in ribosomal RNA (rRNA) synthesis. However, as opposed to CEBPα, hnRNP K's role in RNA stability, rRNA processing, and ribosomal functions is already firmly rooted. Collectively, these results suggest that del(9q) AML dysregulations in hnRNP K and CEBPα expression may have a multi-faceted influence on ribosomal biogenesis and protein translation. Previously, we used animal models and patient samples to demonstrate that Hnrnpk haploinsufficiency alters CEBPa expression, suggesting that reduced hnRNP K expression contributes to decreased CEBPα levels and activity. These findings led us to hypothesize that these distinct, yet interconnected proteins, play a cooperative role in regulating rRNA synthesis in two major ways. Firstly, hnRNP K directly governs rRNA processing and ribosomal loading. And secondly, via hnRNP K's mediated regulation of the nucleolar-localized isoform of CEBPα (p42 isoform), which was recently shown to enhance recruitment of RNA polymerase I to rRNA genes. To explore these relationships, we used AML patient samples, Hnrnpk haploinsufficient animal models, si- and sh- HNRNPK AML cell lines, and biochemical analyses. Initially, RPPA, CyTOF, and western blot analyses were used to investigate how reduced hnRNP K levels affect proteins involved in ribogenesis and protein translation. Here, we observed a direct correlation between hnRNP K expression and defects in proteins and pathways involved in protein translation, such as mTOR, phos-S6, and eEF- and eIF-family members, and CEBPa (log 10pAdj < .0001). Subsequently, we performed polysome assays that revealed 40S and 60S subunit formation was impaired in AML cell lines with reduced hnRNP K levels. Across multiple cell lines, reduced hnRNP K expression directly led to a significant increase in monosomes (unbound/inactive ribosomes; p < 0.01) and a significant decrease in polysome-bound RNA (p < 0.05) . Critically, HNRNPK haploinsufficiency and si- and sh- HNRNPK knock down experiments disrupted the abundance and processing of the 47S transcript (p < 0.05). To understand the impact that hnRNP K has on CEBPα in the context of rRNA biology, we performed co-immunoprecipitation of hnRNP K followed by RNA-seq (RIP-Seq) in human AML cell lines KG-1 and KG-1a. 1076 interacting RNAs were identified, which were strongly enriched in transcription factors implicated in myeloid biology, including CEBPA (log 2pAdj < .0001). Biochemical analyses of sequence-specific motifs conferred direct and functional interactions between hnRNP K and the 5‘ and 3‘ UTRs of the human CEBPA. These interactions support our in vivo findings that del9q AML and Hnrnpk haploinsufficiency lead to reduced CEBPA expression, specifically in the p42 isoform, potentially impacting its role in nucleolar localization and rRNA transcription regulation. Currently, we are evaluating the significance of these interactions at both the biological and translational levels. Using biochemical and cell-based assays, we are evaluating the direct effect that Hnrnpk haploinsufficiency has on rRNA abundance and on the processing of ITS products in the context of CEBPA. Through the use of viability screens, we are examining the efficacy of inhibitors of protein translation, translation initiation, and RNA Pol I on leukemic cells with 9q deletions of reduced hnRNP K expression. Altogether, these findings identify an uncharacterized relationship between del9qAML ( HNRNPK haploinsufficiency), CEBPα expression, and ribogenesis, providing valuable insight towards mechanisms driving this subtype of leukemia. Collectively, these data also suggest that del9q AML represents a novel and currently uncharacterized ribosomopathy.

In Vivo Investigation of the Effect of Dietary Acrylamide and Evaluation of Its Clinical Relevance in Colon Cancer.

