A Metabolic Engineering Approach to Incorporate Modified Pyrimidine

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A Metabolic Engineering Approach to Incorporate Modified Pyrimidine Nucleosides into Cellular RNA Yu Zhang, and Ralph E. Kleiner J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11449 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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Journal of the American Chemical Society

A Metabolic Engineering Approach to Incorporate Modified Pyrimidine Nucleosides into Cellular RNA Yu Zhang & Ralph E. Kleiner* Department of Chemistry, Princeton University, Princeton, NJ 08544, USA

Supporting Information Placeholder ABSTRACT: The incorporation of modified nucleotides

into RNA is a powerful strategy to probe RNA structure and function. While a wide variety of modified nucleotides can be incorporated into RNA in vitro using chemical or enzymatic synthesis, strategies for the metabolic incorporation of artificial nucleotides into cellular RNA are limited, largely due to the incompatibility of modified nucleobases and nucleosides with nucleotide salvage pathways. In this work, we develop a metabolic engineering strategy to facilitate the labeling of cellular RNA with noncanonical pyrimidine nucleosides. First, we use structure-based protein engineering to alter the substrate specificity of uridine-cytidine kinase 2 (UCK2), a key enzyme in the pyrimidine nucleotide salvage pathway. Next, we show that expression of mutant UCK2 in HeLa and U2OS cells is sufficient to enable the incorporation of 5-azidomethyl uridine (5-AmU) into cellular RNA and promotes RNA labeling by other C5-modified pyrimidines. Finally, we apply UCK2-mediated RNA labeling with 5-AmU to study RNA trafficking and turnover during normal and stress conditions and find diminished RNA localization in the cytosol during arsenite stress. Taken together, our study provides a general strategy for the incorporation of modified pyrimidine nucleosides into cellular RNA and expands the chemical toolkit of modified bases for studying dynamic RNA behavior in living cells.

RNA plays a central role in biological processes and understanding its regulation is critical for illuminating normal and disease cell physiology1. The metabolic incorporation of modified nucleotides into cellular RNA is a powerful approach for probing RNA behavior in living cells and can be exploited for drug development and synthetic biology. Several approaches have been developed to allow labeling of cellular RNA with artificial nucleotides. Nucleoside analogs can be activated through nucleotide salvage pathways enabling

RNA synthesis tracking2-6 and protein-RNA crosslinking7-8. Tissue-specific RNA labeling can be achieved by expressing uracil phosphoribosyl transferase9-11 or decaging enzymes12 with nucleobase and nucleoside analogs. Finally, nucleotide salvage pathways can be bypassed by phosphate pro-drugs13, transfection of NTPs14, or by microbial expression of NTP transporters15-16. Despite the promise of these approaches for interrogating RNA biology, the toolbox of modified nucleotides that can be readily incorporated into cellular RNA has remained limited. In particular, uridine analogs with C5 modifications that are larger than an ethynyl group are not efficiently incorporated5, 11. In addition, we lack methods to label cellular RNA with azide-containing pyrimidines, which could be used to track nascent RNA synthesis in living cells through application of the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction17. Of the many requirements for metabolic labeling of cellular RNA, recognition by the nucleotide salvage pathway, a multi-kinase cascade that generates NTPs from nucleotide intermediates, appears to be the most stringent. For pyrimidine nucleosides, salvage relies on the activity of uridine-cytidine kinase (UCK)18, UMPCMP kinase (CMPK)19, and nucleotide diphosphate kinase (NDPK)20. Broadening the substrate specificity of these salvage enzymes presents a promising approach towards developing a diverse set of modified pyrimidine nucleosides to probe RNA function in vivo. In this work, we use structure-based protein engineering to alter the substrate specificity of uridinecytidine kinase 2 (UCK2)18. We identify UCK2 mutant enzymes that can efficiently phosphorylate C5-modified uridine analogs and demonstrate that expression of these mutant enzymes in human cells is sufficient to mediate RNA incorporation of these nucleoside analogs. Finally, we use UCK2-mediated RNA labeling to investigate cellular RNA localization and turnover during normal

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and stress conditions, and to facilitate imaging of RNA in living cells.

Figure 1. Structure-based protein engineering of UCK2. (a) Pyrimidine nucleosides used in this work. (b) Pyrimidine salvage pathway. (c) X-ray crystal structure of UCK2 with UTP. Protein sidechains making interactions with uracil are shown. Tyr65 is colored in teal. (d) Phosphorylation of 5-AmU by recombinant WT and Y65A UCK2 enzymes. Reactions were run for 20 hr at 37 °C. (e) Quantification of 5-AmU phosphorylation by UCK2 enzymes. Data represent the mean value +/- s.d. (n = 3). (f) Quantification of uridine phosphorylation by UCK2 enzymes. Data represent the mean value +/- s.d. (n = 3). (g) Quantification of 5-EU phosphorylation by UCK2 enzymes. Data represent the mean value +/- s.d. (n = 3).

