scholarly journals 5’ UTR recruitment of eIF4GI or DAP5 drives cap-independent translation for a subset of human mRNAs

2018 ◽  
Author(s):  
Solomon A. Haizel ◽  
Usha Bhardwaj ◽  
Ruben L. Gonzalez ◽  
Somdeb Mitra ◽  
Dixie J. Goss
Keyword(s):  
Tumor Hypoxia ◽  
Mrna Translation ◽  
Luciferase Reporter ◽  
Binding Studies ◽  
Mechanistic Insight ◽  
Specific Subset ◽  
Independent Mechanism ◽  
Insight Into ◽  
Independent Translation

AbstractDuring unfavorable human cellular conditions (e.g., tumor hypoxia, viral infection, etc.), canonical, cap-dependent mRNA translation is suppressed. Nonetheless, a subset of physiologically important mRNAs (e.g., HIF-1α, FGF-9, and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eIF4G and its homolog, death associated protein 5 (DAP5), are elevated. Using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, here we demonstrate that eIF4GI and DAP5 specifically bind to the 5’ UTRs of these cap-independently translated mRNAs. Surprisingly, we find 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 show that eIF4GI and/or DAP5 are critical for recruitment of a specific subset of mRNAs to the ribosome and provide mechanistic insight into their cap-independent translation.

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

2020 ◽  
Vol 295 (33) ◽  
pp. 11693-11706 ◽  
Author(s):  
Solomon A. Haizel ◽  
Usha Bhardwaj ◽  
Ruben L. Gonzalez ◽  
Somdeb Mitra ◽  
Dixie J. Goss
Keyword(s):  
Translation Initiation ◽  
Tumor Hypoxia ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Binding Studies ◽  
Specific Subset ◽  
Independent Translation

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.

scholarly journals Cap-independent mRNA translation is upregulated in long-lived endocrine mutant mice

2019 ◽  
Vol 63 (2) ◽  
pp. 123-138 ◽  
Author(s):  
Ulas Ozkurede ◽  
Rishabh Kala ◽  
Cameron Johnson ◽  
Ziqian Shen ◽  
Richard A Miller ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Growth Hormone Receptor ◽  
Protein A ◽  
Mrna Translation ◽  
Mutant Mice ◽  
Mrna Levels ◽  
Healthy Longevity ◽  
Independent Mechanism ◽  
Independent Translation

It has been hypothesized that transcriptional changes associated with lower mTORC1 activity in mice with reduced levels of growth hormone and insulin-like growth factor 1 are responsible for the longer healthy lifespan of these mutant mice. Cell lines and tissues from these mice show alterations in the levels of many proteins that cannot be explained by corresponding changes in mRNAs. Such post-transcriptional modulation may be the result of preferential mRNA translation by the cap-independent translation of mRNA bearing the N6-methyl-adenosine (m6A) modification. The long-lived endocrine mutants – Snell dwarf, growth hormone receptor deletion and pregnancy-associated plasma protein-A knockout – all show increases in the N6-adenosine-methyltransferases (METTL3/14) that catalyze 6-methylation of adenosine (m6A) in the 5′ UTR region of select mRNAs. In addition, these mice have elevated levels of YTH domain-containing protein 1 (YTHDF1), which recognizes m6A and promotes translation by a cap-independent mechanism. Consistently, multiple proteins that can be translated by the cap-independent mechanism are found to increase in these mice, including DNA repair and mitochondrial stress response proteins, without changes in corresponding mRNA levels. Lastly, a drug that augments cap-independent translation by inhibition of cap-dependent pathways (4EGI-1) was found to elevate levels of the same set of proteins and able to render cells resistant to several forms of in vitro stress. Augmented translation by cap-independent pathways facilitated by m6A modifications may contribute to the stress resistance and increased healthy longevity of mice with diminished GH and IGF-1 signals.