Dietary exposure to acrylamide (AA) has been linked with carcinogenicity in the gastrointestinal (GI) tract. However, epidemiologic data on AA intake in relation to cancer risk are limited and contradictory, while the potential cancer-inducing molecular pathways following AA exposure remain elusive. In this study, we collected mechanistic information regarding the induction of carcinogenesis by dietary AA in the colon, using an established animal model. Male Balb/c mice received AA orally (0.1 mg/kg/day) daily for 4 weeks. RNA was extracted from colon tissue samples, followed by RNA sequencing. Comparative transcriptomic analysis between AA and mock-treated groups revealed a set of differentially expressed genes (DEGs) that were further processed using different databases through the STRING-DB portal, to reveal deregulated protein-protein interaction networks. We found that genes implicated in RNA metabolism, processing and formation of the ribosomal subunits and protein translation and metabolism are upregulated in AA-exposed colon tissue; these genes were also overexpressed in human colon adenocarcinoma samples and were negatively correlated with patient overall survival (OS), based on publicly available datasets. Further investigation of the potential role of these genes during the early stages of colon carcinogenesis may shed light into the underlying mechanisms induced by dietary AA exposure.

Dynamics of ribosome composition and ribosomal protein phosphorylation in immune signaling in Arabidopsis thaliana.

In plants, the detection of microbe-associated molecular patterns (MAMPs) induces primary innate immunity by the activation of mitogen-activated protein kinases (MAPKs). We show here that the MAMP-activated MAPK MPK6 not only modulates defense through transcriptional regulation but also via the ribosomal protein translation machinery. To understand the effects of MPK6 on ribosomes and their constituent ribosomal proteins (RPs), polysomes, monosomes and the phosphorylation status of the RPs, MAMP-treated WT and mpk6 mutant plants were analysed. MAMP-activation induced rapid changes in RP composition of monosomes, polysomes and in the 60S ribosomal subunit in an MPK6-specific manner. Phosphoproteome analysis showed that MAMP-activation of MPK6 regulates the phosphorylation status of the P-stalk ribosomal proteins by phosphorylation of RPP0 and the concomitant dephosphorylation of RPP1 and RPP2. These events coincide with a significant decrease in the abundance of ribosome-bound RPP0s, RPP1s and RPP3s in polysomes. The P-stalk is essential in regulating protein translation by recruiting elongation factors. Accordingly, we found that RPP0C mutant plants are compromised in basal resistance to Pseudomonas syringae infection. These data suggest that MAMP-induced defense also involves MPK6-induced regulation of P-stalk proteins, highlighting a new role of ribosomal regulation in plant innate immunity.

MYC is a clinically significant driver of mTOR inhibitor resistance in breast cancer.

Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies.

Cell-free production of fluorescent proteins for the discovery of novel ribosome-targeting antibiotics

Various issues including the overuse of antibiotics has led to the development of threatening multidrug-resistant bacterial strains urging development of novel anti-infectives. One quarter of current clinical phase III antibiotic drug candidates address ribosomal protein translation as a target. Here, we describe an effective cell-free in vitro screening system for inhibitors of bacterial ribosome activity with direct fluorescence read-out. Using ribosomal S30 extracts from Escherichia coli, Salmonella enterica, and Pseudomonas putida, the validity of this system is demonstrated by concentration-dependent inhibition of translation by a set of different classes of translation-targeting drugs. The single-compartment cell-free translation reaction is compatible with multi-well formats. Fluorophore formation of green fluorescent protein or monomeric NeonGreen occurs in an hour time frame without the need of adding reagents for secondary enzymatic detection saving handling time, and prohibiting false positives. As label-free readout, the dose response further allows for IC50 determination in the same setup. Together, we show that cell-free production of fluorescent proteins for the discovery of ribosome-targeting antibiotics is feasible and amenable to high-throughput applications.

Abstract A031: KRAS (G12C) mediated mRNA translation program

Abstract KRAS-dependent activation of mRNA translation signaling is a common feature in cancer however, the KRAS-specific translation landscape and functional proteome are not fully characterized in a genome-wide manner. We show that mutant KRAS drives specific mechanisms of translational control to support cancer growth. We have utilized the technology of ribosome footprinting combined with advanced sequencing methodology to define the translation landscape affected by a specific inhibitor of KRAS, sotorasib that targets G12C mutant KRAS activity. We show that acute treatment of sotorasib inhibits the translation of key oncogenic proteins including MYC and MYC targets. KRAS (G12C) inhibition profoundly affects the translation of ribosomal proteins and translation elongation factors. G12C mutant KRAS remarkably affects the translation of proteins involved in the ROS and DNA repair pathways in cancer. Moreover, these translational changes are mediated through specific RNA motifs and RNA binding proteins. In summary, our study defines the KRAS (G12C) specific regulation of translation and provides the mechanism of KRAS (G12C) mediated regulation of cancer proteome. Citation Format: Kamini Singh. KRAS (G12C) mediated mRNA translation program [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A031.

