Global Rates Of Protein Synthesis Research Articles

Modulation of ribosomal subunit associations by eIF6 is critical for mitotic exit and cancer progression.

Moderating the pool of active ribosomal subunits is critical for maintaining global translation rates. A factor crucial for modulating the 60S ribosomal subunits is eukaryotic translation initiation factor 6. Release of eIF6 from 60S is essential to permit 60S interactions with 40S. Here, using the N106S mutant of eIF6, we show that disrupting eIF6 interaction with 60S leads to an increase in vacant 80S. It further highlights a dichotomy in the anti-association activity of eIF6 that is distinct from its role in 60S biogenesis and shows that the nucleolar localization of eIF6 is not dependent on uL14-BCCIP interactions. Limiting active ribosomal pools markedly deregulates translation especially in mitosis and leads to chromosome segregation defects, mitotic exit delays and mitotic catastrophe. Ribo-Seq analysis of the eIF6-N106S mutant shows a significant downregulation in the translation efficiencies of mitotic factors and specifically transcripts with long 3'UTRs. eIF6-N106S mutation also limits cancer invasion, and this role is correlated with the overexpression of eIF6 only in high-grade invasive cancers suggesting that deregulation of eIF6 is probably not an early event in cancers. Thus, this study highlights the segregation of eIF6 functions and its role in moderating 80S availability for mitotic translation and cancer progression.

Regulation of translation in response to iron deficiency in human cells

Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast Saccharomyces cerevisiae have shown that bulk translation is inhibited during adaptation to iron deficiency, which is consistent with its requirement for ribosome biogenesis and recycling. Although iron deficiency anemia is the most common human nutritional disorder, how iron modulates translation in mammals is poorly understood. Studies during erythropoiesis have shown that iron bioavailability is coordinated with globin synthesis via bulk translation regulation. However, little is known about the control of translation during iron limitation in other tissues. Here, we investigated how iron depletion affects protein synthesis in human osteosarcoma U-2 OS cells. By adding an extracellular iron chelator, we observed that iron deficiency limits cell proliferation, induces autophagy, and decreases the global rate of protein synthesis. Analysis of specific molecular markers indicates that the inhibition of bulk translation upon iron limitation occurs through the eukaryotic initiation factor eIF2α and mechanistic target of rapamycin (mTOR) pathways. In contrast to other environmental and nutritional stresses, iron depletion does not trigger the assembly of messenger ribonucleoprotein stress granules, which typically form upon polysome disassembly.

Abstract LB411: Investigating the dependency of androgen receptor-positive prostate cancer on class I myosins

Abstract Prostate adenocarcinoma is characterized by high expression and activity of the androgen receptor (AR). AR is a nuclear hormone receptor that stimulates anabolic cell metabolism. We hypothesized that the high activity of AR in prostate cancer creates unique dependencies on cellular machinery to support this high rate of anabolism. We observed that androgen treatment of patients with metastatic castration-resistant prostate cancer resulted in increased abundance of MYO1E, a class I myosin that facilitates intracellular vesicular trafficking, in tumor biopsies. Similarly, MYO1E protein increased in AR+ prostate cancer cell lines upon treatment with androgens. Notably, a specific small molecule inhibitor of MYO1E, pentachloropseudilin (PClP), induced cell death of AR-positive prostate cancer cell lines with an IC50 of 2-4uM, but was non-toxic to AR-negative prostate cancer cell lines. Expression of AR in these AR-negative lines conferred sensitivity to PClP, while deletion of AR in an AR-positive line conferred resistance to PClP, indicating that AR expression was required and sufficient for sensitivity of prostate cancer cell lines to PClP. Previous studies have suggested that AR globally increases protein synthesis and proteotoxic stress, and we asked whether this may create a dependency on MYO1E for secretion of protein-containing vesicles to manage proteotoxic stress. Initial experiments suggest that AR increases global rates of protein synthesis, as assessed by puromycin-incorporation assays, and inhibition of MYO1E increases phosphorylation of EIF2A, suggestive of activation of the integrated stress response. Further experiments will determine the essential function of MYO1E in AR-positive prostate cancer. Altogether our studies suggest that the class I myosin MYO1E is an essential protein in AR-positive cell lines and may represent a novel drug target for prostate adenocarcinoma. Citation Format: Anoushka Kadam, Shivani Kumar, Varsha Vakkala, Rajendra Kumar, Angelo M. De Marzo, Edward J. Pearce, Laura A. Sena. Investigating the dependency of androgen receptor-positive prostate cancer on class I myosins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB411.

