Planes, trains, and automobiles: How cells localize their molecules.

In this issue of Molecular Cell, Gasparski et al.1 and Loedige et al.2 reshape our understanding of subcellular gene product localization by highlighting the importance of messenger RNA (mRNA) stability and co-translational mechanisms in mRNA and protein localization.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

A chemoproteomic portrait of the oncometabolite fumarate.

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

Photoinducible Oncometabolite Detection.

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.

A Chemical Signature for Cytidine Acetylation in RNA.

N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease.

Muskelin acts as a substrate receptor of the highly regulated Drosophila CTLH E3 ligase during the maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch (TRAL) are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies. In particular, Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental specificity of the CTLH complex is mediated by multipronged regulation, including transcriptional control by the transcription factor OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate acts as a substrate adaptor for the Drosophila CTLH complex. Although conserved, Muskelin has structural roles in other species, suggesting a surprising functional plasticity. Finally, we find that Muskelin has few targets beyond the three known RNA binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, seemingly with the goal of regulating three important RNA binding proteins.

An Oncometabolite Isomer Rapidly Induces a Pathophysiological Protein Modification.

Metabolites regulate protein function via covalent and noncovalent interactions. However, manipulating these interactions in living cells remains a major challenge. Here, we report a chemical strategy for inducing cysteine S-succination, a nonenzymatic post-translational modification derived from the oncometabolite fumarate. Using a combination of antibody-based detection and kinetic assays, we benchmark the in vitro and cellular reactivity of two novel S-succination "agonists," maleate and 2-bromosuccinate. Cellular assays reveal maleate to be a more potent and less toxic inducer of S-succination, which can activate KEAP1-NRF2 signaling in living cells. By enabling the cellular reconstitution of an oncometabolite-protein interaction with physiochemical accuracy and minimal toxicity, this study provides a methodological basis for better understanding the signaling role of metabolites in disease.

Bioorthogonal oncometabolite ligation.

Dysregulated cellular metabolism is an emerging hallmark of cancer. Improved methods to profile aberrant metabolic activity thus have substantial applications as tools for diagnosis and understanding the biology of malignant tumors. Here we describe the utilization of a bioorthogonal ligation to fluorescently detect the TCA cycle oncometabolite fumarate. This method enables the facile measurement of fumarate hydratase activity in cell and tissue samples, and can be used to detect disruptions in metabolism that underlie the genetic cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The current method has substantial utility for sensitive fumarate hydratase activity profiling, and also provides a foundation for future applications in diagnostic detection and imaging of cancer metabolism.