Integrating transcriptomic and metabolomic analysis to understand muscle qualities of red swamp crayfish (Procambarus clarkii) under transport stress

This study investigated the transport stress (crowding stress and duration) on the physicochemical properties, energy metabolism and antioxidant enzyme activities of the red swamp crayfish (Procambarus clarkii) tail muscle (CTM). Besides, transcriptomic and metabolomic were conducted to elucidate the possible mechanism of CTM alternations during transport stress. The survival rate of crayfish gradually decreased with the external crowding stress and crowding time increasing. The transport stress also led to the increased distance among muscle fibers, water mobility and energy consumption, and the decreased of water holding capacity (WHC), hardness of CTM. The hepatopancreas exhibited more sensitive to crowding stress than muscle. The multi-omics analysis revealed that transport stress could interfere the translation and protein folding functions of ribosomal proteins, fatty acid metabolism and degradation, physiological functions of mitochondria in CTM. This study could provide critical information to increase the understanding of the regulation mechanism of crayfish when subjected to transport stress.

Abstract 5331: Biological assessment of the virus-like drug conjugate AU-011 to specifically target a breadth of human cancer types

Abstract Introduction: AU-011 is a virus-like drug conjugate based on a human papillomavirus (HPV) derived virus-like particle (VLP) conjugated to a light-activated cytotoxic payload (IRDye700DX) currently in Phase II clinical trials for the treatment of primary choroidal melanoma. When activated by near-infrared light, AU-011 has a dual mechanism of action inducing rapid, tumor necrosis resulting in pro-immunogenic cell death and long-term anti-tumor immunity. HPV-derived VLPs bind a wide variety of tumor types via modified heparan sulfate proteoglycans (HSPG) found on the tumor surface. This study explores the breadth of tumor types that could benefit from a targeted treatment intervention with AU-011 and surveys genes and biological pathways that are positively and negatively associated with this targeting. Methods: AU-011 binding, cytotoxicity and HSPG specificity was surveyed in 127 tumor cell lines representing breast, cervical, CNS (glioblastoma, astrocytoma), colon, esophageal, gastric, hematopoietic, lung, liver, melanoma (cutaneous or choroidal), oropharyngeal, ovarian, pancreas, prostate, renal, urothelial and skin cancers, and sarcomas (osteosarcomas, mesotheliomas, retinoblastomas). Publicly available gene expression data for 101 of these cell lines was cross-referenced to identify gene signatures that correlated (either positively or negatively) with AU-011 binding and cytotoxicity. Results: AU-011 activity was observed for every tumor type examined, with some variation across several of the types. HSPG-specific binding was well-conserved across all tumor types tested except for most cells of lymphoid origin which are known to have HSPG deficiency. Collectively the tumor-derived cell lines exhibiting average binding EC50s < 100 pM were ocular cancers (choroidal melanomas and retinoblastomas) and solid tumors including urothelial, bone, breast, cervical, CNS, colon, cutaneous melanoma, esophageal, gastric, liver, lung, skin, oropharyngeal, ovarian, and renal. Urothelial, bone, breast, cervical, CNS, esophageal, gastric, liver, lung, melanoma, skin, ovarian, renal and retinoblastoma all exhibited AU-011 mediated cytotoxicity with an average potency of < 100pM. Correlative gene expression analysis demonstrated a strong association between AU-011 activity and genes involved in epithelial-to-mesenchymal transition, glycosaminoglycan biosynthesis/metabolism, and extracellular matrix interactions. Expression signatures for ribosomal activity and protein translation were negatively associated with AU-011 binding and activity. Conclusions: Collectively these data demonstrate the wide potential applicability of AU-011 to target a number of tumor types, particularly those derived from neural or epithelial lineages. Importantly, a large portion of these tumors are accessible making their AU-011 targeting clinically translatable. Citation Format: Rhonda C. Kines, Nathan R. Fons, Elisabet de los Pinos, John T. Schiller. Biological assessment of the virus-like drug conjugate AU-011 to specifically target a breadth of human cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5331.

