Activity dependent translation in astrocytes dynamically alters the proteome of the perisynaptic astrocyte process

Sapkota, Darshan; Kater, Mandy S.J.; Sakers, Kristina; Nygaard, Kayla R; Liu, Y.; Lake, Allison M.; Khazanchi, Rohan; Khankan, Rana R.; Smit, August B.; Maloney, Susan E.; Verheijen, Mark H. G.; Zhang, Y; Dougherty, Joseph D.

doi:10.1101/2020.04.08.033027

Cited by 8 publications

(10 citation statements)

References 90 publications

(128 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gene expression requires two steps – transcription and translation – which can be regulated independently to allow nuanced, localized, and rapid responses to cellular stimuli. Astrocytes, besides neurons, respond transcriptionally and translationally to bursts of brain activity [ 26 ]. Similarly, although the dynamic modulation of Ras GTPases is critically dependent on their post-translational modifications, burst of redox signaling in astrocytic cultures can induce a transcriptional mechanism.…”

Section: Discussionmentioning

confidence: 99%

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study

2022

View full text Add to dashboard Cite

Background Although the protooncogenes small GTPases Ras are redox-sensitive proteins, how they are regulated by redox signaling in the central nervous system (CNS) is still poorly understood. Alteration in redox-sensitive targets by redox signaling may have myriad effects on Ras stability, activity and localization. Redox-mediated changes in astrocytic RAS may contribute to the control of redox homeostasis in the CNS that is connected to the pathogenesis of many diseases. Results and Methods Here, we investigated the transient physiological induction, at both transcriptional and translational levels, of small GTPases Ras in response to redox stimulation. Cultured astrocytes were treated with hydrogen peroxide as in bolus addition and relative mRNA levels of murine hras and kras genes were detected by qRT-PCR. We found that de novo transcription of hras mRNA in reactive astrocytes is redox-sensitive and mimics the prototypical redox-sensitive gene iNOS. Protein abundance in combination with protein turnover measurements by cycloheximide-chase experiments revealed distinct translation efficiency, GTP-bound enrichment, and protein turnover rates between the two isoforms H-Ras and K-Ras. Conclusion Reports from recent years support a significant role of H-Ras in driving redox processes. Beyond its canonical functions, Ras may impact on the core astrocytic cellular machinery that operates during redox stimulation.

show abstract

Section: Discussionmentioning

confidence: 99%

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study

2022

View full text Add to dashboard Cite

show abstract

“…TRAP was performed on P21 whole brain homogenates as described previously (Heiman et al 2008 and Sapkota et al 2020). RNA was extracted from the input and TRAP samples using the RNA Clean & Concentrator Kit (Zymo Research).…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, assessment of Translational Efficiency (TE), defined empirically as the relative number of ribosomes per transcript using combinations of Ribosome Footprinting and RNA sequencing (Dalal et al, 2017;Ingolia et al, 2009Ingolia et al, , 2011 has suggested that transcript abundance only accounts for 43%-63% of ribosome occupancy across transcripts. Further, post-transcriptional regulation is especially important in the CNS, where both neurons and astrocytes show evidence of complex activity dependent and localized regulation of translation (Sakers et al, 2017;Sapkota et al, 2020). To what extent do variants in UTRs also have substantial impact on final levels of ribosomes on the mRNA?…”

Section: Introductionmentioning

confidence: 99%

TRAP-based allelic translation efficiency imbalance analysis to identify genetic regulation of ribosome occupancy in specific cell typesin vivo

Liu

Fischer

Pierre

et al. 2020

Preprint

View full text Add to dashboard Cite

The alteration of gene expression due to variations in the sequences of transcriptional regulatory elements has been a focus of substantial inquiry in humans and model organisms. However, less is known about the extent to which natural variation contributes to post-transcriptional regulation. Allelic Expression Imbalance(AEI) is a classical approach for studying the association of specific haplotypes with relative changes in transcript abundance. Here, we benchmarked a new TRAP based approach to associate genetic variation with transcript occupancy on ribosomes in specific cell types, to determine if it will allow examination of Allelic Translation Imbalance(ATI), and Allelic Translation Efficiency Imbalance, using as a test case mouse astrocytes in vivo. We show that most changes of the mRNA levels on ribosomes were reflected in transcript abundance, though ~1.5% of transcripts have variants that clearly alter loading onto ribosomes orthogonally to transcript levels. These variants were often in conserved residues and altered sequences known to regulate translation such as upstream ORFs, PolyA sites, and predicted miRNA binding sites. Such variants were also common in transcripts showing altered abundance, suggesting some genetic regulation of gene expression may function through post-transcriptional mechanisms. Overall, our work shows that naturally occurring genetic variants can impact ribosome occupancy in astrocytes in vivo and suggests that mechanisms may also play a role in genetic contributions to disease.

show abstract

“…As TRIM28 was recently shown to regulate antitumor immunity, its role in ARF-mediated immune regulation could warrant further investigation (Lin et al, 2021). With ever-growing data from multiomics analyses being fed into databases like BioGRID, artificial intelligence-aided literature mining tool, such as CompBio (https:// gtac-compbio.wustl.edu/), could facilitate our ability to extract useful information more effectively (Sapkota et al, 2021).…”

Section: Development Of Arf-based Therapeutic Strategies-functional Antagonists Against Arf Deficiencymentioning

confidence: 99%

It’s Getting Complicated—A Fresh Look at p53-MDM2-ARF Triangle in Tumorigenesis and Cancer Therapy

Kung

Weber

2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

Anti-tumorigenic mechanisms mediated by the tumor suppressor p53, upon oncogenic stresses, are our bodies’ greatest weapons to battle against cancer onset and development. Consequently, factors that possess significant p53-regulating activities have been subjects of serious interest from the cancer research community. Among them, MDM2 and ARF are considered the most influential p53 regulators due to their abilities to inhibit and activate p53 functions, respectively. MDM2 inhibits p53 by promoting ubiquitination and proteasome-mediated degradation of p53, while ARF activates p53 by physically interacting with MDM2 to block its access to p53. This conventional understanding of p53-MDM2-ARF functional triangle have guided the direction of p53 research, as well as the development of p53-based therapeutic strategies for the last 30 years. Our increasing knowledge of this triangle during this time, especially through identification of p53-independent functions of MDM2 and ARF, have uncovered many under-appreciated molecular mechanisms connecting these three proteins. Through recognizing both antagonizing and synergizing relationships among them, our consideration for harnessing these relationships to develop effective cancer therapies needs an update accordingly. In this review, we will re-visit the conventional wisdom regarding p53-MDM2-ARF tumor-regulating mechanisms, highlight impactful studies contributing to the modern look of their relationships, and summarize ongoing efforts to target this pathway for effective cancer treatments. A refreshed appreciation of p53-MDM2-ARF network can bring innovative approaches to develop new generations of genetically-informed and clinically-effective cancer therapies.

show abstract

Activity dependent translation in astrocytes dynamically alters the proteome of the perisynaptic astrocyte process

Cited by 8 publications

References 90 publications

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study

TRAP-based allelic translation efficiency imbalance analysis to identify genetic regulation of ribosome occupancy in specific cell typesin vivo

It’s Getting Complicated—A Fresh Look at p53-MDM2-ARF Triangle in Tumorigenesis and Cancer Therapy

Contact Info

Product

Resources

About