In order to develop a platform for incorporating modified nucleosides into cellular RNA, we focused on the uridine analog 5-AmU (Fig. 1a). 5-AmU does not label cellular RNA5, 11, but is ready accepted by cellular RNA polymerases when provided in the triphosphate form14. Therefore, we reasoned that the lack of incorporation of 5-AmU into cellular RNA was due to incompatibility with pyrimidine salvage enzymes (Fig. 1b). Of these enzymes, UCK2, which generates pyrimidine monophosphates from nucleosides, has been proposed to be rate limiting for pyrimidine salvage21. Indeed, analysis of the x-ray crystal structure of UCK2 with the substrate analog UTP22 (Fig. 1c) suggests that an azidomethyl modification at C5 would be poorly accommodated in the enzyme active site.

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To test this hypothesis, we expressed and purified recombinant UCK2 enzyme and measured its activity on uridine and 5-AmU using an HPLC-based assay. Treatment of uridine with UCK2 in the presence of ATP led to complete conversion to UMP in 30 min, while incubation of 5-AmU under the same conditions resulted in 90% after 20 hr, Supplementary Fig. 1), a modified nucleoside commonly used to label cellular RNA. Our results led us to speculate that a mutant version of UCK2 that could efficiently phosphorylate 5-AmU would enable its incorporation into cellular RNA. Towards this end, we generated a panel of enzymes containing substitutions of Tyr65 to smaller amino acids and tested their ability to phosphorylate 5-AmU in vitro. We chose Tyr65 since it abuts the C5 position in the xray crystal structure of UTP with UCK222(Fig. 1c). Remarkably, we observed efficient conversion (84-89% over 20 hr) of 5-AmU to 5-AmU monophosphate with a UCK2 enzyme containing Y65A or Y65G substitutions (Fig. 1d, 1e, Supplementary Fig. 1, 2). In addition, Y65G UCK2 phosphorylates 5-EU more efficiently than does WT UCK2 (Fig. 1g, Supplementary Fig. 1). In contrast, UCK2 containing Y65F, Y65L, or Y65V mutations did not support phosphorylation of 5-AmU and retained high activity on uridine (Fig. 1f). Taken together, our results suggest that Y65G/A mutations create a “hole” in the active site to accommodate the bulky 5-AmU nucleoside. We next transfected mutant UCK2 into HeLa cells to test whether these enzymes would enable incorporation of 5-AmU into cellular RNA. 20 hr post-transfection, cells were treated for 1 hr with 100 µM 5-AmU and RNA incorporation was monitored using fluorescence microscopy after CuAAC with Cy3-alkyne (Fig. 2a). Gratifyingly, cells expressing Y65G or Y65A UCK2 showed clear labeling with 5-AmU (Fig. 2b). In contrast, we observed significantly reduced labeling in untransfected cells and cells transfected with WT UCK2 (Fig. 2b). We were also able to observe similar Y65G UCK2-dependent 5-AmU labeling in U2OS cells (Supplementary Fig. 5), demonstrating the generality of our approach. We further tested whether 5-AmU would enable RNA imaging in live cells. U2OS cells transfected with Y65G UCK2 were treated with 5-AmU and then processed for labeling using SPAAC17 chemistry with a BODIPY-BCN23-24 fluorophore. Gratifyingly, we were able to observe strong nuclear and cytosolic labeling only in cells treated with 5-AmU (Supplementary Fig. 6). To test whether labeling was specific for RNA over DNA, we treated cells with DNA and RNA synthesis inhibitors during 5-AmU labeling. Treatment with


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hydroxyurea did not affect labeling (Supplementary Fig. 7). In contrast, the RNA polymerase inhibitors actinomycin D and -aminitin greatly reduced 5-AmU labeling (Supplementary Fig. 8), indicating that RNA incorporation is the primary route of 5-AmU metabolism. We also used these compounds (Supplementary Fig. 8) together with gel electrophoresis analysis of RNA extracted from 5-AmU treated

with 5-EU (Fig. 3a). In addition, we found that UCK2 expression sensitized cells to 5-fluorouridine (5-FUrd), a cytotoxic nucleoside25 (Fig. 3b, Supplementary Fig. 10, 11). Taken together, our results establish that UCK2 activity is rate limiting for the salvage of C5-modified pyrimidines. We chose to apply RNA labeling with 5-AmU and 5EU to probe RNA synthesis and turnover during the cellular stress response. Mammalian cells undergo substantial changes on the transcriptional level during the integrated stress response26, but how diverse stress conditions affect

Figure 2. UCK2 mutants facilitate the incorporation of 5AmU into cellular RNA. (a) Workflow for detection of 5AmU in cellular RNA. (b) HeLa cells were transfected with UCK2 construct and 20 hr later were treated with 100 µM 5-AmU for 1 hr. After fixation and Cu(I)-catalyzed click chemistry with Cy3-alkyne, 5-AmU incorporation was visualized by fluorescence microscopy. Immunofluorescence was used to confirm UCK2 transfection.