Abstract 3247: Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I

Abstract Increased ribosome biogenesis is a hallmark of cancer and targeting this process with RNA polymerase I (Pol I) inhibitors is a promising strategy for cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme. This approach is effective across many cancer types. However, a heterogeneous response was observed in cancer cell lines emphasizing the need to increase knowledge of factors that underlie the response to this therapeutic strategy. To identify genes that modulate the response of cancer cells to BMH-21, we performed genome-wide CRISPR-Cas9-based positive selection screens in human colorectal carcinoma cells. These screens identified high-confidence hits accounting for BMH-21 drug resistance that included all key positive regulators of the mTORC1 complex. Given that p53 has been identified as a downstream effector of Pol I transcription stress, we conducted the screens in TP53 isogenic cells. Notably, the mTORC1 pathway hits were identified in all screens indicating that the resistance was p53 independent. These findings are particularly striking given that mTOR is a major driver of ribosome biogenesis and cellular translational programs. The findings were validated using chemical and genetic approaches. Torin-1, a catalytic mTOR inhibitor, was found to cause resistance to BMH-21. mTOR signaling pathway knockout and rescue cell lines were generated and tested for changes in the drug responses by growth, viability, colony formation and GFP competition assays. In each case, compromised mTOR activity led to resistance to BMH-21. However, compromised mTOR activity did not abrogate Pol I transcription inhibition by BMH-21. To assess impact on protein translation, we used polysome profiling. BMH-21 treatment was found to cause a severe ribosome biogenesis defect. Surprisingly, mTOR inactivation partially rescued the translation ability under the drug treatment suggesting that this translation is pivotal for cell survival. To uncover factors critical for the survival, we performed Ribo-seq and RNA-seq in the drug-treated BMH-21 sensitive and resistant cells. The profiling results revealed that mTOR inactivation led to elevated translation efficiency of mRNAs encoding ribosomal proteins. These findings suggest that mTOR inactivation evokes compensatory selective translation of ribosomal proteins under severe ribosome biogenesis defect caused by the Pol I inhibition. The findings indicate that mTOR inactivation regulates selective translation as means to bypass Pol I inhibition, and more generally, that maintenance of translational capacity strongly contributes to treatment resistance. These findings reveal an unexpected complication by mTOR inhibitory strategies as well as have implications on exploring drug combinations in cancer. Citation Format: Wenjun Fan, Hester Liu, Stephanie Pitts, Brittany Ford, Rajeshkumar NV, Marikki Laiho. Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3247.

Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome

Dysregulated protein synthesis is a core pathogenic mechanism in Fragile X Syndrome (FX). The mGluR Theory of FX predicts that pathological synaptic changes arise from the excessive translation of mRNAs downstream of mGlu1/5 activation. Here, we use a combination of CA1 pyramidal neuron-specific TRAP-seq and proteomics to identify the overtranslating mRNAs supporting exaggerated mGlu1/5 -induced long-term synaptic depression (mGluR-LTD) in the FX mouse model (Fmr1−/y). Our results identify a significant increase in the translation of ribosomal proteins (RPs) upon mGlu1/5 stimulation that coincides with a reduced translation of long mRNAs encoding synaptic proteins. These changes are mimicked and occluded in Fmr1−/y neurons. Inhibiting RP translation significantly impairs mGluR-LTD and prevents the length-dependent shift in the translating population. Together, these results suggest that pathological changes in FX result from a length-dependent alteration in the translating population that is supported by excessive RP translation.

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis.

Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discover that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which we name Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. We find that TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation.

