Hatchet ribozyme structure and implications for cleavage mechanism Luqian Zhenga,1, Christoph Falschlungerb,1, Kaiyi Huanga, Elisabeth Mairhoferb, Shuguang Yuanc, Juncheng Wangd, Dinshaw J. Pateld,e,2, Ronald Micurab,2, and Aiming Rena,2 aLife Science Institute, Zhejiang University, 310058 Hangzhou, China; bInstitute of Organic Chemistry, Leopold Franzens University, 6020 Innsbruck, Austria; cInstitute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland; dStructural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and eDepartment of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China Contributed by Dinshaw J. Patel, March 29, 2019 (sent for review February 11, 2019; reviewed by Andrej Lupták and Keqiong Ye) Small self-cleaving ribozymes catalyze site-specific cleavage of their mutations on ribozyme activity, with implications for modeling the own phosphodiester backbone with implications for viral genome precatalytic fold and insights into cleavage chemistry. replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which Results adopts a compact pseudosymmetric dimeric scaffold, with each The hatchet ribozyme is composed of four base-paired stems la- monomer stabilized by long-range interactions involving highly con- beled P1–P4, in which P1 and P2 are linked by three highly con- served nucleotides brought into close proximity of the scissile phos- served residues, while P2, P3, and P4 are bridged by two internal phate. Strikingly, the catalytic pocket contains a cavity capable of loops L2 and L3 (Fig. 1A). Most of the highly conserved residues accommodating both the modeled scissile phosphate and its flanking (shown in red rectangles in Fig. 1A) are dispersed and positioned 5′ nucleoside. The resulting modeled precatalytic conformation incor- within loop L2. The cleavage site is located at the 5′ end of stem porates a splayed-apart alignment at the scissile phosphate, thereby P1, a unique feature of the hatchet ribozyme, that contrasts it from providing structure-based insights into the in-line cleavage mecha- the internal cleavage sites observed for the pistol, twister, and nism. We identify a guanine lining the catalytic pocket positioned twister-sister ribozymes (5). The secondary structure of the to contribute to cleavage chemistry. The functional relevance of hatchet ribozyme has been validated from covariation and muta- BIOCHEMISTRY structure-based insights into hatchet ribozyme catalysis is strongly tion studies (23), but insights into the catalytic mechanism require supported by cleavage assays monitoring the impact of selected information on both the tertiary fold and alignment of catalytic nucleobase and atom-specific mutations on ribozyme activity. residues mediating in-line cleavage chemistry. hatchet | ribozyme | cleavage | catalysis | noncoding RNA Crystallization of the Hatchet Ribozyme Product. For crystallization trials, we screened a large number of chemically synthesized atalytic noncoding RNAs, termed ribozymes, are involved in (one- and two-stranded constructs) and in vitro transcribed Cmany vital cellular reactions ranging from tRNA processing constructs of the hatchet ribozyme. For the in vitro transcribed to intron splicing, protein synthesis, and regulation of gene ex- pression (1). Nucleolytic ribozymes are small RNAs that adopt Significance compact folds capable of site-specific cleavage/ligation reactions (2–4). Nine unique nucleolytic ribozymes have been identified to Self-cleaving ribozymes are RNAs that catalyze position-specific date, including recently discovered twister, pistol, twister-sister, cleavage of their phosphodiester backbone. The cleavage site of and hatchet ribozymes that were identified based on application the newly discovered hatchet ribozyme is located at the very 5′ of comparative sequence and structural algorithms (5, 6). The end of its consensus secondary structure motif. Here we report structure/function characterization of such ribozymes would pro- on the 2.1-Å crystal structure of the hatchet ribozyme in the vide mechanistic insights into ribozyme activity and its modulation. product state, which defines its intricate tertiary fold and iden- Nucleolytic ribozymes adopt an SN2-like mechanism that re- tifies key residues lining the catalytic pocket. This in turn has sults