Abstract A145: CBFβ supports global protein translation in osteosarcoma and may provide a new therapeutic target

Abstract Background: Core binding factor beta (CBFβ) is a binding partner to the RUNX family of DNA binding transcription factors (RUNX1-3) and acts as a transcriptional co-activator by allosterically increasing their affinity for DNA. RUNX2 is the master regulator of bone growth and differentiation, and its binding to CBFβ is of particular interest to osteosarcoma (OS) research. Notably, increased expression of each is correlated with poor disease-free and overall survival in numerous sarcomas. CBFβ’s transcriptional role is well-understood but recent research in breast cancer cells proposes a non-canonical role in the regulation of protein translation, influencing translation of hundreds of mRNA transcripts via binding with heterogenous nuclear ribonucleoprotein K (hnRNPK). The binding of CBFβ to hnRNPK or RUNX1 is reportedly mutually exclusive, suggesting possible interplay between the two roles. We hypothesize that CBFβ regulates protein translation in OS, participating in the translation of RUNX2 and other oncoproteins, promoting a malignant phenotype. Furthermore, we propose that disrupting its translational role may result in an antitumor effect. While CBFβ’s transcriptional activities are well-characterized, we endeavored to isolate its translational role and investigate its contribution to OS. Methods: CRISPR/Cas9 was used to generate a CBFβ knockout (KO) U2OS cell line, while site-directed mutagenesis of CBFβ and lentiviral transduction of U2OS CBFβ KO cells was used to generate cell lines expressing FLAG-tagged wild type (WT) mutant CBFβ. Western Blotting (WB) was used to assess changes in protein expression and subcellular localization while RUNX2 mRNA expression was measured via qPCR and RNAseq. CBFβ’s interacting partners were identified by Immunoprecipitation Mass Spectrometry (IP-MS). Cellular electro-thermal shift assay was used to evaluate influences of CBFβ mutations on RUNX2 and hnRNPK binding. The Click-IT system followed by WB and IP-MS were used to assess global protein translation and synthesis rates of specific proteins, respectively. Results: CBFβ and hnRNPK interact in U2OS cells and loss of CBFβ leads to a decrease in global protein translation. This results in decreased RUNX2 protein, but not mRNA, expression. Re-introduction of WT CBFβ caused RUNX2 protein levels to recover while expression of CBFβ with key mutations in the binding interface abrogated this effect. Importantly, we observed this was not due to direct protection of RUNX2 from proteasomal degradation by WT CBFβ. We identified a cohort of proteins that appear to be translationally regulated by CBFβ and investigated the impact of CBFβ mutations on its translational activities and RUNX2 binding. We also identified a list of putative CBFβ protein-protein interactions in human OS cells that are altered by these mutations. Conclusions: CBFβ appears to participate in protein translation in OS, regulating the expression of numerous oncoproteins including RUNX2. Key mutations in CBFβ designed to interrupt RUNX2 binding also affect this non-canonical activity of CBFβ. Citation Format: Nicholas A Oldberg, Dayn R Godinez, Luke A Wittenburg. CBFβ supports global protein translation in osteosarcoma and may provide a new therapeutic target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A145.

Trazodone rescues dysregulated synaptic and mitochondrial nascent proteomes in prion neurodegeneration.

