The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme

Mukherjee, Chandrama; Bakthavachalu, Baskar; Schoenberg, Daniel R.

doi:10.1371/journal.pbio.1001933

Cited by 43 publications

(66 citation statements)

References 36 publications

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“…[10] These interactions require N7 methylation, and unmodified caps have been show to become remethylated in the cytoplasm. [11] We measured the K d values of recombinantly produced eIF4E and the inactive variant DcpS (H277N) for the native and modified cap and found that these proteins do not bind to N7-BP-modified-GpppA (10d), whereas m 7 GpppA is bound, showing a K d value in the sub-micromolar range,w hich is in line with reported values (Figures S40, S41, Table S1). [10d,21] After irradiation and enzymatic remethylation to mimic cellular remethylation, binding to both proteins was fully restored (Figure 4C,D).…”

Section: Angewandte Chemiesupporting

confidence: 81%

“…[10] Due to its importance in biology,wechose the N7 position of guanosine for modification with functional groups with the goal of removing them upon irradiation with light and recover the free guanosine.Inthe 5' cap of mRNAs, the Gb ecomes remethylated in cells. [11] To generate photocaged guanosines in different contexts,t hat is,f rom single nucleosides to long mRNAs,wedevised both achemical and an enzymatic preparative route (Scheme 1). PC groups for nucleosides that do not substitute ah ydrogen atom but instead introduce apositive charge have not been reported to date to the best of our knowledge.…”

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confidence: 99%

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A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

et al. 2019

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Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring‐nitrogen methylation, such as m7G and m1A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α‐hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho‐nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site‐specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m7G and m1A with spatiotemporal control.

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Section: Angewandte Chemiesupporting

confidence: 81%

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confidence: 99%

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

et al. 2019

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“…Although Nck1 and Nck2 share common functions and interacting proteins (38), studies have reported specific functions and exclusive binding partners for Nck proteins (39)(40)(41). In agreement, we showed that Nck1-deficient mice display normal glucose homeostasis (15), and in the current study, we report that Nck2-deficient mice spontaneously develop progressive increased adiposity concomitant with impaired glucose homeostasis, insulin resistance, and hepatic steatosis.…”

Section: Discussionsupporting

confidence: 90%

Nck2 Deficiency in Mice Results in Increased Adiposity Associated With Adipocyte Hypertrophy and Enhanced Adipogenesis

Dusseault

Haider

et al. 2016

Diabetes

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Obesity results from an excessive expansion of white adipose tissue (WAT) from hypertrophy of preexisting adipocytes and enhancement of precursor differentiation into mature adipocytes. We report that Nck2-deficient mice display progressive increased adiposity associated with adipocyte hypertrophy. A negative relationship between the expression of Nck2 and WAT expansion was recapitulated in humans such that reduced Nck2 protein and mRNA levels in human visceral WAT significantly correlate with the degree of obesity. Accordingly, Nck2 deficiency promotes an adipogenic program that not only enhances adipocyte differentiation and lipid droplet formation but also results in dysfunctional elevated lipogenesis and lipolysis activities in mouse WAT as well as in stromal vascular fraction and 3T3-L1 preadipocytes. We provide strong evidence to support that through a mechanism involving primed PERK activation and signaling, Nck2 deficiency in adipocyte precursors is associated with enhanced adipogenesis in vitro and adiposity in vivo. Finally, in agreement with elevated circulating lipids, Nck2-deficient mice develop glucose intolerance, insulin resistance, and hepatic steatosis. Taken together, these findings reveal that Nck2 is a novel regulator of adiposity and suggest that Nck2 is important in limiting WAT expansion and dysfunction in mice and humans.

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“…In contrast to mammals, downstream CAGE tags are absent from the Drosophila transcriptome [15]. This raised the possibility that Drosophila lacks one or more of the biochemical mechanisms needed for downstream capping, and it was this idea that led us to perform the phylogenetic analysis in [6] that identified the basis for this. That analysis identified a proline rich sequence at the C-terminus of mammalian capping enzyme whose absence from Drosophila capping enzyme precludes it is binding to Nck1, and therefore, its ability to participate in cytoplasmic capping.…”

Section: Relationship Of Cage Tags To Recapping Sitesmentioning

confidence: 99%

Uncapped 5′ ends of mRNAs targeted by cytoplasmic capping map to the vicinity of downstream CAGE tags

et al. 2014

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In mammalian transcriptomes approximately 25% of 5’ ends determined by Capped Analysis of Gene Expression (CAGE) map to locations within spliced exons. The current study sought to determine if the cytoplasmic capping complex participates in generating these downstream CAGE tags. 5’-RACE was used to amplify the uncapped ends of target transcripts that accumulate when cytoplasmic capping is blocked. Sequencing of these RACE products mapped the positions of uncapped ends either exactly to or just downstream of archived CAGE tags. These findings support a role for cytoplasmic capping in generating the downstream capped ends identified by CAGE.

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The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme

Abstract: mRNA capping and decapping requires a cytoplasmic complex to maintain and/or restore the 5′ cap on a subset of the mammalian transcriptome; Nck1, an SH2/SH3 adapter, creates a scaffold upon which the cytoplasmic capping complex forms.

Cited by 43 publications

References 36 publications

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

Nck2 Deficiency in Mice Results in Increased Adiposity Associated With Adipocyte Hypertrophy and Enhanced Adipogenesis

Uncapped 5′ ends of mRNAs targeted by cytoplasmic capping map to the vicinity of downstream CAGE tags

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