The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand

Abstract

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8.

Figure 1

Structure of group II intron and its IEP. (a) Secondary and tertiary structure of group II introns. The secondary structural domains (D1–6, left) are color coded to match the tertiary structure of a group II intron lariat RNA (PDB: 4R0D, right), and the open reading frame (ORF) in D4 is indicated. (b) Domain organization of group II intron IEPs showing that IEPs from Eubacterium retale (E.r.) and Roseburia intestinalis (R.i.) contain only the RT domain and X domain. On the crystal structure of the R.i. RT domain (bottom left, PDB: 5HHJ), the dotted oval indicates the expected position of the X domain that is not included in the crystal structure. The IEP from Lactococcus lactics (L.l.) contains DNA binding domain (DBD) and endonuclease (EN) domains following the RT and X domains (bottom right, PDB: 5G2X). A large portion of its EN domain is disordered is not visualized in the structure.

Figure 2

Group II intron IEPs are constructed on a polymerase scaffold. (a) Domain organization of HIV RT and group II intron IEPs. The seven conserved regions in all RTs are boxed and labeled, and insertions in group II intron IEPs are indicated as 2a, 3a, 4a and 7a. Insertions 2a, 3a and 4a are mapped onto the tertiary structures of R.i. (below left, PDB: 5HHJ) and L.l. RT domains (below right, PDB: 5G2X) using the same color code. (b) Comparison of the IEP X domain and polymerase thumb domains. The X (thumb) domain of L.l. IEP (PDB: 5G2X) is compared with the thumb domains of HIV polymerase (PDB: 2HMI) telomerase (TERT, PDB: 3KYL). In all structures, the analogous regions are colored in red, and the rest of thumb domain, if present, is colored in pink. (c) Comparison of insertion 2a in IEPs (PDB: 5HHJ) and the analogous location in HIV reverse transcriptase (PDB: 2HMI). (d) Comparison of insertion 3a in E.r/R.i. (PDB: 5HHJ) with L.l. (PDB: 5G2X) IEPs. The insertion in finger domain (IFD) motif contains two α-helices that are indicated by a black box. (e) Location of insertion 4a. In L.l. IEP (PDB: 5G2X), insertion 4a contacts helix d(iii)a in intron D1. E.r/R.i. IEPs lack insertion 4a. (f) Homology model for the RT domain of human retro-transposon L1. The domain organization of L1 ORF2 and partial sequence alignment between RT domains from R.i. IEP and L1 ORF2 are shown at left. The predicted structure of L1 ORF RT domain is shown at right. It was produced by I-TASSER [40], using the secondary structure topology of the target sequence and restraints from the crystal structure of R.i. RT domain. The model shown here has an expected TM-score of 0.67±0.13 (>0.5 indicates correct topology) and an expected RMSD of 7.3±4.2 Å.

Figure 3

Interaction of an IEP with its cognate group II intron RNA. (a) Overall scaffold of the L.l. IEP-intron complex (PDB: 5G2X). The IEP contacts the intron RNA via 3 anchor points (black boxes). Anchor points 1 and 2 are mediated by the RT domain, whereas anchor point 3 is mediated by the X domain. (b) Structure and sequence conservation of anchor point 3, whereby the the “ti-loop” in the X-domain penetrates deeply into intron D1 within the L.l. IEP-intron complex structure (PDB: 5G2X). The consensus sequence for ti-loop is shown below. (c) Location of the EYSC motif. (d) Potential dimerization of the group II intron IEP. Observed dimerization of the IEP RT domain (right, PDB: 5HHJ). A hypothetical IEP dimerization site within the L.l. IEP-intron complex structure (orange oval, PDB: 5G2X) suggests a location for the addidtional IEP monomer if the IEP were to dimerize within the holoenzyme complex.

Figure 4

Loss of RNA-IEP binding specificity through the course of evolution. (a) Domain organization of group II intron (GII) IEP, spliceosomal Prp8 and group II intron trans-splicing factors in mitochondria (MatR), chloroplasts (MatK) and nuclear (nMat). The shading on RT0 indicates its loss of amino acids associated with RNA binding affinity and/or specificity. MLS represents a mitochondrial localization signal. (b) Electrostatic surface electrostatic potential of the RT0 regions (dotted orange ovals) in group II intron IEP (PDB: 5HHJ) and spliceosomal Prp8 (PDB: 5LJ3). (c) Spliceosomal Prp8 (PDB: 5LJ3), which uses an auxiliary NTD domain (rather than RT0) to interact with snRNA U5.