PMC 20201219 pmc.key 4872110 CC BY-NC no 1 1 10.1093/nar/gkw244 4872110 27084949 4304 9 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 4316 surname:Meyer;given-names:Britta surname:Wurm;given-names:Jan Philip surname:Sharma;given-names:Sunny surname:Immer;given-names:Carina surname:Pogoryelov;given-names:Denys surname:Kötter;given-names:Peter surname:Lafontaine;given-names:Denis L. J. surname:Wöhnert;given-names:Jens surname:Entian;given-names:Karl-Dieter TITLE front 44 2016 0 Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans 0.9939031 protein_type cleaner0 2023-07-04T14:27:44Z MESH: Ribosome biogenesis factor 0.99808204 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9947394 protein_type cleaner0 2023-07-04T12:19:28Z MESH: aminocarboxypropyl transferase 0.9733748 chemical cleaner0 2023-07-04T12:17:53Z CHEBI: 18S rRNA 0.9940844 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast 0.9955108 species cleaner0 2023-07-04T12:18:31Z MESH: humans ABSTRACT abstract 133 The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. 0.996774 taxonomy_domain cleaner0 2023-07-04T12:20:32Z DUMMY: eukaryotic 0.3907103 chemical cleaner0 2023-07-04T12:17:20Z CHEBI: rRNA 0.9956499 protein_state cleaner0 2023-07-04T16:03:17Z DUMMY: conserved 0.9075137 protein_state cleaner0 2023-07-04T14:38:36Z DUMMY: hypermodified 0.9943597 chemical cleaner0 2023-07-04T14:29:32Z CHEBI: nucleotide 0.9960326 chemical cleaner0 2023-07-04T12:18:06Z CHEBI: N1-methyl-N3-aminocarboxypropyl-pseudouridine 0.97784287 chemical cleaner0 2023-07-04T12:18:11Z CHEBI: m1acp3Ψ site SO: cleaner0 2023-07-04T14:28:59Z P-site chemical CHEBI: cleaner0 2023-07-04T14:29:09Z tRNA chemical CHEBI: cleaner0 2023-07-04T12:17:54Z 18S rRNA 0.99749947 chemical cleaner0 2023-07-04T12:30:02Z CHEBI: S-adenosylmethionine 0.99658644 chemical cleaner0 2023-07-04T14:29:37Z CHEBI: aminocarboxypropyl 0.9577856 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.99857605 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9746249 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast 0.9947389 species cleaner0 2023-07-04T12:27:15Z MESH: human protein PR: cleaner0 2023-07-04T14:08:01Z Tsr3 0.59193677 protein_state cleaner0 2023-07-04T16:03:23Z DUMMY: mutants chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp chemical CHEBI: cleaner0 2023-07-04T12:18:51Z 20S pre-rRNA 0.9973525 evidence cleaner0 2023-07-04T15:08:12Z DUMMY: crystal structure 0.9966714 taxonomy_domain cleaner0 2023-07-04T12:34:33Z DUMMY: archaeal 0.985493 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9943765 protein_type cleaner0 2023-07-04T12:19:34Z MESH: SPOUT-class RNA-methyltransferases 0.78219205 site cleaner0 2023-07-04T12:19:43Z SO: SAM binding mode 0.71633095 site cleaner0 2023-07-04T12:19:44Z SO: SAM binding mode 0.99791557 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9964366 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9982572 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9923056 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.7530078 protein_type cleaner0 2023-07-04T12:36:04Z MESH: acp transferase INTRO title_1 1127 INTRODUCTION INTRO paragraph 1140 Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. An increasing number of diseases—so called ribosomopathies—are associated with disturbed ribosome biogenesis. 0.996716 taxonomy_domain cleaner0 2023-07-04T12:20:32Z DUMMY: Eukaryotic 0.99148196 chemical cleaner0 2023-07-04T14:29:53Z CHEBI: small non-coding RNAs 0.99014926 chemical cleaner0 2023-07-04T14:29:57Z CHEBI: ribosomal RNAs 0.9890884 chemical cleaner0 2023-07-04T14:30:00Z CHEBI: rRNAs INTRO paragraph 1435 During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. 0.99573874 taxonomy_domain cleaner0 2023-07-04T12:20:32Z DUMMY: eukaryotic 0.8191487 chemical cleaner0 2023-07-04T12:20:15Z CHEBI: rRNA 0.9955368 chemical cleaner0 2023-07-04T14:30:03Z CHEBI: nucleotides 0.3742851 chemical cleaner0 2023-07-04T14:12:44Z CHEBI: rRNA 0.92576516 ptm cleaner0 2023-07-04T15:56:23Z MESH: methylations 0.75269485 chemical cleaner0 2023-07-04T14:30:08Z CHEBI: ribose 0.55292654 chemical cleaner0 2023-07-04T14:30:15Z CHEBI: uridine 0.94387376 chemical cleaner0 2023-07-04T12:27:44Z CHEBI: pseudouridine 0.85888165 complex_assembly cleaner0 2023-07-04T12:20:49Z GO: small nucleolar ribonucleoprotein particles 0.8105936 complex_assembly cleaner0 2023-07-04T12:20:53Z GO: snoRNPs 0.6118344 chemical cleaner0 2023-07-04T12:21:09Z CHEBI: 18S 0.39413956 chemical cleaner0 2023-07-04T12:21:18Z CHEBI: 25S 0.9883475 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast 0.52910745 chemical cleaner0 2023-07-04T12:21:28Z CHEBI: 28S 0.9891657 species cleaner0 2023-07-04T12:18:31Z MESH: humans 0.29764622 chemical cleaner0 2023-07-04T14:30:19Z CHEBI: rRNAs chemical CHEBI: cleaner0 2023-07-04T12:21:56Z snoRNA 0.9952312 species cleaner0 2023-07-04T12:22:09Z MESH: Saccharomyces cerevisiae chemical CHEBI: cleaner0 2023-07-04T12:17:54Z 18S rRNA 0.81744164 ptm cleaner0 2023-07-04T15:56:27Z MESH: methylations 0.9603707 ptm cleaner0 2023-07-04T15:56:32Z MESH: acetylations chemical CHEBI: cleaner0 2023-07-04T13:52:12Z 3-amino-3-carboxypropyl chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.8290558 ptm cleaner0 2023-07-04T15:56:36Z MESH: methylations chemical CHEBI: cleaner0 2023-07-04T12:23:02Z 25S rRNA 0.9947161 species cleaner0 2023-07-04T12:18:31Z MESH: humans chemical CHEBI: cleaner0 2023-07-04T12:17:55Z 18S rRNA 0.99721646 protein_state cleaner0 2023-07-04T16:03:48Z DUMMY: highly conserved 0.99383414 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast 0.9987739 protein cleaner0 2023-07-04T12:26:28Z PR: ScRrp8 0.9982759 protein cleaner0 2023-07-04T12:26:36Z PR: HsNML 0.9987858 protein cleaner0 2023-07-04T12:26:42Z PR: ScRcm1 0.99836296 protein cleaner0 2023-07-04T12:26:47Z PR: HsNSUN5 0.9988186 protein cleaner0 2023-07-04T12:26:52Z PR: ScNop2 0.9983693 protein cleaner0 2023-07-04T12:26:57Z PR: HsNSUN1 0.9948597 species cleaner0 2023-07-04T12:27:14Z MESH: human chemical CHEBI: cleaner0 2023-07-04T12:22:27Z 28S rRNA INTRO paragraph 2321 Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established. They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell. Importantly, malfunctions of several base modifying enzymes are linked to developmental diseases, aging or tumorigenesis. 0.6964196 chemical cleaner0 2023-07-04T14:30:23Z CHEBI: Ribosomal RNA chemical CHEBI: cleaner0 2023-07-04T12:24:16Z RNA 0.9017562 chemical cleaner0 2023-07-04T12:28:01Z CHEBI: pseudouridines 0.8979499 chemical cleaner0 2023-07-04T12:28:02Z CHEBI: pseudouridines 0.9240885 ptm cleaner0 2023-07-04T15:56:41Z MESH: base methylations ptm MESH: cleaner0 2023-07-04T15:57:20Z ribose methylations 0.5139551 chemical cleaner0 2023-07-04T12:23:53Z CHEBI: rRNA 0.94696105 chemical cleaner0 2023-07-04T14:30:27Z CHEBI: nucleotides 0.5629403 site cleaner0 2023-07-04T15:16:28Z SO: decoding 0.9937131 site cleaner0 2023-07-04T15:16:31Z SO: peptidyl transferase center 0.6016872 chemical cleaner0 2023-07-04T12:24:06Z CHEBI: rRNA 0.6801251 chemical cleaner0 2023-07-04T14:30:34Z CHEBI: rRNA INTRO paragraph 3215 The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B). There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former. The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years. 0.90391713 structure_element cleaner0 2023-07-04T15:58:02Z SO: loop capping helix 31 chemical CHEBI: cleaner0 2023-07-04T12:17:55Z 18S rRNA 0.5255018 residue_name cleaner0 2023-07-04T15:26:56Z SO: uridine 0.9984127 residue_name_number cleaner0 2023-07-04T15:01:12Z DUMMY: U1191 0.9965745 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast 0.9967144 chemical cleaner0 2023-07-04T12:24:41Z CHEBI: 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine 0.9299609 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.8263748 taxonomy_domain cleaner0 2023-07-04T12:24:54Z DUMMY: hamster 0.90488756 protein_state cleaner0 2023-07-04T16:03:57Z DUMMY: conserved in 0.9952035 taxonomy_domain cleaner0 2023-07-04T14:58:46Z DUMMY: eukaryotes 0.7234122 protein_state cleaner0 2023-07-04T14:38:36Z DUMMY: hypermodified 0.99725276 chemical cleaner0 2023-07-04T14:30:39Z CHEBI: nucleotide 0.9953161 site cleaner0 2023-07-04T15:16:37Z SO: P-site 0.38184616 chemical cleaner0 2023-07-04T14:30:43Z CHEBI: tRNA chemical CHEBI: cleaner0 2023-07-04T14:08:48Z snR35 structure_element SO: cleaner0 2023-07-04T14:04:38Z H/ACA 0.9066868 complex_assembly cleaner0 2023-07-04T12:29:16Z GO: snoRNP 0.8301994 chemical cleaner0 2023-07-04T14:30:46Z CHEBI: uridine 0.96616 chemical cleaner0 2023-07-04T12:27:45Z CHEBI: pseudouridine 0.9949054 protein_type cleaner0 2023-07-04T13:56:16Z MESH: SPOUT-class methyltransferase 0.99870765 protein cleaner0 2023-07-04T14:39:37Z PR: Nep1 0.99626595 protein cleaner0 2023-07-04T14:39:44Z PR: Emg1 0.9889643 chemical cleaner0 2023-07-04T12:27:45Z CHEBI: pseudouridine 0.9949991 chemical cleaner0 2023-07-04T14:30:51Z CHEBI: N1-methylpseudouridine ptm MESH: cleaner0 2023-07-04T14:40:35Z Methylation 0.9898722 ptm cleaner0 2023-07-04T15:57:29Z MESH: pseudouridylation 0.6104194 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.99630696 chemical cleaner0 2023-07-04T14:30:59Z CHEBI: N1-methyl-N3-aminocarboxypropyl-pseudouridine 0.9727791 complex_assembly cleaner0 2023-07-04T12:48:58Z GO: 40S 0.98769104 ptm cleaner0 2023-07-04T15:57:32Z MESH: pseudouridylation 0.99729675 chemical cleaner0 2023-07-04T12:30:01Z CHEBI: S-adenosylmethionine 0.9979736 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.90244967 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp gkw244fig1.jpg F1 FIG fig_caption 4441 Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. The asterisk indicates the C1-atom labeled in the 14C-incorporation assay. (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). (C) 14C-acp labeling of 18S rRNAs. Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification. All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545. The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). As a loading control, a structural stop is shown (asterisks). 0.9977913 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.66196173 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.98787004 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast 0.99423885 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.48301774 protein_state cleaner0 2023-07-04T14:38:36Z DUMMY: Hypermodified 0.9966366 chemical cleaner0 2023-07-04T14:31:26Z CHEBI: nucleotide 0.9877445 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.98037624 ptm cleaner0 2023-07-04T15:57:36Z MESH: pseudouridylation 0.99872464 complex_assembly cleaner0 2023-07-04T12:33:33Z GO: snoRNP35 0.810374 ptm cleaner0 2023-07-04T15:57:38Z MESH: N1-methylation 0.96947104 protein_type cleaner0 2023-07-04T13:56:57Z MESH: methyltransferase 0.99881953 protein cleaner0 2023-07-04T14:13:21Z PR: Nep1 0.4573778 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.99871767 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9611237 experimental_method cleaner0 2023-07-04T15:30:01Z MESH: 14C-incorporation assay 0.9936349 experimental_method cleaner0 2023-07-04T15:30:06Z MESH: RP-HPLC 0.862252 evidence cleaner0 2023-07-04T15:08:23Z DUMMY: elution profile 0.99435884 taxonomy_domain cleaner0 2023-07-04T12:18:25Z DUMMY: yeast chemical CHEBI: cleaner0 2023-07-04T12:17:55Z 18S rRNA 0.9959521 chemical cleaner0 2023-07-04T14:36:41Z CHEBI: nucleosides 0.88696 protein_state cleaner0 2023-07-04T14:38:36Z DUMMY: Hypermodified 0.9843954 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.9969202 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.99864334 mutant cleaner0 2023-07-04T12:32:05Z MESH: Δtsr3 mutant MESH: cleaner0 2023-07-04T13:24:04Z Δnep1 Δnop6 0.9283411 chemical cleaner0 2023-07-04T14:36:53Z CHEBI: 14C-acp chemical CHEBI: cleaner0 2023-07-04T12:33:54Z 18S rRNAs 0.9971446 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: Wild type 0.991848 protein_state cleaner0 2023-07-04T12:31:16Z DUMMY: WT 0.59906983 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.9985305 mutant cleaner0 2023-07-04T12:31:47Z MESH: U1191U 0.98280305 chemical cleaner0 2023-07-04T14:36:57Z CHEBI: 14C-acp 0.9748735 chemical cleaner0 2023-07-04T14:37:00Z CHEBI: 14C-acp 0.99853575 mutant cleaner0 2023-07-04T13:24:17Z MESH: U1191A 0.9680972 protein_state cleaner0 2023-07-04T16:04:06Z DUMMY: mutant 0.64080244 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.9984263 mutant cleaner0 2023-07-04T13:24:17Z MESH: U1191A mutant MESH: cleaner0 2023-07-04T12:32:05Z Δtsr3 0.9984206 mutant cleaner0 2023-07-04T12:32:05Z MESH: Δtsr3 chemical CHEBI: cleaner0 2023-07-04T12:38:51Z ethidium bromide 0.6604001 chemical cleaner0 2023-07-04T12:33:54Z CHEBI: 18S rRNAs 0.82524186 experimental_method cleaner0 2023-07-04T15:30:12Z MESH: Primer extension analysis chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.9893718 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.6189569 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.86530745 residue_number cleaner0 2023-07-04T15:32:51Z DUMMY: 1191 mutant MESH: cleaner0 2023-07-04T12:32:05Z Δtsr3 mutant MESH: cleaner0 2023-07-04T12:46:37Z Δtsr3 Δsnr35 0.9750293 experimental_method cleaner0 2023-07-04T15:30:16Z MESH: Primer extension analysis 0.9907601 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.62276906 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.9582116 experimental_method cleaner0 2023-07-04T15:30:21Z MESH: siRNA knockdown 0.99881566 protein cleaner0 2023-07-04T12:30:51Z PR: HsNEP1 0.99794334 protein cleaner0 2023-07-04T13:57:13Z PR: EMG1 0.99872357 protein cleaner0 2023-07-04T12:30:27Z PR: HsTSR3 0.6693486 chemical cleaner0 2023-07-04T15:30:34Z CHEBI: siRNAs 0.8873454 chemical cleaner0 2023-07-04T12:32:52Z CHEBI: siRNA 0.9972561 protein cleaner0 2023-07-04T12:30:26Z PR: HsTSR3 0.67660683 evidence cleaner0 2023-07-04T15:08:32Z DUMMY: primer extension signals INTRO paragraph 6133 Only a few acp transferring enzymes have been characterized until now. During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. 