The unfolded protein response (UPR) is rapidly gaining momentum as a therapeutic target for protein misfolding neurodegenerative diseases, in which its overactivation results in sustained translational repression leading to synapse loss and neurodegeneration. In mouse models of these disorders, from Alzheimer's to prion disease, modulation of the pathway-including by the licensed drug, trazodone-restores global protein synthesis rates with profound neuroprotective effects. However, the precise nature of the translational impairment, in particular the specific proteins affected in disease, and their response to therapeutic UPR modulation are poorly understood. We used non-canonical amino acid tagging (NCAT) to measure de novo protein synthesis in the brains of prion-diseased mice with and without trazodone treatment, in both whole hippocampus and cell-specifically. During disease the predominant nascent proteome changes occur in synaptic, cytoskeletal and mitochondrial proteins in both hippocampal neurons and astrocytes. Remarkably, trazodone treatment for just 2 weeks largely restored the whole disease nascent proteome in the hippocampus to that of healthy, uninfected mice, predominantly with recovery of proteins involved in synaptic and mitochondrial function. In parallel, trazodone treatment restored the disease-associated decline in synapses and mitochondria and their function to wild-type levels. In conclusion, this study increases our understanding of how translational repression contributes to neurodegeneration through synaptic and mitochondrial toxicity via depletion of key proteins essential for their function. Further, it provides new insights into the neuroprotective mechanisms of trazodone through reversal of this toxicity, relevant for the treatment of neurodegenerative diseases via translational modulation.

Gelation of cytoplasmic expanded CAG RNA repeats suppresses global protein synthesis.

RNA molecules with the expanded CAG repeat (eCAGr) may undergo sol-gel phase transitions, but the functional impact of RNA gelation is completely unknown. Here, we demonstrate that the eCAGr RNA may form cytoplasmic gel-like foci that are rapidly degraded by lysosomes. These RNA foci may significantly reduce the global protein synthesis rate, possibly by sequestering the translation elongation factor eEF2. Disrupting the eCAGr RNA gelation restored the global protein synthesis rate, whereas enhanced gelation exacerbated this phenotype. eEF2 puncta were significantly enhanced in brain slices from a knock-in mouse model and from patients with Huntington's disease, which is a CAG expansion disorder expressing eCAGr RNA. Finally, neuronal expression of the eCAGr RNA by adeno-associated virus injection caused significant behavioral deficits in mice. Our study demonstrates the existence of RNA gelation inside the cells and reveals its functional impact, providing insights into repeat expansion diseases and functional impacts of RNA phase transition.

Study on the protective effect of RNA-binding motif protein 3 in mild hypothermia oxygen-glucose deprivation/reoxygenation cell model

Mild hypothermia is proven neuroprotective in clinical practice. While hypothermia leads to the decrease of global protein synthesis rate, it upregulates a small subset of protein including RNA-binding motif protein 3 (RBM3). In this study, we treated mouse neuroblastoma cells (N2a) with mild hypothermia before oxygen-glucose deprivation/reoxygenation (OGD/R) and discovered the decrease of apoptosis rate, down-regulation of apoptosis-associated protein and enhancement of cell viability. Overexpression of RBM3 via plasmid exerted similar effect while silencing RBM3 by siRNAs partially reversed the protective effect exerted by mild hypothermia pretreatment. The protein level of Reticulon 3(RTN3), a downstream gene of RBM3, also increased after mild hypothermia pretreatment. Silencing RTN3 weakened the protective effect of mild hypothermia pretreatment or RBM3 overexpression. Also, the protein level of autophagy gene LC3B increased after OGD/R or RBM3 overexpression while silencing RTN3 decreased this trend. Furthermore, immunofluorescence observed enhanced fluorescence signal of LC3B and RTN3 as well as a large number of overlaps after RBM3 overexpressing. In conclusion, RBM3 plays a cellular protective role by regulating apoptosis and viability via its downstream gene RTN3 in the hypothermia OGD/R cell model and autophagy may participate in it.

Impact of silencing eEF2K expression on the malignant properties of chordoma.

Eukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. eEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer. In our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells. Silencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties. Our findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity.

Regulation of eIF4E guides a unique translational program to control erythroid maturation.

Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.

