Journal: bioRxiv

Article Title: Nucleus translocation of tRNA synthetase mediates late integrated stress response

doi: 10.1101/2020.06.07.138792

Figure Lengend Snippet: Schematic illustration of the classical ISR and the TyrRS-mediated late ISR pathways in parallel. As an immediate response to a wide range of stress signals, the classical ISR pathway is activated by four upstream kinases that phosphorylate eIF2α to subsequently inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. While the classical ISR provides the early response, the tRNA synthetase-based ISR mediates the late response, providing the cells with a second chance to adapt and survive through stress conditions. A variety of stress conditions can be sensed by TyrRS through its intrinsic properties as a tRNA synthetase and its strategically located NLS and be integrated through TyrRS nuclear translocation to effect protective responses against prolonged stress. Similar to the negative feedback regulation instigated in ISR to prevent over-suppression of translation and to restore homeostasis, the tRNA synthetase-mediated response also provides negative feedback mechanisms: The translation inhibition and stress-response genes activation effected by nuclear TyrRS would preserve ATP and amino acid and help alleviate the stressors that stimulate TyrRS nuclear translocation in the first place.

Article Snippet: The following primary antibodies were diluted in 1% milk in TBST prior to usage at the indicated concentration: mouse monoclonal α-Tubulin (1:3000, Cell Signaling Technology, #3873), rabbit monoclonal p-p70 S6K (1:1000, Cell Signaling Technology, #9234), rabbit monoclonal p-4E-BP1 (1:1000, Cell Signaling Technology, #2855), rabbit monoclonal p70 S6K (1:1000, Cell Signaling Technology, #2708), rabbit monoclonal 4E-BP1 (1:1000, Cell Signaling Technology, #9644), rabbit polyclonal p-eIF2α (1:1000, Cell Signaling Technology, #9721), rabbit polyclonal eIF2α (1:1000, Cell Signaling Technology, #9722), rabbit monoclonal ATF-4 (1:1000, Cell Signaling Technology, #11815), rabbit polyclonal SARS (1:1000, made in-house), rabbit polyclonal WARS1 (1:3000, made in-house), mouse monoclonal GARS (1:4000, made in-house), rabbit monoclonal EEF1A1 (1:1000, Cell Signaling Technology, #3586), goat polyclonal RAE1 (1:1000, Abcam, ab36139), rabbit polyclonal HARS (1:1000, Abcam, ab137591), mouse monoclonal V5 (1:5000, Thermo Fisher Scientific, R960CUS), mouse monoclonal HDAC1 (1:1000, Cell Signaling Technology, #5356), mouse monoclonal HDAC2 (1:1000, Cell Signaling Technology, #5113), mouse monoclonal HDAC3 (1:1000, Cell Signaling Technology, #3949), mouse monoclonal RbAp46 (1:1000, NOVUS BIOLOGICALS, OTI5A4), mouse monoclonal RbAp48 (1:1000, NOVUS BIOLOGICALS, 13D10), mouse monoclonal CHD4 (1:1000, Abcam, ab70469), rabbit monoclonal MBD3 (1:1000, Abcam, ab157464), rabbit polyclonal MTA1 (1:1000, Abcam, ab71153), rabbit polyclonal p66a (1:1000, NOVUS BIOLOGICALS, NB100-56643), rabbit polyclonal LSD1 (1:1000, Abcam, ab17721), rabbit polyclonal TRIM28 (1:1000, Abcam, ab10483), rabbit polyclonal caspase 3 (1:1000, Cell Signalling Technology, #9662), rabbit polyclonal cleaved caspase 3 (1:1000, Cell Signaling Technology, #9661), rabbit monoclonal PARP (1:1000, Cell Signaling Technology, #9532), rabbit polyclonal SOD1 (1:1000, Abcam, ab13498), rabbit polyclonal Catalase (1:1000, Abcam, ab16731), rabbit polyclonal Thioredoxin (1:1000, Abcam, ab26320).