0.689649 chemical cleaner0 2023-07-04T12:34:58Z CHEBI: acp 0.9988757 chemical cleaner0 2023-07-04T14:37:17Z CHEBI: wybutosine 0.60589993 chemical cleaner0 2023-07-04T14:37:22Z CHEBI: nucleoside 0.99640274 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic 0.9962257 taxonomy_domain cleaner0 2023-07-04T12:34:33Z DUMMY: archaeal chemical CHEBI: cleaner0 2023-07-04T14:37:48Z phenylalanine chemical CHEBI: cleaner0 2023-07-04T14:38:00Z tRNA 0.9990005 protein cleaner0 2023-07-04T14:13:38Z PR: Tyw2 0.99896145 protein cleaner0 2023-07-04T12:35:12Z PR: Trm12 0.9952987 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99641234 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9981183 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.99501246 taxonomy_domain cleaner0 2023-07-04T12:34:33Z DUMMY: Archaeal 0.9989735 protein cleaner0 2023-07-04T14:13:49Z PR: Tyw2 0.9959848 evidence cleaner0 2023-07-04T15:08:39Z DUMMY: structure protein_type MESH: cleaner0 2023-07-04T13:58:12Z Rossmann-fold (class I) RNA-methyltransferases 0.84350294 site cleaner0 2023-07-04T15:17:12Z SO: SAM-binding mode 0.9315737 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.8116939 chemical cleaner0 2023-07-04T14:38:06Z CHEBI: diphtamide 0.9980925 chemical cleaner0 2023-07-04T12:37:15Z CHEBI: acp 0.9985505 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9903178 residue_name cleaner0 2023-07-04T15:27:03Z SO: histidine 0.99253607 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic 0.9430889 protein_type cleaner0 2023-07-04T13:52:57Z MESH: translation elongation factor 2 INTRO paragraph 6861 In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis. It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted. 0.9967096 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9333779 gene cleaner0 2023-07-04T12:35:31Z GENE: YOR006c 0.99700546 protein_state cleaner0 2023-07-04T16:04:14Z DUMMY: highly conserved 0.99592763 taxonomy_domain cleaner0 2023-07-04T14:58:51Z DUMMY: eukaryotes 0.9961767 taxonomy_domain cleaner0 2023-07-04T12:35:37Z DUMMY: archaea chemical CHEBI: cleaner0 2023-07-04T12:35:58Z 20S pre-rRNA 0.99367166 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA site SO: cleaner0 2023-07-04T12:37:54Z D-site 0.95902884 gene cleaner0 2023-07-04T12:39:20Z GENE: YOR006C protein PR: cleaner0 2023-07-04T14:14:03Z Twenty S rRNA accumulation 3 0.9505563 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 0.9926686 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA INTRO paragraph 7453 Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. 0.99876356 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.99231577 protein_type cleaner0 2023-07-04T12:36:03Z MESH: acp transferase protein_state DUMMY: cleaner0 2023-07-04T14:38:36Z hypermodified 0.99090844 chemical cleaner0 2023-07-04T14:38:16Z CHEBI: nucleotide 0.9904343 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.97093236 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99499077 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.9358644 protein_state cleaner0 2023-07-04T16:04:21Z DUMMY: catalytically defective 0.97263247 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9987662 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9625655 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.92116916 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.9973514 evidence cleaner0 2023-07-04T15:08:47Z DUMMY: crystal structures 0.9961379 taxonomy_domain cleaner0 2023-07-04T12:34:33Z DUMMY: archaeal 0.9986999 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.98849475 protein_type cleaner0 2023-07-04T12:36:16Z MESH: SPOUT-class RNA methyltransferases 0.99062526 protein_type cleaner0 2023-07-04T12:36:04Z MESH: acp transferase 0.9989262 protein cleaner0 2023-07-04T14:14:17Z PR: Tyw2 protein_type MESH: cleaner0 2023-07-04T14:14:42Z Rossmann-fold class of methyltransferase proteins 0.99854136 chemical cleaner0 2023-07-04T12:36:25Z CHEBI: SAM 0.9936279 protein_type cleaner0 2023-07-04T12:36:21Z MESH: methyltransferases 0.9980083 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9983473 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM METHODS title_1 8305 MATERIALS AND METHODS METHODS title_2 8327 Genetic constructions, growth conditions and yeast media METHODS paragraph 8384 Detailed descriptions are available in Supplementary Data. METHODS title_2 8443 Cell culture METHODS paragraph 8456 HCT116(+/+) cells (CCL-247; ATCC) were grown at 37°C in a humidified incubator under 5% CO2 in the McCoy's 5a modified (Sigma-Aldrich)/10% FBS media. All media were supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies). METHODS title_2 8707 DsiRNA inactivation and RT-qPCR METHODS paragraph 8739 Reverse transfection of HCT116 cells, DsiRNA inactivation and RT-qPCR using total human RNA are described in Supplementary Data. METHODS title_2 8868 Sucrose gradient analysis METHODS paragraph 8894 Detailed descriptions for analytical or preparative separations of ribosomal subunits or polysome gradients are provided in Supplementary Data. METHODS title_2 9038 HPLC analysis of 18S rRNA nucleosides METHODS paragraph 9076 40S subunits from 200 ml yeast culture were isolated by sucrose gradient centrifugation in a SW28 rotor as described above, and precipitated with 2.5 vol of 100% ethanol (−20°C over night). Precipitated 40S subunits were dissolved in water and the 18S rRNA was purified via spin columns (Ambion PureLink RNA Mini Kit). RNA fragments were hydrolysed and dephosphorylated as described by Gehrke and Kuo. HPLC analysis of rRNA nucleoside composition was performed using a Supelcosil LC-18S column (Sigma; 250 × 4.6 mm, 5 μm) with a pre-column (4.6 × 20 mm) as previously described. METHODS title_2 9661 14C labeling of 18S rRNA nucleotide Ψ(U)1191 METHODS paragraph 9708 To enhance 14C-labeling, mutants of interest were recombined with a Δmet13 deletion. Resulting strains were cultivated with l-[1-14C]-methionine (Hartmann Analytic, 0.1 mCi/ml, 54 mCi/mmol) as described before. From isotope labeled cells total RNA was isolated with the PureLink RNA Mini Kit (Ambion) after enzymatic cell lysis with zymolyase. Ribosomal RNAs were separated on a 4% denaturing polyacrylamide gel. After ethidium bromide straining gels were dried and analyzed by autoradiography for 3–5 days using a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare). METHODS title_2 10315 Northern blot analysis METHODS paragraph 10338 5 μg of total yeast RNAs extracted with phenol/chloroform were separated on 1.2% agarose gels in BPTE buffer for 16 h at 60V and afterwards transferred to a Biodyne B membrane by vacuum blotting. Oligonucleotides D/A2 or +1-A0 were radiolabeled using γ-[32P]-ATP and T4-polynucleotide kinase and hybridized to the membrane at 37°C. Signals were visualized by phosphoimaging with the Typhoon 9100 (GE Healthcare). RNA extraction from human cells, gel-electrophoresis and northern blotting were performed as described before. METHODS title_2 10867 Primer extension METHODS paragraph 10884 20 pmol of oligonucleotide PE-1191 complementary to yeast 18S rRNA nucleotides 1247–1228 were labeled with 50 μCi γ-[32P]-ATP using T4-polynucleotide kinase, purified via Sephadex G-25 and annealed to 500 ng of 18S rRNA. Primer annealing and reverse transcription were carried out as described by Sharma et al.. After precipitation with ethanol and 3 M NaAc pH 5.2 pellets were washed with 70% ethanol, dried and dissolved in 12 μl formamide loading dye. 2 μl of primer extension samples were separated on sequencing or mini gels which were dried after running and exposed on a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare). METHODS paragraph 11557 Primer extension on human RNA was performed using 5 μg of total RNA with AMV Reverse Transcriptase (Promega) and oligonucleotide PE_1248. Following alkaline hydrolysis, cDNAs were precipitated with ethanol, resuspended in acrylamide loading buffer and separated on a 6% (v/v) denaturing acrylamide gel in 0.5× TBE at 80 W for 1.5 h. After migration, the gels were dried and exposed to Fuji Imaging plates (Fujifilm). The signal was acquired with a Phosphor imager (FLA-7000, Fujifilm). METHODS title_2 12045 Protein detection and localization METHODS paragraph 12080 A description of the western blot detection of HA-fused Tsr3 in yeast crude extracts or sucrose gradients fractions is provided in Supplementary Data. For cellular localization Tsr3 was expressed as N-terminal fusion with yEGFP in a yeast strain encoding for ScNop56-mRFP. Protein localization in exponentially growing cells was visualized using a Leica TCS SP5. METHODS title_2 12443 In vitro SAM binding METHODS paragraph 12464 Purified SsTsr3 protein in 25 mM Tris–HCl pH 7.8 250 mM NaCl was mixed with S-[methyl-14C]-adenosyl-l-methionine (PerkinElmer; 20 μCi/ml, 58 mCi/mmol) and 0–10 mM non-labeled SAM in a binding buffer (50 mM Tris–HCl pH 7.8, 250 mM NaCl) in a total volume of 50 μl and incubated at 30°C for 10 min. Samples were passed over HAWP02500 membrane filters (Millipore) and unbound 14C-SAM was removed by washing three times with 5 ml buffer using a vacuum filtering equipment. Filter bound 14C-SAM was measured by liquid scintillation spectrometry in a Wallac 1401 scintillation counter. METHODS title_2 13053 Protein expression and purification METHODS paragraph 13089 Genes coding for archaeal Tsr3 homologs without any tags were obtained commercially (Genscript) in pET11a vectors and overexpressed in Escherichia coli BL21(DE3). Proteins were purified by a combination of heat shock and appropriate column chromatography steps as described in detail in the Supplementary Data. METHODS title_2 13400 Crystallization, X-ray data collection, structure calculation and refinement METHODS paragraph 13477 Initial hits for VdTsr3 and SsTsr3 were obtained using the Morpheus Screen (Molecular dimensions) and further refined as described in the Supplementary Data. Diffraction data were collected at the Swiss Light Source (Paul Scherer Institut). The structure of VdTsr3 was determined at 1.6 Å by SAD using a selenomethionine derivative. The structure of SsTsr3 was determined at 2.25 Å by molecular replacement using VdTsr3 as the search model. A detailed description of the data collection, processing, structure calculation and refinement procedures can be found in the Supplementary Data and in Supplementary Table S1. Structures were deposited in the Protein Data Bank as entries 5APG (VdTsr3) and 5AP8 (SsTsr3). METHODS title_2 14192 Analytical gel filtration METHODS paragraph 14218 For analytical gel filtration experiments a Sephadex S75 10/300 GL column (GE Healthcare) was used. 100 μl protein samples (25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) with a protein concentration of 150 μM were used. The flow rate was 0.5 ml/min. The column was calibrated using the marker proteins of the LMW gel filtration calibration kit (GE Healthcare). Protein elution was followed by recording the adsorption at a wavelength of λ = 280 nm. METHODS title_2 14691 Fluorescence quenching and fluorescence anisotropy measurements METHODS paragraph 14755 Fluorescence quenching and fluorescence anisotropy measurements were carried out in triplicates at 25°C on a Fluorolog 3 spectrometer (Horiba Jobin Yvon) equipped with polarizers. For fluorescence quenching with SAM, SAH and 5′-methylthioadenosine experiments the tryptophan fluorescence of SsTsr3 (200 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) was excited at 295 nm and emission spectra were recorded from 250 to 450 nm for each titration step. The fluorescence intensity at 351 nm for each titration step was normalized with regard to the fluorescence of the free protein and was used for deriving binding curves. KD's were derived by nonlinear regression with Origin 8.0 (Origin Labs) using Equation (1): (F is the normalized fluorescence intensity, a is the change in fluorescence intensity, c is the ligand concentration and KD is the dissociation constant). METHODS paragraph 15650 5′-Fluoresceine labeled RNAs for fluorescence anisotropy measurements were obtained commercially (Dharmacon), deprotected according to the manufacturer's protocol and the RNA concentration adjusted to 50 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl. Fluoresceine fluorescence was excited at 492 nm and emission was recorded at 516 nm. The data were fitted to Equation (1) (F is the normalized fluorescence anisotropy, a is the change in fluorescence anisotropy). RESULTS title_1 16113 RESULTS RESULTS title_2 16121 Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans 0.97725815 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9920869 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.76246583 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.996209 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99572146 species cleaner0 2023-07-04T12:18:31Z MESH: humans RESULTS paragraph 16202 The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. 