Targeting a Highly Deregulated eIF4F Translation Initiation Complex Sensitises IM-Resistant Cells to Tyrosine Kinase Inhibitors and Effectively Suppresses BCR-ABL1 Protein Expression

Background We previously reported that Abelson helper integration site-1 (AHI-1) is highly deregulated in chronic myeloid leukemia (CML) leukemic stem cells (LSCs) and mediates tyrosine kinase inhibitor (TKI) resistance. AHI-1 physically interacts with BCR-ABL1 and its SH3 domain interacts with BCR-ABL1 substrates, like dynamin-2, which regulate leukemic properties of CML LSCs. However, the molecular and biological roles of AHI-1 and its interacting partners in mediating TKI resistance remain largely unknown. Objective To investigate the molecular functions of AHI-1 and its SH3 domain in regulation of TKI resistance, using advanced antibody microarray analysis. Methods A high content antibody microarray was performed in BCR-ABL1+ cells co-transduced with wild-type (WT) AHI-1 or the deletion of AHI-1 SH3 domain (SH3Δ) with or without imatinib (IM). Changes in antibody signals for protein expression or phosphorylation were determined using limma and pathway enrichment analysis by g:Profiler. eIF4G1 genetic inhibition by lentiviral-mediated shRNA or pharmacological inhibition by SBI-756, was performed in TKI-resistant cells to assay translation initiation activity by proximity ligation assay (PLA), o-propargyl-puromycin (OPP), and polysome profiling. Results The antibody microarray analysis revealed that WT AHI-1 cells have the greatest number of changes in the phospho-proteome and proteome compared to BCR-ABL1+ cells and AHI-1 SH3Δ cells with and without IM. Pathway enrichment analysis identified that the targets with significantly increased differential antibody signal after IM treatment in WT AHI-1 cells were related to the regulation of translation initiation complex (p < 0.0001). Interestingly, our RNA-seq dataset analysis further identified several eukaryotic initiation factor 4F (eIF4F) complex members to be significantly increased in CD34+ CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex (2-fold, p = 0.001). WT AHI-1 cells also showed increased expression and phosphorylation of eIF4G1 (>2-fold) and eIF4B (>2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, and cyclin D3 (a downstream protein of eIF4F translational activity) as compared to BCR-ABL1+ cells, by immunoblotting. These results were similarly demonstrated in IM-resistant cells as compared to IM-sensitive cells (2-5-fold, p < 0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p < 0.0001) and increased TKI sensitivity in IM-resistant cells (p < 0.0021). These cells showed reduced levels of eIF4F complex formation by PLA assay (p = 0.013) and reduced protein expression of cyclin D3 (40%, p = 0.0036) and BCR-ABL1 (70%). Similarly, IM-resistant cells were more sensitive to SBI-756 treatment, an eIF4G1 inhibitor (50% reduction, p = 0.0001) than IM-sensitive cells (30% reduction); these effects were enhanced by a combination of SBI-756 and IM (80%, p < 0.02). Furthermore, SBI-756 treatment reduced PLA foci formation (p = 0.014) and global protein synthesis rates (p < 0.0001). Polysome profiling demonstrated that IM-resistant cells had higher levels of translation activity, which was inhibited by SBI-756 treatment. Most interestingly, SBI-756 treatment reduced protein expression of BCR-ABL1 (70%) and cyclin D3 (40%) in these cells. Conclusion We have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells. eIF4G1 inhibitor treatment sensitises IM-resistant cells to TKI and reduces BCR-ABL1 protein expression, providing potential treatment strategies to overcome TKI resistance.

The histone methyltransferase SETD2 negatively regulates cell size.

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

Structure, Activity and Function of the Dual Protein Lysine and Protein N-Terminal Methyltransferase METTL13.

