While the major architectural features and active-site components of group II introns have been known for almost a decade, information on the individual stages of splicing has been lacking. Recent advances in crystallography and cryo-electron microscopy (cryo-EM) have provided major new insights into the structure of intact lariat introns. Conformational changes that mediate the steps of splicing and retrotransposition are being elucidated, revealing the dynamic, highly coordinated motions that are required for group II intron activity. Finally, these ribozymes can now be viewed in their larger, more natural context as components of holoenzymes that include encoded maturase proteins. These studies expand our understanding of group II intron structural diversity and evolution, while setting the stage for rigorous mechanistic analysis of RNA splicing machines.
Group II intron; RNA structure; maturase; retrotransposition; ribozyme; spliceosome.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Secondary structure and splicing pathways of group II introns. (A) Secondary structure diagrams of three group II introns from class IIC (left), IIB (middle) and IIA (right). The secondary structures were extracted from available tertiary structure models determined by crystallography or cryo-electron microscopy (PDB IDs: 3IGI for IIC, 4R0D for IIB and 5G2X for IIA). The open reading frame (ORF) is shown in D4. Long range interactions are denoted with Greek letters and are color coded. Exon binding sites (EBS) and intron binding sites (IBS) are shown in their respective locations. (B) Primary pathways for group II intron self-splicing. The branching pathway (top) uses the 2’OH group in an adenosine (branch site) in D6 as the first step nucleophile, and the hydrolysis pathway (bottom) uses an external water molecule as the first step nucleophile. Reversibility of each step is indicated by the double arrows.
Tertiary structures of group II introns. (A) Tertiary structures of group II introns from IIC (left), IIB (middle) and IIA (right) (PDB IDs: 4E8K for IIC, 4R0D for IIB and 5G2X for IIA). (B) Novel interactions visualized in the crystal structure of a group IIB intron lariat (PDB ID: 4R0D). Top left: D2 (blue) and D6 (purple) interacts with two “anti-parallel” tetraloop-receptor interactions (η-η′ and π-π′). Top right: D3 (orange) interacts with the base of D5 (red) through the μ-μ′ interaction, which extends the canonical κ-κ′ interaction between D5 (red) and D1 (grey). Bottom: exon recognition in the group IIB intron. The 5′exon recognition pairs with IBS1 (intron binding site 1, in cyan) via the EBS1 (exon binding site 1, in grey) and with IBS2 (purple) via EBS2 (grey). The 3′exon recognition involves only a single base-pair between IBS3 (blue) and EBS3 (black). (C) The β-β′ interaction visualized in the cryo-EM reconstruction of a group IIA intron lariat (PDB ID: 5G2X). Only the D1 portion of the intron lariat is shown. The bottom right corner provides a close-up of the β-β′ kissing loop interaction. (D) Comparison of the core structure of group II introns from different classes.
The stages of group II intron splicing. (A) Individual states along the splicing pathway. During reverse splicing, which occurs during retrotransposition by the intron, DNA is targeted in-trans. Therefore, the double arrows connecting forward and reverse splicing indicate that intron RNA has the same conformation during both types of tranformations, despite the fact that the chemical composition of the nucleic acid target is different. Notably, while reverse-splicing can be initiated by linear intron, it cannot complete the second step of reverse-splicing . (B) Crystal structures of the group II intron active site at specific stages of splicing. The left panel shows the active site of the pre-catalytic state of the first step of splicing (PDB ID: 4FAQ), the middle panel shows the state immediately after the first step of splicing (PDB ID: 4FAR), and the right panel shows the state of a lariat intron before the first step of reverse splicing (coordinates are in the supplementary PDB file in the associated publication ). All phosphorus atoms are colored in orange, and the nucleophilic oxygen is colored in green. The scissile phosphate is shown in a “ball and stick” representation. (C) Occupancy of the group II intron active site at different stages of splicing.
Conformational dynamics during group II intron splicing. (A) Isoforms of the D6 secondary structure. The regions colored in red have alternative conformations during the two steps, and arrows indicate the direction for the base pairs to slide in order to form alternative conformations. (B) Comparison of 5′exon-EBS1 conformation in isolated D1 molecules (PDB ID: 4Y1O) and in the full-length intron (PDB ID: 4FAQ). The left panel shows the full-length intron structure (grey) superimposed with the 5′exo-EBS1 from isolated D1 (green). In the swing state (green), the 3′ end of the 5′exon swings 9.2 Å away, coordinated by the movement of EBS1. (C) Interactions that stabilize D6 in the lariat intron chimera (coordinates were obtained from supplementary PDB file in the associated publication ). D6 is docked by interactions with D1 and J2/3, and this configuration leads to the formation of second step active site. In this way, the 2′-5′ branch formation is coupled to the second step of splicing. (D) 5′exon binding modulates conformation of intron active site. The left panel is the active site of
O.i. group II intron (PDB ID: 4E8M) in the absence of 5′ exon, the middle panel is the active site of O.i.- A.v. chimera group II intron (PDB ID: 5J02) in the presence of 5′ exon, while the right panel is the active site of O.i.- A.v. chimera group II intron (PDB ID: 5J01) in the absence of 5′exon. In the absence of 5′exon, EBS1 is disordered, leading to alternative conformations of ζ′ in D5, A72 in D1C 1 and A106 in λ that interacts with the 2’OH group of residue -2 in the 5′exon.