0.995652 species cleaner0 2023-07-04T14:59:06Z MESH: S. cerevisiae protein_type MESH: cleaner0 2023-07-04T14:59:27Z 18S rRNA acp transferase 0.9912554 experimental_method cleaner0 2023-07-04T15:30:46Z MESH: HPLC 0.7911035 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA RESULTS paragraph 16449 For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3Ψ. In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation. 0.9983985 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 evidence DUMMY: cleaner0 2023-07-04T15:09:17Z HPLC elution profile 0.6619743 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.96492976 chemical cleaner0 2023-07-04T14:40:57Z CHEBI: nucleosides 0.9782849 protein_type cleaner0 2023-07-04T13:58:27Z MESH: pseudouridine-N1 methyltransferase 0.58084774 protein_state cleaner0 2023-07-04T16:04:34Z DUMMY: mutant 0.99856335 mutant cleaner0 2023-07-04T13:24:30Z MESH: Δnep1 0.9661906 experimental_method cleaner0 2023-07-04T15:30:50Z MESH: ESI-MS 0.44394964 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.6568178 chemical cleaner0 2023-07-04T15:33:08Z CHEBI: nucleotide 0.8943901 residue_number cleaner0 2023-07-04T15:33:13Z DUMMY: 1191 0.9051086 experimental_method cleaner0 2023-07-04T15:30:55Z MESH: in vivo14C incorporation assay 0.99287385 chemical cleaner0 2023-07-04T14:41:08Z CHEBI: 1-14C-methionine chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.7277 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.99619067 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.9984763 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9542309 mutant cleaner0 2023-07-04T12:42:59Z MESH: 18S U1191A 0.9941788 protein_state cleaner0 2023-07-04T16:04:37Z DUMMY: mutant 0.9883127 chemical cleaner0 2023-07-04T12:43:07Z CHEBI: 14C-aminocarboxypropyl RESULTS paragraph 17262 As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. Therefore the presence of the acp modification can be directly assessed by primer extension. Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. In contrast, in a Δtsr3 mutant no primer extension stop signal was present at this position. As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). 0.95631903 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9972254 residue_name_number cleaner0 2023-07-04T15:01:18Z DUMMY: U1191 0.9948807 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.6937653 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.96929646 experimental_method cleaner0 2023-07-04T15:31:01Z MESH: primer extension 0.9969904 protein_state cleaner0 2023-07-04T16:04:42Z DUMMY: wild-type 0.99475217 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast evidence DUMMY: cleaner0 2023-07-04T15:10:10Z primer extension stop signal 0.50405174 residue_number cleaner0 2023-07-04T14:41:25Z DUMMY: 1192 0.9984048 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.49695376 protein_state cleaner0 2023-07-04T16:04:47Z DUMMY: mutant 0.9985353 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.69914734 experimental_method cleaner0 2023-07-04T15:31:06Z MESH: deletion 0.98630756 ptm cleaner0 2023-07-04T15:57:43Z MESH: pseudouridylation 0.8456991 ptm cleaner0 2023-07-04T15:57:46Z MESH: N1-methylation 0.91452855 chemical cleaner0 2023-07-04T13:24:53Z CHEBI: acp3U 0.9984842 mutant cleaner0 2023-07-04T13:24:57Z MESH: Δnep1 0.9438199 chemical cleaner0 2023-07-04T12:27:45Z CHEBI: pseudouridine 0.8533305 protein_state cleaner0 2023-07-04T16:04:50Z DUMMY: not methylated 0.82459676 chemical cleaner0 2023-07-04T13:25:20Z CHEBI: acp3Ψ evidence DUMMY: cleaner0 2023-07-04T15:10:27Z primer extension stop signal 0.99689096 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.95136213 mutant cleaner0 2023-07-04T12:46:37Z MESH: Δtsr3 Δsnr35 0.63465166 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.9961945 protein_state cleaner0 2023-07-04T16:04:54Z DUMMY: unmodified 0.5297126 chemical cleaner0 2023-07-04T14:41:17Z CHEBI: U RESULTS paragraph 18125 The Tsr3 protein is highly conserved in yeast and humans (50% identity). Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA. As a control for loading, a structural stop is shown (asterisk, Figure 1E). Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). 0.99776614 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9970249 protein_state cleaner0 2023-07-04T16:04:59Z DUMMY: highly conserved 0.9957001 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99552494 species cleaner0 2023-07-04T12:18:31Z MESH: humans 0.9954537 species cleaner0 2023-07-04T12:27:15Z MESH: Human 0.97678864 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.994372 ptm cleaner0 2023-07-04T14:43:21Z MESH: m1acp3Ψ 0.9758189 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.76458 residue_number cleaner0 2023-07-04T15:33:17Z DUMMY: 1248 0.9573526 experimental_method cleaner0 2023-07-04T15:31:16Z MESH: siRNA-mediated depletion 0.9982256 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 species MESH: cleaner0 2023-07-04T12:27:15Z human 0.92925614 evidence cleaner0 2023-07-04T15:10:37Z DUMMY: acp primer extension arrest 0.37355578 chemical cleaner0 2023-07-04T12:32:53Z CHEBI: siRNA 0.8478326 chemical cleaner0 2023-07-04T12:32:53Z CHEBI: siRNA 0.9939262 experimental_method cleaner0 2023-07-04T15:31:19Z MESH: RT-qPCR 0.48623383 chemical cleaner0 2023-07-04T12:32:53Z CHEBI: siRNA 0.99075913 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 0.39353594 chemical cleaner0 2023-07-04T12:32:53Z CHEBI: siRNA 0.99643326 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.9987041 protein cleaner0 2023-07-04T12:30:27Z PR: HsTsr3 0.9926917 chemical cleaner0 2023-07-04T14:41:46Z CHEBI: RNA 0.9985796 protein cleaner0 2023-07-04T12:30:27Z PR: HsTsr3 chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.89912796 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.91223305 chemical cleaner0 2023-07-04T14:43:53Z CHEBI: nucleotide 0.9950204 ptm cleaner0 2023-07-04T14:43:37Z MESH: Ψ1248 0.94343853 structure_element cleaner0 2023-07-04T15:58:08Z SO: helix 31 0.9956008 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.85411745 experimental_method cleaner0 2023-07-04T15:31:23Z MESH: siRNA-mediated depletion 0.7850956 protein_type cleaner0 2023-07-04T12:44:26Z MESH: Ψ1248 N1-methyltransferase 0.9987066 protein cleaner0 2023-07-04T14:19:15Z PR: Nep1 0.99816966 protein cleaner0 2023-07-04T14:19:24Z PR: Emg1 0.9749694 evidence cleaner0 2023-07-04T15:10:40Z DUMMY: primer extension arrest RESULTS title_2 19249 Phenotypic characterization of Δtsr3 mutants 0.99842864 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 RESULTS paragraph 19298 Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.83371437 chemical cleaner0 2023-07-04T12:17:55Z CHEBI: 18S rRNA 0.9968771 protein_state cleaner0 2023-07-04T16:05:11Z DUMMY: highly conserved 0.9964318 taxonomy_domain cleaner0 2023-07-04T14:58:55Z DUMMY: eukaryotes 0.99618405 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9985261 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9985886 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9986823 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.9297403 ptm cleaner0 2023-07-04T15:57:51Z MESH: pseudouridylation 0.9752127 protein cleaner0 2023-07-04T14:19:42Z PR: Nep1 0.94331276 residue_number cleaner0 2023-07-04T15:01:31Z DUMMY: 1191 0.99091315 mutant cleaner0 2023-07-04T13:27:48Z MESH: Δtsr3 Δnep1 gene GENE: cleaner0 2023-07-04T14:19:51Z nep1 0.9982834 mutant cleaner0 2023-07-04T12:32:33Z MESH: Δnop6 0.95741963 mutant cleaner0 2023-07-04T13:38:41Z MESH: Δtsr3 Δsnr35 Δnep1 0.9960886 protein_state cleaner0 2023-07-04T16:05:13Z DUMMY: unmodified 0.99495137 residue_name_number cleaner0 2023-07-04T15:01:36Z DUMMY: U1191 gkw244fig2.jpg F2 FIG fig_caption 19866 Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. Right gel: Ethidium bromide staining showing 18S and 28S rRNAs. (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. 0.9915274 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.5132569 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 0.9977635 mutant cleaner0 2023-07-04T13:27:59Z MESH: Δtrs3 0.98250157 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.99517316 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 0.60299724 chemical cleaner0 2023-07-04T14:43:58Z CHEBI: siRNAs 0.993353 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99692875 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.99348444 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9944301 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.9975858 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9975426 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.93985236 mutant cleaner0 2023-07-04T12:46:37Z MESH: Δtsr3 Δsnr35 0.9951563 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.8314011 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 0.6921355 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.6743955 protein cleaner0 2023-07-04T13:28:45Z PR: SNR35 0.9975089 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9980921 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.95296794 residue_name_number cleaner0 2023-07-04T15:01:40Z DUMMY: U1191 experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z agar diffusion assays 0.99426323 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99784076 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9782717 protein_state cleaner0 2023-07-04T16:05:28Z DUMMY: deletion mutant 0.5368966 chemical cleaner0 2023-07-04T14:44:02Z CHEBI: paromomycin 0.7071477 chemical cleaner0 2023-07-04T14:44:04Z CHEBI: hygromycin B 0.99770373 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.8474186 experimental_method cleaner0 2023-07-04T15:31:49Z MESH: Northern blot analysis 0.96813726 experimental_method cleaner0 2023-07-04T15:31:53Z MESH: siRNA depletion 0.9944193 protein cleaner0 2023-07-04T12:30:27Z PR: HsTSR3 0.5565004 chemical cleaner0 2023-07-04T14:44:12Z CHEBI: siRNAs 0.9163474 chemical cleaner0 2023-07-04T12:32:53Z CHEBI: siRNA 0.9965947 chemical cleaner0 2023-07-04T14:44:23Z CHEBI: 18SE 0.994317 chemical cleaner0 2023-07-04T14:44:25Z CHEBI: 47S chemical CHEBI: cleaner0 2023-07-04T14:44:45Z 45S pre-RNAs 0.9552973 protein cleaner0 2023-07-04T12:30:27Z PR: HsTSR3 0.9932202 chemical cleaner0 2023-07-04T14:45:09Z CHEBI: 18S chemical CHEBI: cleaner0 2023-07-04T14:45:01Z 28S rRNAs 0.9947326 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9974341 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.94156015 experimental_method cleaner0 2023-07-04T15:31:57Z MESH: fluorescence microscopy mutant MESH: cleaner0 2023-07-04T13:31:12Z GFP-fused Tsr3 experimental_method MESH: cleaner0 2023-07-04T13:30:33Z differential interference contrast experimental_method MESH: cleaner0 2023-07-04T13:30:45Z DIC mutant MESH: cleaner0 2023-07-04T13:29:17Z GFP-Tsr3 mutant MESH: cleaner0 2023-07-04T13:29:35Z Nop56-mRFP mutant MESH: cleaner0 2023-07-04T13:29:53Z GFP-Tsr3 mutant MESH: cleaner0 2023-07-04T13:30:14Z Nop56-mRFP experimental_method MESH: cleaner0 2023-07-04T13:30:46Z DIC 0.9706644 evidence cleaner0 2023-07-04T15:10:47Z DUMMY: Elution profile experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z sucrose gradient separation 0.9942629 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.5915116 complex_assembly cleaner0 2023-07-04T15:51:06Z GO: ribosomal subunits 0.53156686 