Polysome Profiling Analysis of mRNA and Associated Proteins Engaged in Translation

Pringle, Eric S.; McCormick, Craig; Cheng, Zhenyu

doi:10.1002/cpmb.79

Cited by 56 publications

(49 citation statements)

References 13 publications

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“…With knowledge of actinin-localized transcripts, we next performed polysome profiling (Fig. 3i) followed by sequencing (Polysome-seq) 36 to understand if actinin proximity localization related to translation. PCA analysis demonstrated tight clustering of replicates of polysome-bound transcripts compared to controls (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA-binding proteins

Thakar

et al. 2020

Preprint

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1 Actinins are actin cross-linkers that are expressed in nearly all cells and harbor mutations in 2 heritable diseases. We deployed proximity-dependent biotinylation to identify actinin proximity 3 proteins and RNA transcripts in human cardiomyocytes to reveal new functions of the actin 4 cytoskeleton. We identified 285 proximity protein partners including unexpected effectors of 5 RNA-binding and metabolism, and interrogated dynamic partners using a sarcomere assembly 6 model. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein 7 associated with type 2 diabetes, interacted with the rod domain of actinin through its K 8 Homology domain. This interaction was necessary for actinin localization and translation of 9 electron transport chain transcripts, and oxidative phosphorylation. Diminished IGF2BP2 in 10 cardiac microtissues impaired contractile function in accord with diminished metabolic functions. 11 This actinin proximity protein and RNA study expands our functional knowledge of the actin 12 cytoskeleton, and uncovers new modes of metabolic regulation through interactions with RNA-13 binding proteins. 14 15 Main 16 Actinin proteins are ubiquitous spectrin family members that cross-link actin, and are important 17 for a myriad of cellular functions including cell adhesion, migration, contraction, and signaling 1 . 18 The four human actinin isoforms have distinct expression profiles (non-muscle ACTN1 and 19 ACTN4; cardiac muscle ACTN2; and skeletal muscle ACTN3), but share homologous amino 20 acid sequences that are organized into three structural domains-an actin-binding (AB) domain 21 composed of two calponin-homology domains, a central rod domain containing four spectrin-like 22 repeats (SR), and a calmodulin-like domain (CaM) containing two EF hand-like motifs 2 . While it 23is thought that actinin evolved initially to regulate the early eukaryotic actin-based cytoskeleton 3 , 24 it has acquired more elaborate functions in vertebrates, including a mechanical role in the 25 sarcomere, a specialized contractile system required for striated muscle function 3, 4 . Moreover, 21 microtissues with heart failure-associated mutations, twitch force was unaltered by Actinin-BirA* 22 expression (extended data Fig. 1b). We confirmed appropriate localization of Actinin-BirA* to the 23 Z-disk by both co-localization immunofluorescence (Fig.1d) and HA-immunoprecipitation assays 24 (Fig.1e). Actinin-BirA* interacted with the Z-disk protein titin-cap (TCAP), but not the sarcomere 25 M-line protein myomesin [22][23][24][25] , which supported appropriate localization. With the knowledge that 26 Confidential Manuscript 6 Actinin-BirA* functions similarly to unmodified actinin, we next optimized biotin supplementation

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Section: Resultsmentioning

confidence: 99%

Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA-binding proteins

Thakar

et al. 2020

Preprint

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“…After centrifugation at 13,000 × g for 5 min, the lysates were layered onto 10-50% continuous sucrose gradient and centrifuged at 39,000 rpm in a Beckman SW-41Ti rotor for 90 min at 4°C. Each gradient was collected in 12 fractions [57][58][59] . RNA was extracted from all fractions and analyzed by qRT-PCR.…”

Section: Methodsmentioning

confidence: 99%

Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells

et al. 2020

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Gene regulation and metabolism are two fundamental processes that coordinate the selfrenewal and differentiation of neural precursor cells (NPCs) in the developing mammalian brain. However, little is known about how metabolic signals instruct gene expression to control NPC homeostasis. Here, we show that methylglyoxal, a glycolytic intermediate metabolite, modulates Notch signalling to regulate NPC fate decision. We find that increased methylglyoxal suppresses the translation of Notch1 receptor mRNA in mouse and human NPCs, which is mediated by binding of the glycolytic enzyme GAPDH to an AU-rich region within Notch1 3ʹUTR. Interestingly, methylglyoxal inhibits the enzymatic activity of GAPDH and engages it as an RNA-binding protein to suppress Notch1 translation. Reducing GAPDH levels or restoring Notch signalling rescues methylglyoxal-induced NPC depletion and premature differentiation in the developing mouse cortex. Taken together, our data indicates that methylglyoxal couples the metabolic and translational control of Notch signalling to control NPC homeostasis.

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“…Fractions #7-10 correspond to translating mRNAs associated with heavy polysomes. The amount of gene-specific mRNA in each fraction was calculated to obtain the proportion (%) of the target mRNA found in each fraction as described (45,47). As a result, relative abundance and distribution of specific mRNA in 10 fractions were shown for NMA1 ( Fig.…”

Section: Nt-acetylation Is Important For Nmnat Protein Maturationmentioning

confidence: 99%

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+ biosynthetic enzymes in Saccharomyces cerevisiae

Croft

Venkatakrishnan

Raj

et al. 2020

Journal of Biological Chemistry

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NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are incompletely understood. Yeast (Saccharomyces cerevisiae) cells lacking the N-terminal (Nt) protein acetyltransferase complex NatB exhibit an approximate 50% reduction in NAD+ levels and aberrant metabolism of NAD+ precursors, changes that are associated with a decrease in nicotinamide mononucleotide adenylyltransferase (Nmnat) protein levels. Here, we show that this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins and not of other NatB substrates. Nt-acetylation critically regulates protein degradation by the N-end rule pathways, suggesting that the absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t½) of non-Nt-acetylated Nmnats did not significantly differ from those of Nt-acetylated Nmnats. Accordingly, deletion or depletion of the N-end rule pathway ubiquitin E3 ligases in NatB mutants did not restore NAD+ levels. Next, we examined whether the status of Nt-acetylation would affect the translation of Nmnats, finding that the absence of Nt-acetylation does not significantly alter the polysome formation rate on Nmnat mRNAs. However, we observed that NatB mutants have significantly reduced Nmnat protein maturation. Our findings indicate that the reduced Nmnat levels in NatB mutants are mainly due to inefficient protein maturation. Nmnat activities are essential for all NAD+ biosynthesis routes, and understanding the regulation of Nmnat protein homeostasis may improve our understanding of the molecular basis and regulation of NAD+ metabolism.

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Polysome Profiling Analysis of mRNA and Associated Proteins Engaged in Translation

Cited by 56 publications

References 13 publications

Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA-binding proteins

Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA-binding proteins

Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+ biosynthetic enzymes in Saccharomyces cerevisiae

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