Figure 3. UCK2 expression increases 5-EU and 5-FUrd incorporation into RNA. (a) HeLa cells were transfected with UCK2 construct and treated with 100 µM 5-EU for 1 hr. Microscopy was performed as in Figure 2, except using Cy3-azide. (b) HeLa cells containing tetracyclineinducible WT UCK2 were treated with 5-FUrd and cell viability was measured using an MTS-based assay. Plot represents mean normalized cell viability +/- s. d. (n = 5). (c) HeLa cells containing tetracycline-inducible Y65G UCK2 were treated as in (a). Plot represents mean normalized cell viability +/- s. d. (n=3).

cells (Supplementary Fig. 9), to demonstrate that 5AmU is a substrate for RNA polymerase I and II and is incorporated into a broad range of cellular RNAs. We next sought to determine whether UCK2 could facilitate metabolism of other C5-modified pyrimidines. We treated HeLa cells expressing WT or Y65G UCK2 with 5-EU and monitored incorporation by click chemistry with Cy3-azide. Interestingly, expression of WT or Y65G UCK2 enzymes greatly increased labeling

global RNA synthesis and turnover is still largely unknown. To investigate this, we first measured the normal turnover of 5-AmU and 5-EU labeled RNA in a “pulse-chase” experiment. For each nucleoside, we observed a time-dependent decrease in modified transcripts starting at 2 hr post-labeling (Fig. 4a) and continuing until 24 hr, consistent with the kinetics of RNA turnover previously reported for 5-EU2.



Interestingly, we observed different localization patterns for transcripts containing 5-AmU compared to 5-EU. While 5-EU labeling was concentrated in nucleoli and restricted to the nucleus, 5-AmU-labeling originated in the nucleus but could be observed in the nucleus and cytosol at intermediate time points during the chase (Fig. 4a). Next, we measured turnover of 5AmU and 5-EU transcripts during oxidative stress.

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Under these conditions, we observed increased turnover of 5-AmU labeled RNA as early as 2 hr post-labeling (Fig. 4b). Noticeably, 5-AmU signal was absent from the cytosol in cells exposed to arsenite, indicating either altered nuclear export of these transcripts or increased degradation. In contrast, 5-EU signal during arsenite stress was largely unchanged relative to unstressed cells (Fig. 4a and 4b).

Figure 4. Turnover of 5-AmU and 5-EU labeled RNA. (a) HeLa cells or HeLa cells expressing Y65G UCK2 were treated with 1 mM 5-EU or 100 µM 5-AmU for 2 hr, respectively, and then chased with complete medium for the indicated time. Microscopy was performed as in Fig. 2 and 3. (b) Experiment was performed as in (a) with the exception that the chase was performed with complete medium containing 0.5 mM NaAsO2.


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Journal of the American Chemical Society Funding Sources

No competing financial interests have been declared.

We also measured RNA synthesis during stress by exposing cells to heat shock (42 °C) or sodium arsenite during nucleoside treatment. For 5-AmU, we observed no change in labeling during heat shock or arsenite stress (Supplementary Fig. 12). In contrast, while heat shock had little effect on 5-EU labeling, we observed a strong reduction in 5-EU labeling during arsenite stress (Supplementary Fig. 12, 13). Taken together, our results show that arsenite stress affects RNA synthesis and turnover and demonstrates how 5-AmU and 5-EU labeling can be utilized to probe these processes. In this work, we engineer the mammalian pyrimidine salvage pathway to incorporate 5-AmU into cellular RNA. In contrast to 5-EU functionalization, which requires a Cu(I) catalyst, 5-AmU can participate in the SPAAC reaction17, enabling the detection of transcripts in living cells and with minimal RNA degradation27. In addition, our approach provides a method to increase cellular RNA labeling with other C5-modified pyrimidines, allowing treatments to be performed with lower nucleoside concentrations and facilitating labeling experiments that require high temporal resolution. While many enzymes are required to incorporate pyrimidine nucleosides into cellular RNA, we chose to focus our efforts on UCK2. Our work establishes that UCK2 is rate limiting for pyrimidine salvage and that expanding the substrate tolerance of UCK2 alone is sufficient to enable the incorporation of C5-modified pyrimidines into RNA. Our study provides a general strategy for expanding the chemical toolkit of modified pyrimidine nucleosides for metabolic RNA labeling. We envision application of this approach combined with computational protein design and directed evolution strategies to enable the incorporation of diverse nucleotides for use in biorthogonal chemistry28, RNAprotein photocrosslinking29, epitranscriptomics30, and synthetic biology31. Finally, nucleotide salvage is an important pathway for the activation of anti-viral and anti-cancer nucleoside analogs32. Probing the capacity of this system to process non-canonical nucleotides in different biological contexts will have important implications for the design and application of nucleotide therapeutics. ASSOCIATED CONTENT Supporting Information. Supplementary discussion, experimental methods and supplementary figures are available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author

ACKNOWLEDGMENT We thank Vikas Nanda for helpful discussions. We are grateful to Jeremy Baskin for the generous gift of BODIPY-BCN. R. E. K. is a Dale F. Frey Breakthrough Scientist of the Damon Runyon Cancer Research Foundation (DFS #21-16) and a Sidney Kimmel Foundation Scholar. All authors thank Princeton University for financial support.

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A Metabolic Engineering Approach to Incorporate Modified Pyrimidine

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