complex_assembly cleaner0 2023-07-04T15:51:10Z GO: polysomes 0.9763651 experimental_method cleaner0 2023-07-04T15:32:02Z MESH: western blot 0.38005278 chemical cleaner0 2023-07-04T13:32:16Z CHEBI: 3xHA 0.99773884 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.99546224 mutant cleaner0 2023-07-04T12:49:50Z MESH: Tsr3-3xHA 0.9745136 experimental_method cleaner0 2023-07-04T15:32:06Z MESH: SDS-PAGE 0.7641838 protein cleaner0 2023-07-04T14:08:01Z PR: TSR3 protein_state DUMMY: cleaner0 2023-07-04T15:48:29Z fusion 0.99737096 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.7335174 chemical cleaner0 2023-07-04T14:45:13Z CHEBI: 3xHA 0.9918937 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast RESULTS paragraph 21519 The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B). chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.6377991 chemical cleaner0 2023-07-04T15:33:35Z CHEBI: nucleotide 0.9465645 residue_number cleaner0 2023-07-04T15:33:41Z DUMMY: 1191 mutant MESH: cleaner0 2023-07-04T12:32:06Z Δtsr3 0.98296434 gene cleaner0 2023-07-04T14:20:33Z GENE: nep1 0.9572939 protein_state cleaner0 2023-07-04T16:05:41Z DUMMY: mutant 0.99866676 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.99684036 chemical cleaner0 2023-07-04T14:45:21Z CHEBI: paromomycin 0.9956605 chemical cleaner0 2023-07-04T14:45:26Z CHEBI: hygromycin B 0.99802125 chemical cleaner0 2023-07-04T14:46:17Z CHEBI: G418 0.9970824 chemical cleaner0 2023-07-04T14:46:19Z CHEBI: cycloheximide 0.5927976 chemical cleaner0 2023-07-04T14:46:23Z CHEBI: paromomycin chemical CHEBI: cleaner0 2023-07-04T14:46:40Z hygromycin B mutant MESH: cleaner0 2023-07-04T12:46:31Z Δtsr3 Δsnr35 RESULTS paragraph 22073 In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. A minor effect on 20S rRNA accumulation was also observed for Δsnr35, but - probably due to different strain backgrounds – to a weaker extent than described earlier. In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). 0.9893476 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99783283 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 mutant MESH: cleaner0 2023-07-04T12:46:37Z Δtsr3 Δsnr35 chemical CHEBI: cleaner0 2023-07-04T12:48:25Z 20S pre-rRNA 0.994905 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.8788376 chemical cleaner0 2023-07-04T14:46:48Z CHEBI: 20S rRNA 0.9984927 mutant cleaner0 2023-07-04T12:32:25Z MESH: Δsnr35 0.98830533 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.78692925 experimental_method cleaner0 2023-07-04T15:34:08Z MESH: depletion of 0.9969137 protein cleaner0 2023-07-04T12:30:27Z PR: HsTsr3 0.98755056 species cleaner0 2023-07-04T12:27:15Z MESH: human chemical CHEBI: cleaner0 2023-07-04T12:48:26Z 20S pre-rRNA 0.9749642 chemical cleaner0 2023-07-04T14:46:53Z CHEBI: 18S-E 0.9874466 protein cleaner0 2023-07-04T14:08:01Z PR: Tsr3 0.9903704 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.9830163 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.99749655 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.98898333 complex_assembly cleaner0 2023-07-04T14:47:21Z GO: 35S protein PR: cleaner0 2023-07-04T16:06:14Z Tsr3 0.9809749 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.92224807 complex_assembly cleaner0 2023-07-04T14:47:31Z GO: 47S 0.9139037 complex_assembly cleaner0 2023-07-04T14:47:39Z GO: 45S 0.9626368 experimental_method cleaner0 2023-07-04T15:34:12Z MESH: Northern blot RESULTS paragraph 23037 Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). 0.8237121 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.9034129 protein cleaner0 2023-07-04T14:08:02Z PR: TSR3 0.94708794 complex_assembly cleaner0 2023-07-04T12:48:57Z GO: 40S complex_assembly GO: cleaner0 2023-07-04T12:49:13Z 60S 0.8403921 mutant cleaner0 2023-07-04T12:46:37Z MESH: Δtsr3 Δsnr35 0.63479966 evidence cleaner0 2023-07-04T15:19:48Z DUMMY: polysome profiles complex_assembly GO: cleaner0 2023-07-04T15:12:16Z 80S ribosomes 0.8691986 complex_assembly cleaner0 2023-07-04T12:49:13Z GO: 60S 0.6724853 complex_assembly cleaner0 2023-07-04T12:48:58Z GO: 40S RESULTS title_2 23496 Cellular localization of Tsr3 in S. cerevisiae 0.9898936 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9955408 species cleaner0 2023-07-04T14:59:36Z MESH: S. cerevisiae RESULTS paragraph 23543 Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E). Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. 0.98735833 experimental_method cleaner0 2023-07-04T15:34:16Z MESH: Fluorescence microscopy protein_state DUMMY: cleaner0 2023-07-04T14:10:17Z GFP-tagged 0.99816704 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9956487 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9889811 protein cleaner0 2023-07-04T14:25:18Z PR: Nop56 chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.9948225 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.94876456 complex_assembly cleaner0 2023-07-04T12:48:58Z GO: 40S 0.8942659 experimental_method cleaner0 2023-07-04T15:34:20Z MESH: polysome gradient separation 0.99817866 mutant cleaner0 2023-07-04T12:49:50Z MESH: Tsr3-3xHA evidence DUMMY: cleaner0 2023-07-04T15:34:54Z distribution on a density gradient 0.9985233 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.8188459 complex_assembly cleaner0 2023-07-04T15:52:58Z GO: pre-40S subunits 0.988572 experimental_method cleaner0 2023-07-04T15:35:02Z MESH: pre-ribosome affinity purifications RESULTS title_2 24341 Structure of Tsr3 0.9653325 evidence cleaner0 2023-07-04T15:19:53Z DUMMY: Structure 0.99821293 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 RESULTS paragraph 24359 Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). 0.99540585 species cleaner0 2023-07-04T14:59:40Z MESH: S. cerevisiae 0.8784227 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.99813855 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.99666363 taxonomy_domain cleaner0 2023-07-04T12:35:38Z DUMMY: archaea 0.91221446 protein_type cleaner0 2023-07-04T14:27:49Z MESH: Tsr3-like proteins 0.99677074 taxonomy_domain cleaner0 2023-07-04T12:34:33Z DUMMY: archaeal 0.9985812 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.99671614 taxonomy_domain cleaner0 2023-07-04T12:35:38Z DUMMY: archaea 0.9966486 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast RESULTS paragraph 24699 To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B). protein PR: cleaner0 2023-07-04T14:08:02Z Tsr3 0.9229415 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.9971113 protein_state cleaner0 2023-07-04T16:06:21Z DUMMY: highly conserved 0.9294225 experimental_method cleaner0 2023-07-04T15:35:08Z MESH: expressed 0.99860305 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.9922924 protein_state cleaner0 2023-07-04T16:06:24Z DUMMY: mutant 0.9922793 experimental_method cleaner0 2023-07-04T15:35:13Z MESH: primer extension 0.9610331 experimental_method cleaner0 2023-07-04T15:35:16Z MESH: Northern blotting experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z truncations 0.6994033 residue_range cleaner0 2023-07-04T15:42:34Z DUMMY: 45 aa experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z truncations 0.7585292 residue_range cleaner0 2023-07-04T15:42:53Z DUMMY: 76 aa chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.9966659 protein_state cleaner0 2023-07-04T16:06:28Z DUMMY: full-length chemical CHEBI: cleaner0 2023-07-04T12:50:34Z 20S pre-RNA protein PR: cleaner0 2023-07-04T14:08:02Z Tsr3 0.73044395 residue_range cleaner0 2023-07-04T15:42:56Z DUMMY: 90 aa 0.8059091 residue_range cleaner0 2023-07-04T15:42:59Z DUMMY: 46 aa gkw244fig3.jpg F3 FIG fig_caption 25394 Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a Δtsr3 deletion strain. (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. 0.9951761 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9630516 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp chemical CHEBI: cleaner0 2023-07-04T12:17:56Z 18S rRNA 0.94555557 protein cleaner0 2023-07-04T14:08:02Z PR: TSR3 protein PR: cleaner0 2023-07-04T14:08:02Z TSR3 0.9973393 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.990005 experimental_method cleaner0 2023-07-04T15:35:22Z MESH: Primer extension analysis chemical CHEBI: cleaner0 2023-07-04T12:17:56Z 18S rRNA chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.99398273 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast protein PR: cleaner0 2023-07-04T14:08:02Z TSR3 0.33643785 experimental_method cleaner0 2023-07-04T15:35:29Z MESH: deletions 0.9236323 residue_range cleaner0 2023-07-04T15:43:08Z DUMMY: 36 0.8441494 residue_range cleaner0 2023-07-04T15:43:11Z DUMMY: 45 experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z deletions 0.9539571 residue_range cleaner0 2023-07-04T15:43:24Z DUMMY: 43 0.8959835 residue_range cleaner0 2023-07-04T15:43:27Z DUMMY: 76 evidence DUMMY: cleaner0 2023-07-04T15:20:24Z primer extension stop 0.99560976 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.99218893 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.87861127 residue_range cleaner0 2023-07-04T15:43:30Z DUMMY: 37–223 0.8790083 residue_range cleaner0 2023-07-04T15:43:32Z DUMMY: 46–223 0.9892195 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9662222 residue_range cleaner0 2023-07-04T15:43:36Z DUMMY: 46–270 0.9844143 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type evidence DUMMY: cleaner0 2023-07-04T15:21:09Z primer extension block 0.9899976 experimental_method cleaner0 2023-07-04T15:35:32Z MESH: Northern blot 0.88851964 chemical cleaner0 2023-07-04T12:48:26Z CHEBI: 20S pre-rRNA chemical CHEBI: cleaner0 2023-07-04T15:43:53Z 20S rRNA 0.99354035 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9015322 residue_range cleaner0 2023-07-04T15:43:40Z DUMMY: 46–270 0.99736816 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z deletion protein PR: cleaner0 2023-07-04T14:08:02Z Tsr3 0.8548698 residue_range cleaner0 2023-07-04T15:43:56Z DUMMY: 37–223 0.69136864 residue_range cleaner0 2023-07-04T15:43:59Z DUMMY: 46–223 RESULTS paragraph 26504 Thus, the archaeal homologs correspond to the functional core of Tsr3. In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. 0.99613106 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.9906142 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9757283 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 taxonomy_domain DUMMY: cleaner0 2023-07-04T12:53:34Z thermophilic archaea 0.9104248 experimental_method cleaner0 2023-07-04T15:35:36Z MESH: crystallization 0.9963098 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.9979527 protein cleaner0 2023-07-04T14:21:07Z PR: Nep1 0.9948228 chemical cleaner0 2023-07-04T14:47:49Z CHEBI: N1-methyl-N3-acp-pseudouridine 0.99405485 evidence cleaner0 2023-07-04T15:21:14Z DUMMY: crystals 0.9711174 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9944575 taxonomy_domain cleaner0 2023-07-04T12:53:09Z DUMMY: crenarchaeal 0.994915 species cleaner0 2023-07-04T12:52:13Z MESH: Vulcanisaeta distributa 0.99615246 protein cleaner0 2023-07-04T12:52:36Z PR: VdTsr3 0.99521303 species cleaner0 2023-07-04T12:52:19Z MESH: Sulfolobus solfataricus 0.99852824 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9984504 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9986474 protein cleaner0 2023-07-04T12:52:48Z PR: SsTsr3 0.99852055 protein cleaner0 2023-07-04T12:52:42Z PR: ScTsr3 0.98933065 structure_element cleaner0 2023-07-04T15:58:31Z SO: core region 0.99867487 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.9788138 residue_range cleaner0 2023-07-04T15:44:20Z DUMMY: 46–223 0.9926 species cleaner0 2023-07-04T12:52:24Z MESH: S. solfataricus 0.9942549 chemical cleaner0 2023-07-04T14:47:54Z CHEBI: nucleotide structure_element SO: cleaner0 2023-07-04T15:58:50Z loop capping helix 31 0.7716433 chemical cleaner0 2023-07-04T12:52:59Z CHEBI: 16S rRNA 0.9940617 species cleaner0 2023-07-04T12:52:30Z MESH: V. distributa RESULTS paragraph 27381 Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). The structure of VdTsr3 can be divided into two domains (Figure 4A). The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. Both domains are tightly packed against each other. Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. Thus, the VdTsr3 structure contains a deep trefoil knot. The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. 