Experimental evolution links post-transcriptional regulation to Leishmania fitness gain.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

Changes to the amino acid profile and proteome of the tropical freshwater microalga Chlorella sp. in response to copper stress

Contamination of freshwaters is increasing globally, with microalgae considered one of the most sensitive taxa to metal pollution. Here, we used 72 h bioassays to explore the biochemical effects of copper (Cu) on the amino acid (AA) profile and proteome of Chlorella sp. and advance our understanding of the molecular changes that occur in algal cells during exposure to environmentally realistic Cu concentrations. The Cu concentrations required to inhibit algal growth rate by 10% (EC10) and 50% (EC50) were 1.0 (0.7–1.2) µg L−1 and 2.0 (1.9–2.4) µg L−1, respectively. The AA profile of Chlorella sp. showed increases in glycine and decreases in isoleucine, leucine, valine, and arginine, with increasing Cu. Proteomic analysis revealed the modulation of several proteins involved in energy production pathways, including: photosynthesis, carbon fixation, glycolysis, and oxidative phosphorylation, which likely assists in meeting increased energy demands under Cu-stressed conditions. Copper exposure also caused up-regulation of cellular processes and signalling proteins, and the down-regulation of proteins related to ribosomal structure and protein translation. These changes in biomolecular pathways have direct effects on the AA profile and total protein content and provide an explanation for the observed changes in amino acid profile, cell growth and morphology. This study shows the complex mode of action of Cu on Chlorella under environmentally realistic Cu concentrations and highlights several potential biomarkers for future investigations.

Reuterin in the healthy gut microbiome suppresses colorectal cancer growth through altering redox balance.

Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restrictscolon tumor growth, increases tumor reactive oxygen species, and decreases protein translation invivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.

Early rRNA processing is a stress-dependent regulatory event whose inhibition maintains nucleolar integrity.

The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.

Flaccidoxide Induces Apoptosis Through Down-regulation of PI3K/AKT/mTOR/p70S6K Signaling in Human Bladder Cancer Cells.

Urothelial carcinoma (UC) is the most common type of genitourinary cancer with high incidence and mortality rates in men. In this study, we used the BFTC-905 and T24 bladder cancer cell lines as in vitro models to investigate the pathways involved in flaccidoxide-induced apoptosis. We utilized MTT assays, colony assays, wound-healing assays and fluorescence with TUNEL to confirm the cytotoxicity of flaccidoxide in bladder cancer cell lines. Potential proliferative and apoptotic molecular mechanisms were evaluated by western blotting. The expression of anti-apoptotic proteins Bcl-2 and phosphorylated Bad (p-Bad) was attenuated with an increasing flaccidoxide concentration, while the expression of proapoptotic proteins Bax, Bad, cleaved caspase-3, cleaved caspase-9 and cleaved PARP-1 was found increased. Additionally, phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) in the PI3K/AKT/mTOR pathway was reduced, leading to a reduction in the phosphorylation of downstream 70-kDa ribosomal protein S6 kinase 1 (p70S6K), S6 ribosomal protein (S6) and eukaryotic translation initiation factor 4B (eIF4B). However, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein phosphorylation was increased due to attenuation of the upstream phosphorylation of mTOR protein. Flaccidoxide-induced apoptosis in BFTC-905 and T24 cells is mediated by mitochondrial dysfunction and down-regulation the PI3K/AKT/mTOR/p70S6K signaling pathway.

Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus.

In addition to ribosomal protein synthesis and protein translation, ribosomal proteins also participate in tumorigenesis and tumor progression, immune responses, and viral replication. Here, we show that ribosomal protein L13 (RPL13) participates in the antiviral immune response induced by foot-and-mouth disease virus (FMDV), inhibiting FMDV replication. The overexpression of RPL13 promoted the induction and activation of the promoters of the nuclear factor-κB (NF-κB) and interferon-β (IFN-β) genes, and the expression and protein secretion of the antiviral factor IFN-β and proinflammatory cytokine interleukin-6 (IL-6). The knockdown of RPL13 had the opposite effects. We also found that the FMDV 3Cpro protease interacts with RPL13, and that its activity reduces the expression of RPL13, thus antagonizing the RPL13-mediated antiviral activity. This study extends our knowledge of the extraribosomal functions of ribosomal proteins and provides new scientific information on cellular antiviral defenses and virus-antagonizing mechanisms.