Autophagy is an essential catabolic process responsible for recycling of intracellular material and preserving cellular fidelity. Key to the autophagy pathway is the ubiquitin‐like conjugation system ...mediating lipidation of Atg8 proteins and their anchoring to autophagosomal membranes. While regulation of autophagy has been characterized at the level of transcription, protein interactions and post‐translational modifications, its translational regulation remains elusive. Here we describe a role for the conserved eukaryotic translation initiation factor 5A (eIF5A) in autophagy. Identified from a high‐throughput screen, we find that eIF5A is required for lipidation of LC3B and its paralogs and promotes autophagosome formation. This feature is evolutionarily conserved and results from the translation of the E2‐like ATG3 protein. Mechanistically, we identify an amino acid motif in ATG3 causing eIF5A dependency for its efficient translation. Our study identifies eIF5A as a key requirement for autophagosome formation and demonstrates the importance of translation in mediating efficient autophagy.
Synopsis
Eukaryotic translation initiation factor 5A (eIF5A) is required for lipidation of ATG8 proteins and autophagosome formation. This results from eIF5A‐mediated translation of the E2‐like ATG3 protein, which contains an amino acid motif causing eIF5A dependency for its efficient translation.
A high‐throughput screen identifies eIF5A as a translational requirement of autophagy.
eIF5A facilitates autophagosome formation by facilitating translation of ATG3.
A tri‐peptide motif in ATG3 causes eIF5A dependency for its efficient translation.
Induction of autophagy causes enhanced association of eIF5A with the ribosome.
eIF5A is required for lipidation of ATG8 proteins and autophagosome formation. This results from eIF5A‐mediated translation of the E2‐like ATG3 protein, which contains an amino acid motif causing eIF5A dependency for its efficient translation.
Abstract
Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four ...different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system.
The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of ...their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.
miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is ...particularly interesting considering its conserved location in the Hox cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a miR-10a knock out mouse. Here we show that, in the Apc(min) mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10(+/+) and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that Lpo upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.
MicroRNAs are small regulatory RNAs found in multicellular organisms where they post-transcriptionally regulate gene expression. In animals, microRNAs bind mRNAs via incomplete base pairings making ...the identification of microRNA targets inherently difficult. Here, we present a detailed method for experimental identification of microRNA targets based on affinity purification of tagged microRNAs associated with their targets.
micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either ...negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.
Prostate cancers show a slow progression from a local lesion (primary tumor) to a metastatic and hormone-resistant phenotype. After an initial step of hyperplasia, in a high percentage of cases a ...neoplastic transformation event occurs that, less frequently, is followed by epithelial to mesenchymal transition and invasion of healthy tissues (usually bones). MicroRNA-203 (miR-203) is a tumor suppressor microRNA often silenced in different malignancies. Here, we show that miR-203 is downregulated in clinical primary prostatic tumors compared to normal prostate tissue, and in metastatic prostate cancer cell lines compared to normal epithelial prostatic cells. Overexpression of miR-203 in brain or bone metastatic prostate cell lines (DU145 and PC3) is sufficient to induce a mesenchymal to epithelial transition with inhibition of cell proliferation, migration and invasiveness. We have identified CKAP2, LASP1, BIRC5, WASF1, ASAP1 and RUNX2 as new miR-203 direct target mRNAs involved in these events. Therefore, miR-203 could be a potentially new prognostic marker and therapeutic target in metastatic prostate cancer.
RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin ...leading to the synthesis of 2′,5′-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis.
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•RNase L activation leads to a ribotoxic stress response•The stress response requires the ribosome-associated MAP3K ZAKα•Phosphorylation of ZAKα, MAP2Ks, JNK, and p38α leads to induction of proinflammatory genes•The stress response triggered by RNase L culminates in apoptosis
In response to viral double-stranded RNA, OAS enzymes produce 2′,5′-oligoadenylates, known as 2-5As, which activate RNase L. J. Xi et al. show that RNase L activity triggers ZAKα to phosphorylate MAP2Ks that signal to JNK and p38α, leading to inflammatory signaling and apoptosis.
This article contrasts the intentions and outcomes of the publicly instigated and supported urban renewal of Copenhagen's Inner Vesterbro district. Apart from physically upgrading the decaying ...buildings, the municipality's aim was to include the inhabitants in the urban renewal process and, seemingly, to prevent the dislocation of people from the neighbourhood. However, due to ambiguous policies, the workings of the property market and the lack of sufficient deflecting mechanisms, middle-class inhabitants are now replacing the high concentration of socioeconomically vulnerable people that characterised Vesterbro before the urban renewal. This process may appear 'gentle', but it is nonetheless an example of how state and market interact to produce gentrification with 'traumatic' consequences for individuals and the city as a socially just space.
Impairment of protein translation can cause stalling and collision of ribosomes and is a signal for the activation of ribosomal surveillance and rescue pathways. Despite clear evidence that ribosome ...collision occurs stochastically at a cellular and organismal level, physiologically relevant sources of such aberrations are poorly understood. Here we show that a burst of the cellular signaling molecule nitric oxide (NO) reduces translational activity and causes ribosome collision in human cell lines. This is accompanied by activation of the ribotoxic stress response, resulting in ZAKα-mediated activation of p38 and JNK kinases. In addition, NO production is associated with ZNF598-mediated ubiquitination of the ribosomal protein RPS10 and GCN2-mediated activation of the integrated stress response, which are well-described responses to the collision of ribosomes. In sum, our work implicates a novel role of NO as an inducer of ribosome collision and activation of ribosomal surveillance mechanisms in human cells.
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