0.894146 evidence cleaner0 2023-07-04T15:21:18Z DUMMY: Crystals 0.99865985 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9334132 evidence cleaner0 2023-07-04T15:21:21Z DUMMY: crystals 0.99864453 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9986304 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.98529214 experimental_method cleaner0 2023-07-04T15:35:47Z MESH: crystallized protein_state DUMMY: cleaner0 2023-07-04T16:06:55Z in complex with protein_state DUMMY: cleaner0 2023-07-04T16:07:13Z endogenous species MESH: cleaner0 2023-07-04T12:53:57Z E. coli 0.9981237 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9985421 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9599628 evidence cleaner0 2023-07-04T15:21:24Z DUMMY: crystals 0.9970573 protein_state cleaner0 2023-07-04T16:07:19Z DUMMY: apo 0.97736746 evidence cleaner0 2023-07-04T15:21:27Z DUMMY: structure 0.99853814 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9950263 experimental_method cleaner0 2023-07-04T15:35:50Z MESH: single-wavelength anomalous diffraction phasing 0.99373865 experimental_method cleaner0 2023-07-04T15:35:53Z MESH: Se-SAD 0.98817676 chemical cleaner0 2023-07-04T14:47:58Z CHEBI: Se 0.97762144 chemical cleaner0 2023-07-04T14:48:01Z CHEBI: selenomethionine chemical CHEBI: cleaner0 2023-07-04T14:48:38Z seleno-substituted SAM 0.9897954 evidence cleaner0 2023-07-04T15:21:29Z DUMMY: structure 0.9986041 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9906043 experimental_method cleaner0 2023-07-04T15:35:57Z MESH: molecular replacement 0.9984681 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9881765 evidence cleaner0 2023-07-04T15:21:33Z DUMMY: structure 0.9985281 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.97266805 structure_element cleaner0 2023-07-04T12:54:49Z SO: N-terminal domain 0.9462447 residue_range cleaner0 2023-07-04T15:44:24Z DUMMY: 1–92 0.98937815 structure_element cleaner0 2023-07-04T12:54:54Z SO: α/β-structure 0.9480546 structure_element cleaner0 2023-07-04T12:54:57Z SO: five-stranded all-parallel β-sheet 0.99170226 structure_element cleaner0 2023-07-04T12:55:01Z SO: β5↑ 0.7989434 structure_element cleaner0 2023-07-04T12:55:04Z SO: β3↑ 0.96559316 structure_element cleaner0 2023-07-04T12:55:06Z SO: β4↑ 0.9663431 structure_element cleaner0 2023-07-04T12:55:08Z SO: β1↑ 0.79176414 structure_element cleaner0 2023-07-04T12:55:10Z SO: β2↑ 0.89726985 structure_element cleaner0 2023-07-04T12:55:12Z SO: loops 0.9906088 structure_element cleaner0 2023-07-04T12:55:15Z SO: β1 0.9852632 structure_element cleaner0 2023-07-04T12:55:21Z SO: β2 0.9770098 structure_element cleaner0 2023-07-04T12:55:33Z SO: β3 0.9747893 structure_element cleaner0 2023-07-04T12:55:27Z SO: β4 0.9883571 structure_element cleaner0 2023-07-04T12:55:27Z SO: β4 0.9778195 structure_element cleaner0 2023-07-04T12:55:41Z SO: β5 0.99453574 structure_element cleaner0 2023-07-04T12:56:03Z SO: α-helices 0.98398674 structure_element cleaner0 2023-07-04T12:56:06Z SO: α1 0.98732066 structure_element cleaner0 2023-07-04T12:56:12Z SO: α2 0.9914119 structure_element cleaner0 2023-07-04T12:56:17Z SO: α3 0.9466435 structure_element cleaner0 2023-07-04T12:56:26Z SO: loop 0.99244314 structure_element cleaner0 2023-07-04T12:55:22Z SO: β2 0.9890349 structure_element cleaner0 2023-07-04T12:55:34Z SO: β3 0.99597985 structure_element cleaner0 2023-07-04T12:56:28Z SO: 310-helix 0.78040475 structure_element cleaner0 2023-07-04T12:56:30Z SO: Helices 0.99200433 structure_element cleaner0 2023-07-04T12:56:07Z SO: α1 0.9761588 structure_element cleaner0 2023-07-04T12:56:13Z SO: α2 0.9830198 structure_element cleaner0 2023-07-04T12:56:34Z SO: five-stranded β-sheet 0.9943243 structure_element cleaner0 2023-07-04T12:56:18Z SO: α3 0.9390106 structure_element cleaner0 2023-07-04T12:56:37Z SO: β-sheet 0.9730159 structure_element cleaner0 2023-07-04T12:56:39Z SO: C-terminal domain 0.97484416 residue_range cleaner0 2023-07-04T15:44:28Z DUMMY: 93–184 0.98369145 structure_element cleaner0 2023-07-04T12:56:42Z SO: globular all α-helical structure 0.9930827 structure_element cleaner0 2023-07-04T12:56:45Z SO: α-helices 0.97653323 structure_element cleaner0 2023-07-04T12:56:48Z SO: α4 to α9 0.9843185 structure_element cleaner0 2023-07-04T12:56:54Z SO: C-terminal domain 0.93844694 residue_range cleaner0 2023-07-04T15:44:31Z DUMMY: 92 aa 0.99063116 structure_element cleaner0 2023-07-04T12:57:00Z SO: loop 0.992326 structure_element cleaner0 2023-07-04T12:57:02Z SO: β-strand 0.98643595 structure_element cleaner0 2023-07-04T12:55:34Z SO: β3 0.9944263 structure_element cleaner0 2023-07-04T12:57:05Z SO: α-helix 0.49424106 structure_element cleaner0 2023-07-04T12:56:13Z SO: α2 structure_element SO: cleaner0 2023-07-04T12:54:38Z N-terminal domain 0.9982253 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9945821 evidence cleaner0 2023-07-04T15:21:36Z DUMMY: structure 0.8641489 structure_element cleaner0 2023-07-04T12:57:07Z SO: deep trefoil knot 0.99427176 evidence cleaner0 2023-07-04T15:21:39Z DUMMY: structure 0.9986442 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99742305 protein_state cleaner0 2023-07-04T16:07:22Z DUMMY: apo 0.9985202 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9943686 evidence cleaner0 2023-07-04T15:21:42Z DUMMY: RMSD 0.9889154 evidence cleaner0 2023-07-04T15:21:45Z DUMMY: structure structure_element SO: cleaner0 2023-07-04T12:57:29Z α-helix structure_element SO: cleaner0 2023-07-04T12:57:43Z α8 protein_state DUMMY: cleaner0 2023-07-04T16:07:46Z absence of structure_element SO: cleaner0 2023-07-04T12:58:00Z α-helix structure_element SO: cleaner0 2023-07-04T12:58:10Z α9 0.99849546 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 gkw244fig4.jpg F4 FIG fig_caption 29350 Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. The bound S-adenosylmethionine is shown in a stick representation and colored by atom type. A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. Vertical lines indicate the elution volumes of molecular weight markers. Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. 0.99868983 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9898712 protein_type cleaner0 2023-07-04T13:58:35Z MESH: SPOUT-class RNA methyltransferases 0.98756117 evidence cleaner0 2023-07-04T15:21:51Z DUMMY: X-ray structure 0.9984108 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9932092 structure_element cleaner0 2023-07-04T15:58:55Z SO: β-strands 0.9937828 structure_element cleaner0 2023-07-04T15:58:59Z SO: α-helices 0.9317618 structure_element cleaner0 2023-07-04T12:54:50Z SO: N-terminal domain 0.99329805 structure_element cleaner0 2023-07-04T15:59:02Z SO: α-helices 0.97738254 structure_element cleaner0 2023-07-04T12:56:54Z SO: C-terminal domain 0.9962704 chemical cleaner0 2023-07-04T12:30:02Z CHEBI: S-adenosylmethionine 0.8493673 structure_element cleaner0 2023-07-04T15:59:06Z SO: topological knot 0.9957776 evidence cleaner0 2023-07-04T15:21:55Z DUMMY: structure 0.9981481 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9955183 evidence cleaner0 2023-07-04T15:21:57Z DUMMY: structure 0.9939426 experimental_method cleaner0 2023-07-04T15:36:03Z MESH: Structural superposition 0.97521293 evidence cleaner0 2023-07-04T12:59:20Z DUMMY: X-ray structures 0.99852186 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.99706334 protein_state cleaner0 2023-07-04T16:07:59Z DUMMY: SAM-bound 0.99863017 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99736446 protein_state cleaner0 2023-07-04T16:08:02Z DUMMY: apo 0.99600226 structure_element cleaner0 2023-07-04T15:59:09Z SO: α-helix 0.52897996 structure_element cleaner0 2023-07-04T12:59:08Z SO: α8 0.99844724 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99594086 structure_element cleaner0 2023-07-04T15:59:13Z SO: α-helix 0.66921145 structure_element cleaner0 2023-07-04T12:59:01Z SO: α9 0.9980621 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9956155 species cleaner0 2023-07-04T12:58:29Z MESH: S. pombe 0.99886984 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 0.98729503 protein_type cleaner0 2023-07-04T13:58:56Z MESH: SPOUT-class RNA methyltransferase 0.9988362 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9872602 experimental_method cleaner0 2023-07-04T15:36:06Z MESH: superposition 0.9986639 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.99880326 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 0.9941336 evidence cleaner0 2023-07-04T12:59:20Z DUMMY: X-ray structures 0.9899495 experimental_method cleaner0 2023-07-04T15:36:10Z MESH: Analytical gel filtration 0.7014155 evidence cleaner0 2023-07-04T15:22:56Z DUMMY: profiles 0.99863523 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.99863416 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.6680443 oligomeric_state cleaner0 2023-07-04T15:59:23Z DUMMY: monomeric 0.92951286 species cleaner0 2023-07-04T12:58:35Z MESH: Vd 0.9925858 species cleaner0 2023-07-04T12:52:14Z MESH: Vulcanisaeta distributa 0.97483873 species cleaner0 2023-07-04T12:58:44Z MESH: Ss 0.9946715 species cleaner0 2023-07-04T12:52:20Z MESH: Sulfolobus solfataricus RESULTS paragraph 30665 Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. However, no structural similarity to an RLI-domain was detectable. This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3). experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z Structure predictions 0.9984465 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9907023 structure_element cleaner0 2023-07-04T15:59:34Z SO: RLI domain 0.8769801 structure_element cleaner0 2023-07-04T15:59:45Z SO: bacterial like’ ferredoxin fold 0.7951602 protein_state cleaner0 2023-07-04T16:08:09Z DUMMY: conserved 0.99081624 residue_name cleaner0 2023-07-04T12:59:30Z SO: cysteine 0.9892566 structure_element cleaner0 2023-07-04T15:59:50Z SO: RLI-domain 0.9950836 experimental_method cleaner0 2023-07-04T15:37:01Z MESH: alanine replacement mutations 0.9880019 residue_name cleaner0 2023-07-04T12:59:31Z SO: cysteine 0.9986821 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 RESULTS paragraph 31085 The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. Most SPOUT-class RNA-methyltransferases are homodimers. A notable exception is Trm10. Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. 0.9482632 structure_element cleaner0 2023-07-04T13:00:51Z SO: β-strand topology 0.9970552 structure_element cleaner0 2023-07-04T13:00:42Z SO: trefoil knot 0.9952211 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.99835443 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.991478 protein_type cleaner0 2023-07-04T13:00:23Z MESH: SPOUT-class RNA-methyltransferase 0.9869234 experimental_method cleaner0 2023-07-04T15:37:04Z MESH: DALI search 0.9954552 protein_type cleaner0 2023-07-04T13:00:15Z MESH: tRNA methyltransferase 0.9988763 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 evidence DUMMY: cleaner0 2023-07-04T15:02:13Z DALI Z-score 0.6525595 residue_name_number cleaner0 2023-07-04T15:02:48Z DUMMY: G9 0.58903307 residue_name_number cleaner0 2023-07-04T15:02:54Z DUMMY: A9 0.99626356 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.99567163 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic 0.9963192 chemical cleaner0 2023-07-04T14:48:45Z CHEBI: tRNAs 0.99841475 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.99876726 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9794103 structure_element cleaner0 2023-07-04T13:00:48Z SO: β-sheet element 0.99884915 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 0.97613883 structure_element cleaner0 2023-07-04T13:00:46Z SO: β-strand 0.98917216 structure_element cleaner0 2023-07-04T12:55:22Z SO: β2 0.99724126 structure_element cleaner0 2023-07-04T13:00:39Z SO: trefoil knot 0.9988411 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 0.99886334 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9988117 protein cleaner0 2023-07-04T14:21:38Z PR: Nep1 0.9986298 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9943409 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.98864883 protein_type cleaner0 2023-07-04T13:00:26Z MESH: SPOUT-class of RNA methyltransferases 0.9988576 protein cleaner0 2023-07-04T14:21:45Z PR: Nep1 0.9988325 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 evidence DUMMY: cleaner0 2023-07-04T15:02:13Z DALI Z-score 0.9988111 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.99880946 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 0.9966035 protein_type cleaner0 2023-07-04T12:19:35Z MESH: SPOUT-class RNA-methyltransferases 0.9852149 oligomeric_state cleaner0 2023-07-04T15:00:04Z DUMMY: homodimers 0.99888307 protein cleaner0 2023-07-04T12:58:54Z PR: Trm10 0.991344 experimental_method cleaner0 2023-07-04T15:37:08Z MESH: Gel filtration 0.9986413 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9987723 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9127686 oligomeric_state cleaner0 2023-07-04T15:00:07Z DUMMY: monomeric 0.9987997 protein cleaner0 2023-07-04T12:58:55Z PR: Trm10 RESULTS paragraph 32231 So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B). 