scholarly journals SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

2021 ◽  
Author(s):  
Scott B Biering ◽  
Francielle Tramontini Gomes de Sousa ◽  
Laurentia V. Tjang ◽  
Felix Pahmeier ◽  
Richard Ruan ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Barrier Dysfunction ◽  
Vascular Leak ◽  
Endothelial Barrier Dysfunction ◽  
Mechanistic Insight ◽  
Starting Point ◽  
Signaling Axis ◽  
Insight Into

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

scholarly journals Mechanistic insight into DsyB/DSYB, key enzymes in marine dimethylsulfoniopropionate synthesis

2021 ◽  
Author(s):  
Jonathan Todd ◽  
Chun-Yang Li ◽  
Jason Crack ◽  
Simone Newton-Payne ◽  
Andrew Murphy ◽  
...  
Keyword(s):  
Marine Algae ◽  
Nucleophilic Attack ◽  
Mechanistic Insight ◽  
Signalling Molecule ◽  
Major Nutrient ◽  
Methyltransferase Gene ◽  
Key Enzyme ◽  
Algae And Bacteria ◽  
Insight Into

Abstract Marine algae and bacteria produce eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth’s surface oceans every year. DMSP is an anti-stress compound and, once released into the environment, a major nutrient, signalling molecule and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria. The S-directed S-adenosylmethionine-dependent methyltransferase (SAM-MT) 4-methylthio-2-hydroxybutyrate (MTHB) S-methyltransferase, encoded by the dsyB/DSYB gene, is the key enzyme of this pathway, generating S-adenosylhomocysteine (SAH) and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB). dsyB/DSYB, present in most DMSP-producing bacteria and haptophyte and dinoflagellate algae with the highest known DMSP concentrations, is shown to be far more abundant and transcribed in marine environments than any other known DMSP synthesis pathway S-methyltransferase gene. Furthermore, we demonstrate in vitro activity of the bacterial DsyB enzyme from Nisaea denitrificans, and provide its crystal structure in complex with SAM and SAH-MTHB, which together provide the first mechanistic insights into a DMSP synthesis enzyme. Structural and mutational analyses imply that DsyB adopts a novel mechanism, distinct from any previously reported SAM-MT, in which the DsyB residue Tyr142 activates the sulfur atom of MTHB for nucleophilic attack on the SAM methyl group. Sequence analysis suggests that this mechanism is common to all bacterial DsyB enzymes and also, importantly, eukaryotic DSYB enzymes from e.g., algae that are the major DMSP producers in Earth’s surface oceans.

scholarly journals Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Suk Min Jang ◽  
Catherine Lachance ◽  
Wenyi Mi ◽  
Jie Lyu ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Memory Impairment ◽  
Target Genes ◽  
Epigenetic Mark ◽  
Mechanistic Insight ◽  
Genomic Analyses ◽  
Insight Into

Abstract Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

scholarly journals Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

2019 ◽  
Author(s):  
Tugsan Tezil ◽  
Manish Chamoli ◽  
Che-Ping Ng ◽  
Roman P. Simon ◽  
Victoria J. Butler ◽  
...  
Keyword(s):  
Small Molecules ◽  
Physiological Function ◽  
Nordihydroguaiaretic Acid ◽  
Mechanistic Insight ◽  
Epigenetic Regulator ◽  
Novel Target ◽  
Cellular Thermal Shift Assay ◽  
Insight Into ◽  
In Cells

AbstractAging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

scholarly journals Down-Regulation of miR-212-5p Facilitates Homocysteine-Induced Hepatocytes Apoptosis Via Targeting PSMD10

2020 ◽  
Author(s):  
Huiping Zhang ◽  
Kun Xiao ◽  
Shengchao Ma ◽  
Long Xu ◽  
Ning Ding ◽  
...  
Keyword(s):  
Liver Injury ◽  
Er Stress ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Over Expression ◽  
Direct Binding ◽  
Induced Apoptosis ◽  
Insight Into