Structures of group II intron maturases. (A) Structures and electrostatic surfaces of group II intron maturases. In the top row, left panel shows the crystal structure of the RT domain of a group IIC intron maturase (PDB ID: 5HHJ), and the right panel shows the cryo-EM structure of the RT domain of a group IIA intron maturase (LtrA, PDBID: 5G2Y). Their corresponding electrostatic surfaces are shown in the bottom row. (B) The overall architecture of a group II intron RNP solved by cryo-EM (PDB ID: 5G2Y), showing insertion of the Ti-loop into D1. (C) The protein-RNA interactions that are may influence catalysis, with RNA and protein domains annotated. (D) The maturase RT domain forms a dimer in crystals and solution. Crystal structure of a maturase RT domain dimer (PDB ID: 5HHJ) is shown (left panel). This RT dimer is stabilized by an extended interface (cyan, right).
Comparison of group II introns and the yeast spliceosome structures (A) Comparison of the catalytic RNA secondary structures in the active sites of the group II intron (left) and spliceosome (right). Dots indicate base-pairs while arrows indicate triple helix interactions. (B) Comparison of the RNA tertiary structure within the group II intron (PDB ID: 4FAR) and spliceosome (PDB ID: 5LJ3, C-complex) active-sites. Both structures represent the state immediately after the first step of splicing. (C) Comparison of the structure of protein components in group II introns and the spliceosome. Left panel is the crystal structure of RT domain (finger and palm) from a group IIC intron (PDB ID: 5HHJ), middle panel is the cryo-EM structure of the maturase from a group IIA intron (PDB ID: 5G2Y), and the right panel is the crystal structure of the large domain of spliceosomal protein Prp8 (PDB ID: 4I43). Domain organization of group II intron maturase and spliceosomal Prp8 are shown in the bottom left corner. RT: reverse transcriptase. IFD: insertion in finger domain. DBD: DNA binding domain. EN: endonuclease. (D) Comparing position of X/thumb domain in the active sites of the group II intron and the spliceosome. The EN domain is not shown.
The mechanism of splicing as told by group II introns: Ancestors of the spliceosome.
Biochim Biophys Acta Gene Regul Mech. 2019 Nov-Dec;1862(11-12):194390. doi: 10.1016/j.bbagrm.2019.06.001. Epub 2019 Jun 13.
Biochim Biophys Acta Gene Regul Mech. 2019.
Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA.
Cell. 2019 Jul 25;178(3):612-623.e12. doi: 10.1016/j.cell.2019.06.035.
Group II Intron Self-Splicing.
Annu Rev Biophys. 2016 Jul 5;45:183-205. doi: 10.1146/annurev-biophys-062215-011149.
Annu Rev Biophys. 2016.
B2 and ALU retrotransposons are self-cleaving ribozymes whose activity is enhanced by EZH2.
Proc Natl Acad Sci U S A. 2020 Jan 7;117(1):415-425. doi: 10.1073/pnas.1917190117. Epub 2019 Dec 23.
Proc Natl Acad Sci U S A. 2020.
Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing.
Nucleic Acids Res. 2019 Dec 2;47(21):11430-11440. doi: 10.1093/nar/gkz965.
Nucleic Acids Res. 2019.
31665419 Free PMC article.
Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulations.
J Struct Biol. 2019 Jun 1;206(3):267-279. doi: 10.1016/j.jsb.2019.03.004. Epub 2019 Mar 15.
J Struct Biol. 2019.
30880083 Free PMC article.
Small molecules that target group II introns are potent antifungal agents.
Nat Chem Biol. 2018 Dec;14(12):1073-1078. doi: 10.1038/s41589-018-0142-0. Epub 2018 Oct 15.
Nat Chem Biol. 2018.
30323219 Free PMC article.
Contribution of Mobile Group II Introns to
Sinorhizobium meliloti Genome Evolution.
Front Microbiol. 2018 Apr 4;9:627. doi: 10.3389/fmicb.2018.00627. eCollection 2018.
Front Microbiol. 2018.
29670598 Free PMC article.
Research Support, N.I.H., Extramural
RNA, Catalytic / chemistry*
RNA, Catalytic / metabolism