0.99843997 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9985026 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9970258 chemical cleaner0 2023-07-04T14:48:49Z CHEBI: RNA 0.98358846 chemical cleaner0 2023-07-04T14:48:52Z CHEBI: nucleotide 0.99894077 protein cleaner0 2023-07-04T14:22:01Z PR: Tyw2 0.98726386 chemical cleaner0 2023-07-04T14:48:55Z CHEBI: wybutosine nucleotides 0.9973935 chemical cleaner0 2023-07-04T14:48:59Z CHEBI: tRNAs 0.99888283 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9989672 protein cleaner0 2023-07-04T14:22:10Z PR: Tyw2 0.9862101 structure_element cleaner0 2023-07-04T13:01:04Z SO: all-parallel β-sheet 0.60714936 structure_element cleaner0 2023-07-04T13:01:10Z SO: knot structure 0.99880314 protein cleaner0 2023-07-04T14:22:17Z PR: Tyw2 0.9827729 protein_type cleaner0 2023-07-04T13:01:07Z MESH: class-I-or Rossmann-fold class of methyltransferases RESULTS title_2 32732 Cofactor binding of Tsr3 0.9985978 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 RESULTS paragraph 32757 The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C). In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B). 0.99810785 site cleaner0 2023-07-04T15:17:20Z SO: SAM-binding site 0.9987777 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.924005 structure_element cleaner0 2023-07-04T16:00:03Z SO: N- and C-terminal domains 0.9967799 structure_element cleaner0 2023-07-04T16:00:07Z SO: trefoil knot 0.9960068 protein_type cleaner0 2023-07-04T12:19:35Z MESH: SPOUT-class RNA-methyltransferases 0.84465826 chemical cleaner0 2023-07-04T14:49:03Z CHEBI: adenine 0.9990513 residue_name_number cleaner0 2023-07-04T15:03:01Z DUMMY: L93 0.9896722 structure_element cleaner0 2023-07-04T12:55:42Z SO: β5 0.99908054 residue_name_number cleaner0 2023-07-04T15:03:04Z DUMMY: Y108 0.9925528 structure_element cleaner0 2023-07-04T16:00:13Z SO: loop 0.99131435 structure_element cleaner0 2023-07-04T12:55:42Z SO: β5 0.6410846 structure_element cleaner0 2023-07-04T16:00:24Z SO: N-terminal 0.9918751 structure_element cleaner0 2023-07-04T16:00:31Z SO: α4 0.8204628 structure_element cleaner0 2023-07-04T12:56:55Z SO: C-terminal domain 0.6286845 chemical cleaner0 2023-07-04T14:49:06Z CHEBI: adenine 0.99832124 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9990734 residue_name_number cleaner0 2023-07-04T15:03:07Z DUMMY: L45 0.99424934 structure_element cleaner0 2023-07-04T12:55:34Z SO: β3 0.99873954 residue_name_number cleaner0 2023-07-04T15:03:10Z DUMMY: P47 0.99907047 residue_name_number cleaner0 2023-07-04T15:03:12Z DUMMY: W73 0.9933528 structure_element cleaner0 2023-07-04T12:56:18Z SO: α3 0.73925114 structure_element cleaner0 2023-07-04T12:54:50Z SO: N-terminal domain 0.9990564 residue_name_number cleaner0 2023-07-04T15:03:15Z DUMMY: L93 0.99907124 residue_name_number cleaner0 2023-07-04T15:03:19Z DUMMY: L110 0.9804981 structure_element cleaner0 2023-07-04T16:00:38Z SO: loop 0.99220556 structure_element cleaner0 2023-07-04T12:55:42Z SO: β5 0.9943011 structure_element cleaner0 2023-07-04T16:00:41Z SO: α4 0.99905497 residue_name_number cleaner0 2023-07-04T15:03:24Z DUMMY: A115 0.9954548 structure_element cleaner0 2023-07-04T16:00:44Z SO: α5 0.8100722 structure_element cleaner0 2023-07-04T12:56:55Z SO: C-terminal domain chemical CHEBI: cleaner0 2023-07-04T14:49:27Z ribose 0.99836034 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9990386 residue_name_number cleaner0 2023-07-04T15:03:28Z DUMMY: I69 0.99780697 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.99834704 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.99905115 residue_name_number cleaner0 2023-07-04T15:03:32Z DUMMY: T19 0.9845276 structure_element cleaner0 2023-07-04T12:56:07Z SO: α1 0.9990582 residue_name_number cleaner0 2023-07-04T15:03:34Z DUMMY: T112 0.98914295 structure_element cleaner0 2023-07-04T16:00:49Z SO: α4 structure_element SO: cleaner0 2023-07-04T12:56:55Z C-terminal domain 0.99828535 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9978372 site cleaner0 2023-07-04T15:17:30Z SO: hydrophobic pocket 0.9990553 residue_name_number cleaner0 2023-07-04T15:03:39Z DUMMY: W73 0.9990553 residue_name_number cleaner0 2023-07-04T15:03:41Z DUMMY: A76 0.9903403 structure_element cleaner0 2023-07-04T12:56:18Z SO: α3 0.999012 residue_name_number cleaner0 2023-07-04T15:03:44Z DUMMY: W73 0.9969107 protein_state cleaner0 2023-07-04T16:08:14Z DUMMY: highly conserved 0.7578435 protein_type cleaner0 2023-07-04T14:27:55Z MESH: Tsr3 proteins 0.99897087 residue_name_number cleaner0 2023-07-04T15:03:48Z DUMMY: A76 0.728626 chemical cleaner0 2023-07-04T14:49:33Z CHEBI: amino acids 0.9963153 protein_type cleaner0 2023-07-04T13:59:17Z MESH: RNA-methyltransferases 0.99891496 protein cleaner0 2023-07-04T12:58:55Z PR: Trm10 0.99788874 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9983612 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM protein_state DUMMY: cleaner0 2023-07-04T16:12:29Z Tsr3-bound gkw244fig5.jpg F5 FIG fig_caption 34453 SAM-binding by Tsr3. (A) Close-up view of the SAM-binding pocket of VdTsr3. Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow. Hydrogen bonds are indicated by dashed lines. (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation. Atoms are colored as in (A). A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). (E) Binding of 14C-labeled SAM to SsTsr3. Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. Addition of unlabeled SAM competes with the binding of labeled SAM. A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). chemical CHEBI: cleaner0 2023-07-04T12:36:26Z SAM 0.9984106 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9982338 site cleaner0 2023-07-04T15:17:35Z SO: SAM-binding pocket 0.9986333 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.93889225 chemical cleaner0 2023-07-04T14:49:39Z CHEBI: sulfur 0.9985293 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.84584075 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9984308 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.8229357 protein_state cleaner0 2023-07-04T16:08:21Z DUMMY: bound to 0.99847895 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9986192 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.99353164 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.9986284 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9983114 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.86399364 protein_state cleaner0 2023-07-04T16:08:25Z DUMMY: bound to 0.9903197 protein_type cleaner0 2023-07-04T13:59:28Z MESH: RNA methyltransferase 0.9988348 protein cleaner0 2023-07-04T12:58:55Z PR: Trm10 0.99634093 protein_state cleaner0 2023-07-04T16:08:31Z DUMMY: Bound 0.99854654 chemical cleaner0 2023-07-04T12:36:26Z CHEBI: SAM 0.9953718 evidence cleaner0 2023-07-04T15:23:13Z DUMMY: X-ray structure complex_assembly GO: cleaner0 2023-07-04T15:56:05Z Trm10/SAH 0.9987343 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM chemical CHEBI: cleaner0 2023-07-04T14:50:00Z SAM 0.99836856 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.98396105 evidence cleaner0 2023-07-04T15:23:18Z DUMMY: Tryptophan fluorescence quenching curves 0.99866307 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9976242 chemical cleaner0 2023-07-04T14:50:05Z CHEBI: 5′-methyl-thioadenosine 0.9985287 chemical cleaner0 2023-07-04T13:02:55Z CHEBI: SAH chemical CHEBI: cleaner0 2023-07-04T14:50:38Z 14C-labeled SAM 0.99834895 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99848664 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM protein_state DUMMY: cleaner0 2023-07-04T16:08:57Z presence of 0.99842 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9985732 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9985936 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9953957 mutant cleaner0 2023-07-04T13:34:09Z MESH: W66A 0.7282947 protein_state cleaner0 2023-07-04T16:09:01Z DUMMY: mutant 0.9984365 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99610424 residue_name_number cleaner0 2023-07-04T15:03:58Z DUMMY: W73 0.9983192 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9986083 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.97968125 experimental_method cleaner0 2023-07-04T15:37:32Z MESH: Primer extension chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.99094594 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.8286558 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.9967998 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.99414736 mutant cleaner0 2023-07-04T13:34:14Z MESH: Tsr3-S62D 0.96076906 mutant cleaner0 2023-07-04T13:34:17Z MESH: -E111A 0.98010665 mutant cleaner0 2023-07-04T13:34:19Z MESH: –W114A 0.8035865 chemical cleaner0 2023-07-04T12:48:26Z CHEBI: 20S pre-rRNA 0.99793786 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.8783016 experimental_method cleaner0 2023-07-04T15:37:43Z MESH: northern blot protein_state DUMMY: cleaner0 2023-07-04T13:34:46Z 3xHA tagged 0.3629817 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.71937597 protein_state cleaner0 2023-07-04T13:35:01Z DUMMY: mutants 0.9967206 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.92499804 experimental_method cleaner0 2023-07-04T15:37:46Z MESH: western blot RESULTS paragraph 36003 Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. 0.9926367 evidence cleaner0 2023-07-04T15:23:25Z DUMMY: Binding affinities 0.99860746 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9977343 chemical cleaner0 2023-07-04T14:50:45Z CHEBI: 5′-methylthioadenosin 0.9984421 chemical cleaner0 2023-07-04T13:02:56Z CHEBI: SAH 0.99856395 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99246246 experimental_method cleaner0 2023-07-04T15:37:49Z MESH: tryptophan fluorescence quenching 0.99861467 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.8481856 protein_state cleaner0 2023-07-04T16:09:07Z DUMMY: stable 0.9949457 protein_state cleaner0 2023-07-04T16:09:12Z DUMMY: SAM-free 0.99857163 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9594057 protein_state cleaner0 2023-07-04T16:09:15Z DUMMY: bound 0.9980363 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9939552 evidence cleaner0 2023-07-04T15:23:29Z DUMMY: KD 0.9500855 evidence cleaner0 2023-07-04T15:23:32Z DUMMY: SAM-KD's 0.99625754 protein_type cleaner0 2023-07-04T13:59:48Z MESH: SPOUT-class methyltransferases 0.99783933 chemical cleaner0 2023-07-04T14:50:48Z CHEBI: 5′-methylthioadenosin chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.9985222 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9977331 chemical cleaner0 2023-07-04T14:50:53Z CHEBI: S-adenosylhomocysteine 0.998492 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.6109229 evidence cleaner0 2023-07-04T15:23:39Z DUMMY: affinity 0.5327044 evidence cleaner0 2023-07-04T15:23:43Z DUMMY: KD 0.99848396 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9978147 site cleaner0 2023-07-04T15:17:52Z SO: hydrophobic pocket 0.99859685 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9986084 chemical cleaner0 2023-07-04T12:37:16Z CHEBI: acp 0.99764055 site cleaner0 2023-07-04T15:18:02Z SO: substrate binding pocket RESULTS paragraph 37081 Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). 