Techniques: Translocation Assay, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: Nucleus translocation of tRNA synthetase mediates late integrated stress response

doi: 10.1101/2020.06.07.138792

Figure Lengend Snippet: Schematic illustration of the classical ISR and the TyrRS-mediated late ISR pathways in parallel. As an immediate response to a wide range of stress signals, the classical ISR pathway is activated by four upstream kinases that phosphorylate eIF2α to subsequently inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. While the classical ISR provides the early response, the tRNA synthetase-based ISR mediates the late response, providing the cells with a second chance to adapt and survive through stress conditions. A variety of stress conditions can be sensed by TyrRS through its intrinsic properties as a tRNA synthetase and its strategically located NLS and be integrated through TyrRS nuclear translocation to effect protective responses against prolonged stress. Similar to the negative feedback regulation instigated in ISR to prevent over-suppression of translation and to restore homeostasis, the tRNA synthetase-mediated response also provides negative feedback mechanisms: The translation inhibition and stress-response genes activation effected by nuclear TyrRS would preserve ATP and amino acid and help alleviate the stressors that stimulate TyrRS nuclear translocation in the first place.

Article Snippet: The following primary antibodies were diluted in 1% milk in TBST prior to usage at the indicated concentration: mouse monoclonal α-Tubulin (1:3000, Cell Signaling Technology, #3873), rabbit monoclonal p-p70 S6K (1:1000, Cell Signaling Technology, #9234), rabbit monoclonal p-4E-BP1 (1:1000, Cell Signaling Technology, #2855), rabbit monoclonal p70 S6K (1:1000, Cell Signaling Technology, #2708), rabbit monoclonal 4E-BP1 (1:1000, Cell Signaling Technology, #9644), rabbit polyclonal p-eIF2α (1:1000, Cell Signaling Technology, #9721), rabbit polyclonal eIF2α (1:1000, Cell Signaling Technology, #9722), rabbit monoclonal ATF-4 (1:1000, Cell Signaling Technology, #11815), rabbit polyclonal SARS (1:1000, made in-house), rabbit polyclonal WARS1 (1:3000, made in-house), mouse monoclonal GARS (1:4000, made in-house), rabbit monoclonal EEF1A1 (1:1000, Cell Signaling Technology, #3586), goat polyclonal RAE1 (1:1000, Abcam, ab36139), rabbit polyclonal HARS (1:1000, Abcam, ab137591), mouse monoclonal V5 (1:5000, Thermo Fisher Scientific, R960CUS), mouse monoclonal HDAC1 (1:1000, Cell Signaling Technology, #5356), mouse monoclonal HDAC2 (1:1000, Cell Signaling Technology, #5113), mouse monoclonal HDAC3 (1:1000, Cell Signaling Technology, #3949), mouse monoclonal RbAp46 (1:1000, NOVUS BIOLOGICALS, OTI5A4), mouse monoclonal RbAp48 (1:1000, NOVUS BIOLOGICALS, 13D10), mouse monoclonal CHD4 (1:1000, Abcam, ab70469), rabbit monoclonal MBD3 (1:1000, Abcam, ab157464), rabbit polyclonal MTA1 (1:1000, Abcam, ab71153), rabbit polyclonal p66a (1:1000, NOVUS BIOLOGICALS, NB100-56643), rabbit polyclonal LSD1 (1:1000, Abcam, ab17721), rabbit polyclonal TRIM28 (1:1000, Abcam, ab10483), rabbit polyclonal caspase 3 (1:1000, Cell Signalling Technology, #9662), rabbit polyclonal cleaved caspase 3 (1:1000, Cell Signaling Technology, #9661), rabbit monoclonal PARP (1:1000, Cell Signaling Technology, #9532), rabbit polyclonal SOD1 (1:1000, Abcam, ab13498), rabbit polyclonal Catalase (1:1000, Abcam, ab16731), rabbit polyclonal Thioredoxin (1:1000, Abcam, ab26320).

