Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

Sas‐Chen, Aldema; Thomas, Justin M.; Matzov, Donna; Taoka, Masato; Nance, Kellie D.; Nir, Ronit; Bryson, Keri; Shachar, Ran; Liman, Geraldy L. S.; Burkhart, Brett W.; Gamage, Supuni Thalalla; Nobe, Yuko; Briney, Chloe A.; Levy, Michaella; Fuchs, Ryan T.; Robb, G. Brett; Hartmann, Jesse; Sharma, Sunny; Lin, Qishan; Florens, Laurence; Washburn, Michael P.; Isobe, Toshiaki; Santangelo, Thomas J.; Shalev-Benami, Moran; Meier, Jordan L.; Schwartz, Schraga

doi:10.1038/s41586-020-2418-2

Cited by 186 publications

(182 citation statements)

References 80 publications

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“…Our rationale was since many acetyltransferases bind to positively charged substrates (such as histone tails and protein N-termini), incorporation of a negative charge near the active site may serve as an effective molecular recognition discriminant or, alternatively, facilitate the selective enrichment of acetyltransferases that recognize negatively charged substrates such as amino acids and RNA. 18,19 Based on this design, we synthesized two CoA analogues, CoA-(Ahx) 4 or CoA-D-(Ahx) 3 , using a previously reported route 20 from CoA and the cognate bromoacetamide peptide. Following HPLC purification and quantification, the two CoA analogues were separately coupled to NHS-Sepharose via the epsilon amine of a C-terminal lysine residue, giving access to affinity resins 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

Jing

Montaño

Levy

et al. 2020

Preprint

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Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the Coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based Sepharose pulldown resins differentiated by a single negatively charged residue, and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of ‘probe selectivity’ versus traditional ‘competitive chemical proteomics’. This reveals the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of pre-clinical therapeutic agents.

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Section: Resultsmentioning

confidence: 99%

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

Jing

Montaño

Levy

et al. 2020

Preprint

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“…In addition, methylations on the base and/or the ribose affect the hydration shells in complex ways ). Thiolation of U or C, acetylation of C, and isomerization of U to Ψ, can stabilize the 3'-endo sugar conformation, fill in space and enhance stacking power or base pairing (Davis 1995;Kawai et al 1992;Larsen et al 2015;Sas-Chen et al 2020). High-resolution crystallographic structures would be necessary to apprehend the effects of such complex modification scaffolds.…”

Section: Discussionmentioning

confidence: 99%

Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea

Wolff

Villette

Zumsteg

et al. 2020

RNA

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To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.

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“…Similarly, the examination of cryo-EM map of T. kodakarensis 70S ribosome at 16 Å resolution (Armache et al, 2013) also reveals the electron density that would be consistent with the absence of H98 and nucleotide extensions circularizing the 5´ and 3´ ends of the 23S rRNA ( Figure 5F). Recently, high resolution (2.5-3.0 Å) cryo-EM structures of T. kodakarensis 70S ribosome were reported (Sas- Chen et al, 2020), where structural models for H98 are lacking due to the lack of density for H98. Re-analysis of the cryo-EM map, including low-pass filtering, revealed additional density extending from the 5´ and 3´ ends (Supplementary Figures S2A,B), analogous to that observed in the previous T. kodakarensis 70S ribosome structure (Armache et al, 2013).…”

Section: H98 Is Located At the Surface Of The Large Subunit And Is Opmentioning

confidence: 99%

The 23S Ribosomal RNA From Pyrococcus furiosus Is Circularly Permuted

et al. 2020

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Synthesis and assembly of ribosomal components are fundamental cellular processes and generally well-conserved within the main groups of organisms. Yet, provocative variations to the general schemes exist. We have discovered an unusual processing pathway of pre-rRNA in extreme thermophilic archaea exemplified by Pyrococcus furiosus. The large subunit (LSU) rRNA is produced as a circularly permuted form through circularization followed by excision of Helix 98. As a consequence, the terminal domain VII that comprise the binding site for the signal recognition particle is appended to the 5´ end of the LSU rRNA that instead terminates in Domain VI carrying the Sarcin-Ricin Loop, the primary interaction site with the translational GTPases. To our knowledge, this is the first example of a true post-transcriptional circular permutation of a main functional molecule and the first example of rRNA fragmentation in archaea.

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Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

Cited by 186 publications

References 80 publications

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea

The 23S Ribosomal RNA From Pyrococcus furiosus Is Circularly Permuted

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