0.9705291 mutant cleaner0 2023-07-04T13:35:40Z MESH: W66A experimental_method MESH: cleaner0 2023-07-04T13:35:36Z mutation 0.99866235 residue_name_number cleaner0 2023-07-04T15:04:06Z DUMMY: W73 0.9982755 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9987612 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.6666495 evidence cleaner0 2023-07-04T15:23:48Z DUMMY: SAM-binding 0.98855877 experimental_method cleaner0 2023-07-04T15:37:56Z MESH: filter binding assay 0.99703217 protein_state cleaner0 2023-07-04T12:31:11Z DUMMY: wild type 0.8402462 experimental_method cleaner0 2023-07-04T15:38:13Z MESH: W to A mutation 0.9988821 residue_name_number cleaner0 2023-07-04T15:04:11Z DUMMY: W114 0.9987276 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 protein_type MESH: cleaner0 2023-07-04T12:36:04Z acp transferase 0.99874383 residue_name_number cleaner0 2023-07-04T15:04:14Z DUMMY: T19 chemical CHEBI: cleaner0 2023-07-04T12:36:27Z SAM 0.9686928 experimental_method cleaner0 2023-07-04T15:38:21Z MESH: mutations 0.9987029 residue_name_number cleaner0 2023-07-04T15:04:22Z DUMMY: T17 0.9986645 residue_name_number cleaner0 2023-07-04T15:04:24Z DUMMY: T19 0.99822766 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9717845 residue_name cleaner0 2023-07-04T15:27:23Z SO: A 0.97430634 residue_name cleaner0 2023-07-04T15:27:26Z SO: D 0.99270034 evidence cleaner0 2023-07-04T15:23:52Z DUMMY: SAM-binding affinity 0.9986903 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.87185836 evidence cleaner0 2023-07-04T15:23:55Z DUMMY: KD 0.98011154 experimental_method cleaner0 2023-07-04T15:38:24Z MESH: mutation 0.9986607 residue_name_number cleaner0 2023-07-04T15:04:16Z DUMMY: S62 0.99876213 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.9630524 residue_name cleaner0 2023-07-04T15:27:31Z SO: D 0.96706134 residue_name cleaner0 2023-07-04T15:27:34Z SO: A chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.96824175 experimental_method cleaner0 2023-07-04T15:38:27Z MESH: primer extension analysis RESULTS paragraph 37802 The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). chemical CHEBI: cleaner0 2023-07-04T12:37:16Z acp 0.99884063 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9921693 chemical cleaner0 2023-07-04T12:27:45Z CHEBI: pseudouridine 0.9988368 residue_name_number cleaner0 2023-07-04T15:04:28Z DUMMY: D70 0.99746466 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.99062866 structure_element cleaner0 2023-07-04T12:55:27Z SO: β4 0.9985306 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9653026 protein_state cleaner0 2023-07-04T16:09:22Z DUMMY: conserved as 0.9061018 residue_name cleaner0 2023-07-04T15:27:46Z SO: D 0.91264516 residue_name cleaner0 2023-07-04T15:27:49Z SO: E 0.99593264 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.9953687 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic 0.9853893 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9630044 experimental_method cleaner0 2023-07-04T15:38:32Z MESH: Mutations 0.9986683 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.93534106 residue_name cleaner0 2023-07-04T15:27:59Z SO: A 0.99817395 residue_name_number cleaner0 2023-07-04T15:04:34Z DUMMY: D63 0.99293053 evidence cleaner0 2023-07-04T15:24:07Z DUMMY: SAM-binding affinity evidence DUMMY: cleaner0 2023-07-04T15:24:16Z KD 0.96987814 experimental_method cleaner0 2023-07-04T15:38:37Z MESH: mutation 0.9986234 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.99509156 mutant cleaner0 2023-07-04T13:03:48Z MESH: E111A protein_type MESH: cleaner0 2023-07-04T12:36:04Z acp transferase RESULTS title_2 38456 RNA-binding of Tsr3 0.9985703 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 RESULTS paragraph 38476 Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. Its negatively charged sulfate group might mimic an RNA backbone phosphate. Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z Analysis of the electrostatic surface properties 0.99870753 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9920173 site cleaner0 2023-07-04T15:18:08Z SO: positively charged surface patches 0.99826443 site cleaner0 2023-07-04T15:18:12Z SO: SAM-binding site 0.99828744 site cleaner0 2023-07-04T15:18:16Z SO: RNA-binding site 0.6921352 chemical cleaner0 2023-07-04T14:51:02Z CHEBI: MES 0.99710965 evidence cleaner0 2023-07-04T15:24:55Z DUMMY: crystal structure 0.99869245 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9235827 protein_state cleaner0 2023-07-04T16:09:32Z DUMMY: complexed to 0.99885 residue_name_number cleaner0 2023-07-04T15:04:48Z DUMMY: K22 0.9862877 structure_element cleaner0 2023-07-04T16:00:56Z SO: helix 0.91419095 structure_element cleaner0 2023-07-04T12:56:07Z SO: α1 0.8520645 chemical cleaner0 2023-07-04T14:51:05Z CHEBI: sulfate 0.9982864 chemical cleaner0 2023-07-04T14:51:10Z CHEBI: RNA 0.99472684 structure_element cleaner0 2023-07-04T16:01:02Z SO: Helix 0.7284624 structure_element cleaner0 2023-07-04T12:56:07Z SO: α1 0.9987287 residue_name_number cleaner0 2023-07-04T15:04:52Z DUMMY: K17 0.9987393 residue_name_number cleaner0 2023-07-04T15:04:54Z DUMMY: R25 0.98964345 structure_element cleaner0 2023-07-04T16:01:08Z SO: loop 0.99876547 residue_name_number cleaner0 2023-07-04T15:04:57Z DUMMY: R9 0.9933456 structure_element cleaner0 2023-07-04T16:01:11Z SO: helix 0.7019198 structure_element cleaner0 2023-07-04T12:56:18Z SO: α3 0.99877614 residue_name_number cleaner0 2023-07-04T15:05:00Z DUMMY: K74 0.9987398 residue_name_number cleaner0 2023-07-04T15:05:03Z DUMMY: R75 0.99875665 residue_name_number cleaner0 2023-07-04T15:05:06Z DUMMY: K82 0.9987343 residue_name_number cleaner0 2023-07-04T15:05:08Z DUMMY: R85 0.9987583 residue_name_number cleaner0 2023-07-04T15:05:11Z DUMMY: K87 0.9972134 protein_state cleaner0 2023-07-04T16:09:36Z DUMMY: conserved 0.9950637 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.99446905 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic 0.9987722 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.91891503 structure_element cleaner0 2023-07-04T12:56:55Z SO: C-terminal domain 0.993428 structure_element cleaner0 2023-07-04T16:01:14Z SO: α-helices 0.9057671 structure_element cleaner0 2023-07-04T16:01:18Z SO: α5 0.9876522 structure_element cleaner0 2023-07-04T16:01:21Z SO: α7 0.98904145 experimental_method cleaner0 2023-07-04T15:38:43Z MESH: triple mutation 0.99426585 protein_state cleaner0 2023-07-04T16:09:50Z DUMMY: conserved 0.9986034 residue_name_number cleaner0 2023-07-04T15:05:14Z DUMMY: R60 0.9987668 residue_name_number cleaner0 2023-07-04T15:05:17Z DUMMY: K65 0.9987972 residue_name_number cleaner0 2023-07-04T15:05:19Z DUMMY: R131 0.9349181 residue_name cleaner0 2023-07-04T15:28:11Z SO: A 0.99876946 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 protein_type MESH: cleaner0 2023-07-04T12:36:04Z acp transferase gkw244fig6.jpg F6 FIG fig_caption 39635 RNA-binding of Tsr3. (A) Electrostatic charge distribution on the surface of VdTsr3. Surface areas colored in blue are positively charged whereas red areas are negatively charged. SAM is shown in a stick representation. Also shown in stick representation is a negatively charged MES ion. Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. (C) Binding of SsTsr3 to RNA. 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). Oligo-U9-RNA was used for comparison (black). The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. 0.99881566 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.99865013 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.9986539 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.86521053 chemical cleaner0 2023-07-04T14:51:17Z CHEBI: MES 0.6367843 protein_state cleaner0 2023-07-04T16:10:01Z DUMMY: Conserved chemical CHEBI: cleaner0 2023-07-04T14:51:36Z amino acids 0.9185542 structure_element cleaner0 2023-07-04T16:01:27Z SO: helix 31 chemical CHEBI: cleaner0 2023-07-04T14:00:32Z rRNAs 0.99534893 species cleaner0 2023-07-04T14:59:47Z MESH: S. cerevisiae 0.9950297 species cleaner0 2023-07-04T12:52:25Z MESH: S. solfataricus protein_state DUMMY: cleaner0 2023-07-04T14:38:37Z hypermodified 0.99140173 chemical cleaner0 2023-07-04T14:51:42Z CHEBI: nucleotide 0.994428 species cleaner0 2023-07-04T12:52:25Z MESH: S. solfataricus protein_state DUMMY: cleaner0 2023-07-04T14:38:37Z hypermodified 0.9902706 chemical cleaner0 2023-07-04T14:51:46Z CHEBI: nucleotide 0.99886656 protein cleaner0 2023-07-04T14:23:09Z PR: NEP1 0.99883705 protein cleaner0 2023-07-04T14:08:02Z PR: TSR3 0.9968796 chemical cleaner0 2023-07-04T14:51:49Z CHEBI: N1-methyl-N3-acp-pseudouridine 0.99873513 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9947417 chemical cleaner0 2023-07-04T14:51:53Z CHEBI: RNA chemical CHEBI: cleaner0 2023-07-04T14:53:25Z fluoresceine chemical CHEBI: cleaner0 2023-07-04T14:52:18Z RNA 0.97259057 protein_state cleaner0 2023-07-04T16:10:05Z DUMMY: native 0.79190457 oligomeric_state cleaner0 2023-07-04T13:36:35Z DUMMY: 20mer 0.7217474 protein_state cleaner0 2023-07-04T16:10:09Z DUMMY: stabilized 0.8586677 oligomeric_state cleaner0 2023-07-04T13:36:47Z DUMMY: 20mer_GC structure_element SO: cleaner0 2023-07-04T16:01:44Z helix 31 chemical CHEBI: cleaner0 2023-07-04T14:00:55Z rRNA 0.99525017 species cleaner0 2023-07-04T12:52:25Z MESH: S. solfataricus 0.7044766 experimental_method cleaner0 2023-07-04T15:38:51Z MESH: titrated with increasing amounts 0.9987336 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.74837214 chemical cleaner0 2023-07-04T14:52:35Z CHEBI: fluoresceine 0.9626857 evidence cleaner0 2023-07-04T15:25:14Z DUMMY: fluorescence anisotropy 0.8108233 evidence cleaner0 2023-07-04T15:25:18Z DUMMY: binding curve 0.5670902 oligomeric_state cleaner0 2023-07-04T13:37:00Z DUMMY: 20mer 0.8563897 oligomeric_state cleaner0 2023-07-04T13:37:11Z DUMMY: 20mer_GC 0.99646825 chemical cleaner0 2023-07-04T14:52:39Z CHEBI: Oligo-U9-RNA 0.8098977 oligomeric_state cleaner0 2023-07-04T13:37:23Z DUMMY: 20mer_GC 0.98672026 chemical cleaner0 2023-07-04T14:52:43Z CHEBI: RNA 0.8701082 experimental_method cleaner0 2023-07-04T15:39:51Z MESH: titrated 0.9987534 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.99848866 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.4945502 protein_state cleaner0 2023-07-04T15:40:04Z DUMMY: Mutants 0.99845564 protein cleaner0 2023-07-04T12:52:43Z PR: ScTsr3 0.99465525 residue_name_number cleaner0 2023-07-04T15:05:31Z DUMMY: R60 0.99793184 residue_name_number cleaner0 2023-07-04T15:05:32Z DUMMY: K65 0.99827754 residue_name_number cleaner0 2023-07-04T15:05:35Z DUMMY: R131 0.9979954 residue_name_number cleaner0 2023-07-04T15:05:37Z DUMMY: K17 0.9981592 residue_name_number cleaner0 2023-07-04T15:05:40Z DUMMY: K22 0.998417 residue_name_number cleaner0 2023-07-04T15:05:42Z DUMMY: R91 0.9984724 protein cleaner0 2023-07-04T12:52:37Z PR: VdTsr3 0.7232749 experimental_method cleaner0 2023-07-04T15:40:11Z MESH: expressed 0.99725455 mutant cleaner0 2023-07-04T12:32:06Z MESH: Δtsr3 0.98106325 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.64899063 evidence cleaner0 2023-07-04T15:25:23Z DUMMY: primer extension stop 0.99662733 protein_state cleaner0 2023-07-04T12:31:12Z DUMMY: wild type experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z Combination of the three point mutations 0.9958204 mutant cleaner0 2023-07-04T13:37:27Z MESH: R60A 0.9970393 mutant cleaner0 2023-07-04T13:37:30Z MESH: K65A 0.997195 mutant cleaner0 2023-07-04T13:37:32Z MESH: R131A chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.5971787 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA RESULTS paragraph 41195 In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. 0.99782616 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9878314 experimental_method cleaner0 2023-07-04T15:40:24Z MESH: titrated 0.9984755 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 0.9882069 protein_state cleaner0 2023-07-04T16:10:28Z DUMMY: RNase-free chemical CHEBI: cleaner0 2023-07-04T14:53:44Z fluoresceine 0.99855906 chemical cleaner0 2023-07-04T14:53:51Z CHEBI: RNA 0.9866698 evidence cleaner0 2023-07-04T15:25:31Z DUMMY: affinity 0.9863573 experimental_method cleaner0 2023-07-04T15:40:28Z MESH: fluorescence anisotropy measurements 0.99838126 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 protein_state DUMMY: cleaner0 2023-07-04T16:10:48Z apo 0.9739899 protein_state cleaner0 2023-07-04T16:10:51Z DUMMY: bound 0.7132142 oligomeric_state cleaner0 2023-07-04T14:54:04Z DUMMY: 20mer 0.99687374 chemical cleaner0 2023-07-04T14:54:08Z CHEBI: RNA 0.964221 structure_element cleaner0 2023-07-04T16:01:48Z SO: helix 31 0.9939218 species cleaner0 2023-07-04T12:52:25Z MESH: S. solfataricus chemical CHEBI: cleaner0 2023-07-04T12:52:59Z 16S rRNA 0.8649587 evidence cleaner0 2023-07-04T15:26:21Z DUMMY: KD 0.9732021 structure_element cleaner0 2023-07-04T16:01:51Z SO: hairpin 0.9424103 oligomeric_state cleaner0 2023-07-04T13:37:48Z DUMMY: 20mer-GC 0.55589414 evidence cleaner0 2023-07-04T15:26:24Z DUMMY: KD 0.99726105 chemical cleaner0 2023-07-04T14:54:13Z CHEBI: oligoU-RNA 0.98908454 protein_state cleaner0 2023-07-04T16:10:59Z DUMMY: bound 0.9814804 evidence cleaner0 2023-07-04T15:25:36Z DUMMY: affinity 0.99879575 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9905457 evidence cleaner0 2023-07-04T15:25:48Z DUMMY: RNA-affinity 0.99852633 protein cleaner0 2023-07-04T12:52:49Z PR: SsTsr3 evidence DUMMY: cleaner0 2023-07-04T15:25:45Z KD 0.8882387 oligomeric_state cleaner0 2023-07-04T13:38:02Z DUMMY: 20mer-GC chemical CHEBI: cleaner0 2023-07-04T13:38:18Z RNA DISCUSS title_1 41928 DISCUSSION DISCUSS paragraph 41939 U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). This makes it unique in eukaryotic rRNA modification. The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. 