METTL13 (also known as eEF1A-KNMT and FEAT) is a dual methyltransferase reported to target the N-terminus and Lys55 in the eukaryotic translation elongation factor 1 alpha (eEF1A). METTL13-mediated methylation of eEF1A has functional consequences related to translation dynamics and include altered rate of global protein synthesis and translation of specific codons. Aberrant regulation of METTL13 has been linked to several types of cancer but the precise mechanisms are not yet fully understood. In this article, the current literature related to the structure, activity, and function of METTL13 is systematically reviewed and put into context. The links between METTL13 and diseases, mainly different types of cancer, are also summarized. Finally, key challenges and opportunities for METTL13 research are pinpointed in a prospective outlook.

Cooperative methylation of human tRNA3Lys at positions A58 and U54 drives the early and late steps of HIV-1 replication.

Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N1-methyladenosine modification at tRNA position 58 (m1A58); however, the role of m1A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m1A in HIV-1 infection. We confirmed that tRNA3Lys m1A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m1A58-deficient tRNA3Lys instead of m1A58-modified tRNA3Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2′-O-dimethyluridine at U54 of tRNA3Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNALys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m1A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.

A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation.

Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.

Overexpression of ARHGAP30 suppresses growth of cervical cancer cells by downregulating ribosome biogenesis.

We aimed to identify whether Rho GTPase activating proteins (RhoGAPs) were downregulated in cervical cancers and might be targeted to reduce the growth of cervical cancer using the GEO database and immunohistochemical analysis to identified changes in transcription and protein levels. We analyzed their proliferation, clone formation ability, and their growth as subcutaneous tumors in mice. To detect ARHGAP30 localization in cells, immunofluorescence assays were conducted. Mass spectrometry combined with immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co‐immunoprecipitation assays. Western‐blot and q‐PCR were applied to analyze candidate binding proteins that were associated with ribosome biogenesis. Puromycin incorporation assay was used to detect the global protein synthesis rate. We identified that ARHGAP30 was the only downregulated RhoGAP and was related to the survival of cervical cancer patients. Overexpression of ARHGAP30 in cervical cancer cells inhibited cell proliferation and migration. ARHGAP30 immunoprecipitated proteins were enriched in the ribosome biogenesis process. ARHGAP30 was located in the nucleous and interacted with nucleolin (NCL). Overexpression of ARHGAP30 inhibited rRNA synthesis and global protein synthesis. ARHGAP30 overexpression induced the ubiquitination of NCL and decreased its protein level in Hela cells. The function of ARHGAP30 on cervical cancer cell ribosome biogenesis and proliferation was independent of its RhoGAP activity as assessed with a RhoGAP‐deficient plasmid of ARHGAP30R55A. Overall, the findings revealed that ARHGAP30 was frequently downregulated and associated with shorter survival of cervical cancer patients. ARHGAP30 may suppress growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of NCL.

Translational regulation in the brain by TDP-43 phase separation.

The in vivo physiological function of liquid-liquid phase separation (LLPS) that governs non-membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-deficient murine TDP-43. LLPS-deficient TDP-43 mice demonstrate impaired neuronal function and behavioral abnormalities specifically related to brain function. Brain neurons of these mice, however, did not show TDP-43 proteinopathy or neurodegeneration. Instead, the global rate of protein synthesis was found to be greatly enhanced by TDP-43 LLPS loss. Mechanistically, TDP-43 LLPS ablation increased its association with PABPC4, RPS6, RPL7, and other translational factors. The physical interactions between TDP-43 and translational factors relies on a motif, the deletion of which abolished the impact of LLPS-deficient TDP-43 on translation. Our findings show a specific physiological role for TDP-43 LLPS in the regulation of brain function and uncover an intriguing novel molecular mechanism of translational control by LLPS.

IRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation.

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

Assessing Protein Synthesis and Degradation Rates in Arabidopsis thaliana Using Amino Acid Analysis.