Abstract Background: Increasing evidences supported that elevated homocysteine (Hcy) levels contribute to cell apoptosis is implicated in the pathogenesis of liver injury, it correlates with liver disease severity. However, the underlying mechanism of apoptosis in Hcy-mediated liver injury remains obscure. Results: In this study, we found that homocysteine increases ER stress-mediated apoptosis and aggravates liver injury through up-regulation of PSMD10 expression in cbs+/- mice mice fed with high methionine diet and hepatocytes treated with homocysteine in vitro. Knockdown of PSMD10 expression remarkably reduced ER stress or apoptosis-associated protein in hepatocytes exposed to homocysteine. Moreover, bioinformatics analysis revealed that PSMD10 is a potential target gene of miR-212-5p, and luciferase reporter assay also confirmed that miR-212-5p negatively regulated PSMD10 expression by direct binding to its 3’-UTR regions. Subsequently, over-expression of miR-212-5p inhibited ER stress-mediated hepatocytes apoptosis though targeting PSMD10, all of which were abrogated by knockdown of miR-212-5p expression. Further study showed that the interaction between PSMD10 and GRP78 accelerated ER stress-mediated hepatic apoptosis induced by homocysteine. Conclusion: Taken together, these results demonstrated that down-regulation of miR-212-5p facilitates homocysteine-induced hepatocytes apoptosis via targeting PSMD10, which provides novel insight into the mechanism of homocysteine induced apoptosis in liver injury.

scholarly journals A Trypanosoma brucei orphan kinesin employs a convergent microtubule organization strategy to complete cytokinesis

2021 ◽  
Author(s):  
Thomas E Sladewski ◽  
Paul C Campbell ◽  
Neil Billington ◽  
Alexandra D'Ordine ◽  
Christopher L de Graffenried
Keyword(s):  
Trypanosoma Brucei ◽  
Biophysical Properties ◽  
Microtubule Organization ◽  
Division Plane ◽  
Mechanistic Insight ◽  
Organization Strategy ◽  
Cytoskeletal Elements ◽  
Order Structures ◽  
Insight Into

Many single-celled eukaryotes have complex cell morphologies defined by cytoskeletal elements comprising microtubules arranged into higher-order structures. Trypanosoma brucei (T. brucei) cell polarity is mediated by a parallel array of microtubules that underlie the plasma membrane and define the auger-like shape of the parasite. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the division plane and is essential for the completion of cytokinesis. To gain mechanistic insight into how this novel kinesin functions to complete cleavage furrow ingression, we characterized the biophysical properties of the KLIF motor domain in vitro. We found that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked in an antiparallel orientation are translocated relative to one another by KLIF, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we found that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new posterior, which is a unique requirement for the completion of cytokinesis in T. brucei.

scholarly journals Mitochondrial TRAK adaptors coordinate dynein and kinesin motility

2021 ◽  
Author(s):  
John T. Canty ◽  
Andrew Hensley ◽  
Ahmet Yildiz
Keyword(s):  
Neuronal Activity ◽  
Coiled Coil ◽  
Mitochondrial Transport ◽  
Coordinated Action ◽  
Coiled Coil Domain ◽  
Mechanistic Insight ◽  
Docking Protein ◽  
Insight Into

In neurons, mitochondria are transported to distal regions for supplying energy and buffer calcium. Mitochondrial transport is mediated by Miro and TRAK adaptors that recruit kinesin and dynein-dynactin. To understand how mitochondria are transported by these opposing motors and stalled at regions with elevated calcium levels, we reconstituted the mitochondrial transport machinery in vitro. We show that the coiled-coil domain of TRAK activates dynein-dynactin motility, but kinesin requires an additional factor to efficiently transport Miro/TRAK. Unexpectedly, TRAK adaptors that recruit both motors move towards the plus-end, whereas kinesin is excluded from binding TRAK transported by dynein-dynactin. The assembly and motility of the transport machinery are not affected by calcium. Instead, the mitochondrial docking protein syntaphilin is sufficient to oppose the forces generated by kinesin and stall the motility. Our results provide mechanistic insight into how mitochondria are transported by the coordinated action of motors and statically anchored to regions with high neuronal activity.

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