0.9954489 residue_name_number cleaner0 2023-07-04T15:05:48Z DUMMY: U1191 0.5176854 protein_state cleaner0 2023-07-04T14:38:37Z DUMMY: hypermodified 0.9957968 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.4961639 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.99721104 protein_state cleaner0 2023-07-04T16:11:05Z DUMMY: strongly conserved 0.996487 taxonomy_domain cleaner0 2023-07-04T14:59:00Z DUMMY: eukaryotes 0.99650055 chemical cleaner0 2023-07-04T12:24:42Z CHEBI: 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine 0.9825304 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ structure_element SO: cleaner0 2023-07-04T14:04:37Z H/ACA complex_assembly GO: cleaner0 2023-07-04T12:29:22Z snoRNP 0.9979875 protein cleaner0 2023-07-04T14:25:34Z PR: snR35 0.9965995 protein cleaner0 2023-07-04T14:23:24Z PR: Nep1 0.9951173 protein cleaner0 2023-07-04T14:23:31Z PR: Emg1 0.99738914 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.99688345 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic 0.399971 chemical cleaner0 2023-07-04T14:54:17Z CHEBI: rRNA 0.9253921 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.84618723 structure_element cleaner0 2023-07-04T16:01:56Z SO: helix 31 chemical CHEBI: cleaner0 2023-07-04T12:17:56Z 18S rRNA structure_element SO: cleaner0 2023-07-04T16:02:39Z helices 18, 24, 34 and 44 0.9734116 chemical cleaner0 2023-07-04T14:54:23Z CHEBI: acp3U 0.99549747 species cleaner0 2023-07-04T13:05:16Z MESH: Haloferax volcanii 0.9920008 chemical cleaner0 2023-07-04T14:54:41Z CHEBI: nucleotides 0.9965738 taxonomy_domain cleaner0 2023-07-04T12:35:38Z DUMMY: archaea DISCUSS paragraph 42689 As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases. In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions. 0.9975333 protein cleaner0 2023-07-04T14:08:02Z PR: TSR3 0.8691466 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.99790925 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.89228064 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.9943726 species cleaner0 2023-07-04T12:27:15Z MESH: human 0.9943803 taxonomy_domain cleaner0 2023-07-04T12:34:34Z DUMMY: archaeal 0.9955882 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9956197 evidence cleaner0 2023-07-04T15:26:31Z DUMMY: structure 0.99729 protein_type cleaner0 2023-07-04T14:01:04Z MESH: SPOUT-class RNA methyltransferases 0.98861283 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.99829775 site cleaner0 2023-07-04T15:18:21Z SO: SAM-binding pocket 0.99807334 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.9986235 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.99667263 chemical cleaner0 2023-07-04T14:54:47Z CHEBI: RNA protein_type MESH: cleaner0 2023-07-04T13:06:51Z Rossmann-fold Tyw2 acp transferase 0.99843293 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9988778 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.99789596 site cleaner0 2023-07-04T15:18:26Z SO: hydrophobic pocket 0.9977308 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.99758387 chemical cleaner0 2023-07-04T12:27:45Z CHEBI: pseudouridine 0.9967475 protein_type cleaner0 2023-07-04T14:01:13Z MESH: RNA methyltransferase 0.9989367 protein cleaner0 2023-07-04T12:58:55Z PR: Trm10 0.9985428 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9958383 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.96537554 protein_type cleaner0 2023-07-04T14:01:18Z MESH: SAM-dependent acp transferase 0.995602 protein_type cleaner0 2023-07-04T14:01:21Z MESH: SAM-dependent methyltransferases 0.9983296 site cleaner0 2023-07-04T15:18:30Z SO: SAM-binding pocket 0.86682975 protein_type cleaner0 2023-07-04T12:36:04Z MESH: acp transferase 0.9942889 protein_type cleaner0 2023-07-04T14:01:25Z MESH: methyltransferases 0.9989317 protein cleaner0 2023-07-04T14:23:41Z PR: Nep1 0.99891746 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9979054 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.9988238 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.99848574 chemical cleaner0 2023-07-04T14:54:55Z CHEBI: RNA 0.9985832 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.9387523 complex_assembly cleaner0 2023-07-04T15:53:03Z GO: pre-ribosomal particles 0.6695276 complex_assembly cleaner0 2023-07-04T15:53:08Z GO: ribosomal subunit 0.96781635 experimental_method cleaner0 2023-07-04T15:40:50Z MESH: sucrose gradient analysis 0.99829954 protein cleaner0 2023-07-04T14:08:02Z PR: Tsr3 0.8778073 complex_assembly cleaner0 2023-07-04T15:53:12Z GO: pre-ribosome DISCUSS paragraph 44433 In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein. Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing. 0.50096446 chemical cleaner0 2023-07-04T14:56:08Z CHEBI: rRNAs 0.9946498 protein cleaner0 2023-07-04T14:08:03Z PR: Tsr3 protein_type MESH: cleaner0 2023-07-04T14:01:55Z small subunit rRNA methyltransferases 0.9988682 protein cleaner0 2023-07-04T14:25:50Z PR: Dim1 0.9988501 protein cleaner0 2023-07-04T14:25:53Z PR: Bud23 0.9988293 protein cleaner0 2023-07-04T14:23:56Z PR: Nep1 0.99859124 protein cleaner0 2023-07-04T14:24:03Z PR: Emg1 0.66774464 chemical cleaner0 2023-07-04T14:56:12Z CHEBI: rRNA chemical CHEBI: cleaner0 2023-07-04T14:02:13Z pre-RNA 0.90541875 protein cleaner0 2023-07-04T14:08:03Z PR: Tsr3 protein_state DUMMY: cleaner0 2023-07-04T15:29:19Z SAM-binding 0.6111105 protein_state cleaner0 2023-07-04T15:29:42Z DUMMY: cysteine mutations chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.8981632 chemical cleaner0 2023-07-04T12:17:56Z CHEBI: 18S rRNA 0.81034946 chemical cleaner0 2023-07-04T14:24:14Z CHEBI: rRNA 0.7638087 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.84768337 chemical cleaner0 2023-07-04T14:56:16Z CHEBI: pre-rRNA DISCUSS paragraph 45169 Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. experimental_method MESH: melaniev@ebi.ac.uk 2023-07-06T15:24:22Z structural, functional, and CRAC (cross-linking and cDNA analysis) experiments complex_assembly GO: cleaner0 2023-07-04T15:41:59Z 40S subunits 0.99610144 experimental_method cleaner0 2023-07-04T15:41:34Z MESH: cryo-EM 0.9612105 protein_state cleaner0 2023-07-04T16:11:17Z DUMMY: late 0.94691247 complex_assembly cleaner0 2023-07-04T15:53:18Z GO: pre-40S subunits 0.9573801 complex_assembly cleaner0 2023-07-04T15:53:26Z GO: pre-40S 0.98362994 complex_assembly cleaner0 2023-07-04T15:53:29Z GO: pre-40S particles chemical CHEBI: cleaner0 2023-07-04T14:56:36Z 20S rRNA 0.8642103 protein_type cleaner0 2023-07-04T14:26:48Z MESH: non-ribosomal proteins 0.993677 protein_type cleaner0 2023-07-04T14:26:51Z MESH: D-site endonuclease 0.9986266 protein cleaner0 2023-07-04T14:25:57Z PR: Nob1 0.99859494 protein cleaner0 2023-07-04T14:26:00Z PR: Tsr1 0.9910274 protein_type cleaner0 2023-07-04T14:26:55Z MESH: GTPase 0.9984989 protein cleaner0 2023-07-04T14:26:02Z PR: Rio2 0.9762112 site cleaner0 2023-07-04T15:18:42Z SO: mRNA channel 0.9976265 site cleaner0 2023-07-04T15:18:44Z SO: initiator tRNA binding site DISCUSS paragraph 45789 After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors. Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. 0.9948684 chemical cleaner0 2023-07-04T14:56:41Z CHEBI: GTP 0.9979454 site cleaner0 2023-07-04T15:18:52Z SO: decoding site chemical CHEBI: cleaner0 2023-07-04T12:48:26Z 20S pre-rRNA 0.82476866 protein cleaner0 2023-07-04T13:09:12Z PR: Nob1 0.8359637 site cleaner0 2023-07-04T15:18:58Z SO: site D 0.8337764 complex_assembly cleaner0 2023-07-04T15:53:33Z GO: pre-40S 0.6900945 complex_assembly cleaner0 2023-07-04T15:53:36Z GO: 60S subunits 0.98824817 complex_assembly cleaner0 2023-07-04T15:53:39Z GO: 80S-like particles 0.9983012 protein cleaner0 2023-07-04T13:09:10Z PR: eIF5B complex_assembly GO: cleaner0 2023-07-04T15:54:02Z 40S subunit 0.73086596 protein_type cleaner0 2023-07-04T14:27:00Z MESH: termination factor 0.9985043 protein cleaner0 2023-07-04T13:09:15Z PR: Rli1 0.965042 protein_type cleaner0 2023-07-04T13:09:19Z MESH: ATPase 0.98697394 complex_assembly cleaner0 2023-07-04T15:54:07Z GO: 80S-like complex 0.98814327 protein_state cleaner0 2023-07-04T16:11:28Z DUMMY: mature complex_assembly GO: cleaner0 2023-07-04T15:54:25Z 40S subunit DISCUSS paragraph 46413 Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.9643731 complex_assembly cleaner0 2023-07-04T15:54:32Z GO: pre-40S subunit chemical CHEBI: cleaner0 2023-07-04T14:57:00Z 20S pre-rRNAs 0.96552056 complex_assembly cleaner0 2023-07-04T15:54:35Z GO: pre-40S subunits protein_type MESH: cleaner0 2023-07-04T14:27:20Z ribosome assembly factors 0.9985605 protein cleaner0 2023-07-04T13:08:13Z PR: Tsr1 0.9984236 protein cleaner0 2023-07-04T13:08:15Z PR: Ltv1 0.9982217 protein cleaner0 2023-07-04T13:08:18Z PR: Enp1 0.99821365 protein cleaner0 2023-07-04T13:08:21Z PR: Rio2 protein_state DUMMY: cleaner0 2023-07-04T16:11:55Z acp modified 0.96248454 complex_assembly cleaner0 2023-07-04T15:54:38Z GO: pre-40S subunits 0.99809605 protein cleaner0 2023-07-04T13:09:05Z PR: Nob1 0.99777716 protein cleaner0 2023-07-04T13:09:02Z PR: Rio1 0.8839221 complex_assembly cleaner0 2023-07-04T15:54:42Z GO: 60S subunits 0.78486603 complex_assembly cleaner0 2023-07-04T15:54:45Z GO: 80S-like particles chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.99625635 protein_state cleaner0 2023-07-04T16:12:04Z DUMMY: mature 0.7907378 complex_assembly cleaner0 2023-07-04T15:54:48Z GO: 40S subunits chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.99692446 residue_name_number cleaner0 2023-07-04T15:06:37Z DUMMY: m1Ψ1191 0.9954157 protein cleaner0 2023-07-04T13:09:07Z PR: Rio2 0.9807246 complex_assembly cleaner0 2023-07-04T15:54:51Z GO: pre-40S particle DISCUSS paragraph 47079 These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.94139224 chemical cleaner0 2023-07-04T14:57:42Z CHEBI: pre-20S rRNA chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.9972498 protein cleaner0 2023-07-04T14:26:08Z PR: Rio2 0.9969145 site cleaner0 2023-07-04T15:19:18Z SO: decoding site site SO: cleaner0 2023-07-04T14:58:25Z D-site 0.99823534 protein cleaner0 2023-07-04T14:26:11Z PR: Nob1 chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.99523795 protein cleaner0 2023-07-04T14:26:15Z PR: Rio2 0.842754 experimental_method cleaner0 2023-07-04T15:42:06Z MESH: CRAC analysis 0.995242 protein cleaner0 2023-07-04T14:26:18Z PR: Rio2 0.957628 structure_element cleaner0 2023-07-04T16:02:48Z SO: helix 31 0.99894506 residue_name_number cleaner0 2023-07-04T15:06:43Z DUMMY: Ψ1191 chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.9965959 protein cleaner0 2023-07-04T14:26:22Z PR: Rio2 0.52873844 protein cleaner0 2023-07-04T14:08:03Z PR: Tsr3 0.89228296 structure_element cleaner0 2023-07-04T16:02:50Z SO: helix 31 chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.9940521 protein cleaner0 2023-07-04T14:26:25Z PR: Rio2 chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.9988901 residue_name_number cleaner0 2023-07-04T15:06:48Z DUMMY: m1Ψ1191 chemical CHEBI: cleaner0 2023-07-04T14:57:58Z 20S rRNA 0.99593145 protein cleaner0 2023-07-04T14:26:28Z PR: Rio2 0.9363694 structure_element cleaner0 2023-07-04T16:02:53Z SO: helix 31 0.92609435 chemical cleaner0 2023-07-04T14:58:14Z CHEBI: pre-rRNA DISCUSS paragraph 47812 In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. 0.9977992 protein cleaner0 2023-07-04T14:08:03Z PR: Tsr3 0.8789575 chemical cleaner0 2023-07-04T12:37:17Z CHEBI: acp 0.9624326 protein_state cleaner0 2023-07-04T14:38:37Z DUMMY: hypermodified 0.8184547 chemical cleaner0 2023-07-04T12:18:12Z CHEBI: m1acp3Ψ 0.8651114 chemical cleaner0 2023-07-04T14:58:30Z CHEBI: nucleotide 0.65205365 residue_number cleaner0 2023-07-04T15:33:46Z DUMMY: 1191 0.9899032 taxonomy_domain cleaner0 2023-07-04T12:18:26Z DUMMY: yeast 0.49469864 residue_number cleaner0 2023-07-04T15:06:57Z DUMMY: 1248 0.987317 species cleaner0 2023-07-04T12:18:32Z MESH: humans chemical CHEBI: cleaner0 2023-07-04T12:17:56Z 18S rRNA 0.9969258 taxonomy_domain cleaner0 2023-07-04T12:20:33Z DUMMY: eukaryotic chemical CHEBI: cleaner0 2023-07-04T15:55:21Z small ribosomal subunit rRNA chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp complex_assembly GO: cleaner0 2023-07-04T15:55:41Z pre-40S subunit chemical CHEBI: cleaner0 2023-07-04T12:37:17Z acp 0.9980171 site cleaner0 2023-07-04T15:19:27Z SO: decoding site chemical CHEBI: cleaner0 2023-07-04T12:48:26Z 20S pre-rRNA 0.84141666 site cleaner0 2023-07-04T15:19:33Z SO: D-site 0.9965247 evidence cleaner0 2023-07-04T15:26:40Z DUMMY: structural data 0.61613 chemical cleaner0 2023-07-04T12:36:27Z CHEBI: SAM 0.9981494 site cleaner0 2023-07-04T15:19:39Z SO: ligand binding pocket 0.958201 protein_type cleaner0 2023-07-04T14:27:25Z MESH: SAM-binding enzymes KEYWORD title_1 48587 ACCESSION NUMBERS KEYWORD paragraph 48605 Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes PDB 5APG (VdTsr3/SAM-complex) and PDB 5AP8 (SsTsr3). SUPPL title_1 48759 Supplementary Material SUPPL title_1 48782 SUPPLEMENTARY DATA SUPPL paragraph 48801 Supplementary Data are available at NAR Online. ACK_FUND title_1 48849 FUNDING ACK_FUND paragraph 48857 DFG grant [En134/9-1]; SFB 902 (Molecular Principles of RNA-based Regulation); DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]; European Community's Seventh Framework Programme [FP7/2007-2013] under BioStruct-X [283570]; Goethe University and the State of Hesse; EMBO long-term fellowship [ALTF 644-2014 to S.S.]; Research in the Lab of DLJL is supported by the Université Libre de Bruxelles (ULB); Fonds National de la Recherche (F.R.S./FNRS); Walloon Region [DGO6]; Fédération Wallonie-Bruxelles; European Research Development Fund (ERDF). Funding for open access charge: DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]. 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