Plants continually synthesize and degrade proteins, for example, to adjust protein content during development or during adaptation to new environments. In order to estimate global protein synthesis and degradation rates in plants, we developed a relatively simple and inexpensive method using a combination of 13 CO2 labeling and mass spectrometry-based analyses. Arabidopsis thaliana plants are subjected to a 24-hr 13 CO2 pulse followed by a 4-day 12 CO2 chase. Soluble alanine and serine from total protein and glucose from cell wall material are analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and their 13 C enrichment (%) is estimated. The rate of protein synthesis during the 13 CO2 pulse experiment is defined as the rate of incorporation of labeled amino acids into proteins normalized by a correction factor for incomplete enrichment in free amino acid pools. The rate of protein degradation is estimated as the difference between the rate of protein synthesis and the relative growth rate calculated using the 13 C enrichment of glucose from cell wall material. Degradation rates are also estimated from the 12 CO2 pulse experiment. The following method description includes setting up and performing labeling experiments, preparation and measurement of samples, and calculation steps. In addition, an R script is provided for the calculations. 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Setting up the 13 CO2 labeling system and stable isotope labeling of Arabidopsis thaliana rosette leaves Basic Protocol 2: Extraction of soluble amino acids for GC-TOF-MS analysis Basic Protocol 3: Preparation of amino acids from total protein for GC-TOF-MS analysis Basic Protocol 4: Preparation of sugars from cell wall material for GC-TOF-MS analysis Basis Protocol 5: GC-TOF-MS analysis of 13 C-labeled samples and estimation of 13 C enrichment (%) Basis Protocol 6: Estimation of protein synthesis and degradation rates.

Maintenance of muscle mass in adult male mice is independent of testosterone.

Testosterone is considered a potent anabolic agent in skeletal muscle with a well-established role in adolescent growth and development in males. However, the role of testosterone in the regulation of skeletal muscle mass and function throughout the lifespan has yet to be fully established. While some studies suggest that testosterone is important for the maintenance of skeletal muscle mass, an understanding of the role this hormone plays in young, adult, and old males with normal and low serum testosterone levels is lacking. We investigated the role testosterone plays in the maintenance of muscle mass by examining the effect of orchiectomy-induced testosterone depletion in C57Bl6 male mice at ages ranging from early postnatal through old age (1.5-, 5-, 12-, and 24-month old mice). Following 28 days of testosterone depletion, we assessed mass and fiber cross-sectional-area (CSA) of the tibialis anterior, gastrocnemius, and quadriceps muscles. In addition, we measured global rates of protein synthesis and degradation using the SuNSET method, western blots, and enzyme activity assays. Twenty-eight days of testosterone depletion resulted in reduced muscle mass in the two youngest cohorts, but had no effect in the two oldest cohorts. Mean CSA decreased only in the youngest cohort and only in the tibialis anterior muscle. Testosterone depletion resulted in a general increase in proteasome activity at all ages. No change in protein synthesis was detected at the terminal time point. These data suggest that within physiological serum concentrations, testosterone may not be critical for the maintenance of muscle mass in mature male mice; however, in young mice testosterone is crucial for normal growth.

Examining Myc-Dependent Translation Changes in Cellular Homeostasis and Cancer.

A central component of Myc's role as a master coordinator of energy metabolism and biomass accumulation is its ability to increase the rate of protein synthesis, driving cell cycle progression, and proliferation. Importantly, Myc-induced alterations in both global and specific mRNA translation is a key determinant of Myc's oncogenic function. Herein, we provide five assays to enable researchers to measure global protein synthesis changes, to identify the translatome uniquely regulated by Myc and to investigate the mechanisms generating the tailored Myc translation network. Metabolic labeling of cells with 35S-containing methionine and cysteine in culture and O-propargyl-puromycin (OP-Puro) incorporation in vivo are presented as methods to measure the overall rate of global protein synthesis. Isolation of polysome-associated mRNAs followed by quantitative real-time PCR (qRT-PCR) and the toeprint assay enable the detection of altered translation of specific mRNAs and isoforms, and visualization of differential ribosomal engagement at start codons uniquely mediated by Myc activation, respectively. Finally, the translation initiation reporter assay is utilized to uncover the molecular mechanism mediating altered translation initiation of a specific mRNA. Together, the protocols detailed in this chapter can be used to illuminate how and to what degree Myc-dependent regulation of translation influences homeostatic cellular functions as well as tumorigenesis.