in site-specific phosphodiester bond cleavage. In general, an allowed us to propose a model of the precatalytic state structure ′ ′ activated 2 -OH of the ribose 5 to the scissile phosphate adopts and a role in catalysis for a conserved guanine. This study ′ an in-line alignment to target the adjacent to-be-cleaved P-O5 therefore provides a structure-based platform toward an im- phosphodiester bond, resulting in formation of 2′,3′-cyclic proved understanding of the catalytic mechanism of hatchet phosphate and 5′-OH groups. To date, X-ray crystallographic ribozymes. structural studies on the hammerhead (7, 8), hairpin (9, 10), GlmS (11, 12), hepatitis delta virus (HDV) (13, 14), Varkud satellite Author contributions: R.M. and A.R. designed research; L.Z., C.F., K.H., E.M., S.Y., J.W., (15), and pistol ribozymes (16, 17) have defined the overall RNA R.M., and A.R. performed research; D.J.P., R.M., and A.R. analyzed data; and D.J.P., R.M., fold, the catalytic pocket arrangement, the in-line alignment, and and A.R. wrote the paper. the key residues that contribute to the cleavage reaction. By Reviewers: A.L., University of California, Irvine; and K.Y., Chinese Academy of Sciences. contrast, there is less clarity to date on the cleavage mechanism of The authors declare no conflict of interest. twister (18–20) and twister-sister (21, 22) ribozymes, given distinct This open access article is distributed under Creative Commons Attribution-NonCommercial- catalytic conformations reported from structural studies for these NoDerivatives License 4.0 (CC BY-NC-ND). ribozymes, and a resolution must await additional structural Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6JQ5 for HT_GAAA structure and studies of transition state vanadate mimics of these ribozymes. 6JQ6 for HT_UUCG structure). We now report on structural studies of the hatchet ribozyme 1L.Z. and C.F. contributed equally to this work. product supplemented by structure-based cleavage assays moni- 2To whom correspondence may be addressed. Email: [email protected], ronald.micura@ toring the impact of selected nucleobase and atom-specific muta- uibk.ac.at, or [email protected]. tions on ribozyme activity. These structure/function studies identify This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the tertiary fold of the ribozyme, the alignment of conserved resi- 1073/pnas.1902413116/-/DCSupplemental. dues lining the catalytic pocket, and the impact of site-specific Published online May 14, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1902413116 PNAS | May 28, 2019 | vol. 116 | no. 22 | 10783–10791 Downloaded by guest on October 1, 2021 A BEF CG50 A9 U A CG50 P4 A U U A G10 A U P4 P4 A U L1 U A A U 40 C G U A U U A A 40 C G U L3 U G8 A U A U U39 U L3 UA UU A 60 L3 10 A U P3 C G U U 10 U A 60 P1 U7 A11 A G A G UG C AG A C G P3C G G L1 A 30 G A A G L2 G G U G P3 UU G L1 A 30 G C C G C G U A G A U U UU G L2 G U A G CC AAA A G C U G A A A U 70 C G U A U A 5 G U7 P1 U A P1 G CC A U P2 C G A U U A U A C G 80 A U P2 L2 P2 U A A U U A A U C 80 C AA 1 C G UUC 1 82 U A A11 20 C AA A U C -1 3 20 82 5 3 C H A36 P2 A57 P1 U37 U58 U13 L1 A12 I P3 P4 L2 G10 L3 A38 D G8 U39 minor groove interaction Mol A Mol B minor groove interaction A11 10 A A 10’ A U P1 20 80 UA G A C A G U A A C A C A U G U C U G U A C A C A A U G A C A G A A G G U U AG C A A C A U G U G G U U A C U G UG U G U C A U U - G - J 1 30 P2 1 A A C A A U59 C A A - U P4 40 P3 U A A U U U 70 G U17 A G C U A A U C A U A U C G G U U G G U G G C U A U A C U A A U C A A A A G A U U A G U A U A G U C AAC C A A C U G A U A U G A U U A G A 50 L3 U 60 G L2 G G G U A3 Mol A’ Fig. 1. Schematic and tertiary structure of the hatchet ribozyme. (A) The predicted secondary structure of the env10 hatchet ribozyme. The sequence is color coded according to helical segments observed in the tertiary structure. The highly conserved residues are shown in red rectangles. (B) A schematic repre- sentation of hatchet ribozyme product secondary structure highlighting long-range interactions. (C) The tertiary fold of the HT-GAAA hatchet ribozyme product dimer. The red thick dashed line divided the dimer structure as two new monomer molecules termed A′ and B′.(D) A schematic representation of the tertiary structure of HT-GAAA hatchet ribozyme product dimer.