Techniques: Translocation Assay, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: Nucleus translocation of tRNA synthetase mediates late integrated stress response

doi: 10.1101/2020.06.07.138792

Figure Lengend Snippet: A) TyrRS represses histone H3 acetylation on its target sites. The enrichment of histone H3 acetylation was determined by chromatin IP using α-H3K27Ac antibody followed by qPCR. n=3, biological replicates, one way Student’s t test. Vector: control, YARS: TyrRS overexpression. B) TyrRS interacts with HDAC1 and other factors in the NuRD complex. Co-immunoprecipitation using a-TyrRS antibody followed by Western blot analysis to detect proteins involved in the NuRD complex. Vector: control, YARS: TyrRS overexpression. C) TyrRS recruits HDAC1 to its target sites on protein translation-related genes in TyrRS overexpressing cells. Enrichment of HDAC1 was detected by chromatin IP using α-HDAC1 antibody followed by qPCR. n=3, biological replicates, one way Student’s t test. Vector: control, YARS: TyrRS overexpression. D) Recruitment of TRIM28 at multiple TyrRS target sites (YARS, WARS, and GARS) in TyrRS overexpressing HEK293 cells. Enrichment of TRIM28 was detected by chromatin IP using α-TRIM28 antibody followed by qPCR. n=3, biological replicates, one way Student’s t test. Vector: control, YARS: TyrRS overexpression. E) Increased occupancy of CHD4, a component of the NuRD complex factor, at multiple TyrRS target sites (YARS, SARS, EEF1A1, and RAE1) in TyrRS overexpressing HEK293 cells. Enrichment of CHD4 was detected by chromatin IP using α-CHD4 antibody followed by qPCR. n=3, biological replicates, one way Student’s t test. Vector: control, YARS: TyrRS overexpression. F) Schematic illustration of how TyrRS responds to oxidative stress to repress transcription of protein translation-related genes.

Article Snippet: The following primary antibodies were diluted in 1% milk in TBST prior to usage at the indicated concentration: mouse monoclonal α-Tubulin (1:3000, Cell Signaling Technology, #3873), rabbit monoclonal p-p70 S6K (1:1000, Cell Signaling Technology, #9234), rabbit monoclonal p-4E-BP1 (1:1000, Cell Signaling Technology, #2855), rabbit monoclonal p70 S6K (1:1000, Cell Signaling Technology, #2708), rabbit monoclonal 4E-BP1 (1:1000, Cell Signaling Technology, #9644), rabbit polyclonal p-eIF2α (1:1000, Cell Signaling Technology, #9721), rabbit polyclonal eIF2α (1:1000, Cell Signaling Technology, #9722), rabbit monoclonal ATF-4 (1:1000, Cell Signaling Technology, #11815), rabbit polyclonal SARS (1:1000, made in-house), rabbit polyclonal WARS1 (1:3000, made in-house), mouse monoclonal GARS (1:4000, made in-house), rabbit monoclonal EEF1A1 (1:1000, Cell Signaling Technology, #3586), goat polyclonal RAE1 (1:1000, Abcam, ab36139), rabbit polyclonal HARS (1:1000, Abcam, ab137591), mouse monoclonal V5 (1:5000, Thermo Fisher Scientific, R960CUS), mouse monoclonal HDAC1 (1:1000, Cell Signaling Technology, #5356), mouse monoclonal HDAC2 (1:1000, Cell Signaling Technology, #5113), mouse monoclonal HDAC3 (1:1000, Cell Signaling Technology, #3949), mouse monoclonal RbAp46 (1:1000, NOVUS BIOLOGICALS, OTI5A4), mouse monoclonal RbAp48 (1:1000, NOVUS BIOLOGICALS, 13D10), mouse monoclonal CHD4 (1:1000, Abcam, ab70469), rabbit monoclonal MBD3 (1:1000, Abcam, ab157464), rabbit polyclonal MTA1 (1:1000, Abcam, ab71153), rabbit polyclonal p66a (1:1000, NOVUS BIOLOGICALS, NB100-56643), rabbit polyclonal LSD1 (1:1000, Abcam, ab17721), rabbit polyclonal TRIM28 (1:1000, Abcam, ab10483), rabbit polyclonal caspase 3 (1:1000, Cell Signalling Technology, #9662), rabbit polyclonal cleaved caspase 3 (1:1000, Cell Signaling Technology, #9661), rabbit monoclonal PARP (1:1000, Cell Signaling Technology, #9532), rabbit polyclonal SOD1 (1:1000, Abcam, ab13498), rabbit polyclonal Catalase (1:1000, Abcam, ab16731), rabbit polyclonal Thioredoxin (1:1000, Abcam, ab26320).

Techniques: Chromatin Immunoprecipitation, Plasmid Preparation, Control, Over Expression, Immunoprecipitation, Western Blot