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Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution

Nature Structural & Molecular Biology volume 23, pages 558–565 (2016)Cite this article

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Abstract

Group II introns are self-splicing ribozymes that are essential in many organisms, and they have been hypothesized to share a common evolutionary ancestor with the spliceosome. Although structural similarity of RNA components supports this connection, it is of interest to determine whether associated protein factors also share an evolutionary heritage. Here we present the crystal structures of reverse transcriptase (RT) domains from two group II intron–encoded proteins (maturases) from Roseburia intestinalis and Eubacterium rectale, obtained at 1.2-Å and 2.1-Å resolution, respectively. These domains are more similar in architecture to the spliceosomal Prp8 RT-like domain than to any other RTs, and they share substantial similarity with flaviviral RNA polymerases. The RT domain itself is sufficient for binding intron RNA with high affinity and specificity, and it is contained within an active RT enzyme. These studies provide a foundation for understanding structure–function relationships within group II intron–maturase complexes.

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Figure 1: Overall structure and active site of the R.i. RT domain.
Figure 2: Comparison of the group II intron maturase RT domain with related structures.
Figure 3: The E.r. RT domain binds RNA with high affinity and specificity.
Figure 4: Dimerization interface of the R.i. RT domain.
Figure 5: E.r. RT forms a dimer in solution in the absence or presence of D4A RNA.

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Acknowledgements

The authors acknowledge E. Folta-Stogniew at the Biophysics Resource of the Keck Facility at Yale University for performing the SEC-MALS experiments. Additionally, we thank F. Liu for kindly providing the plasmid, radiolabeled primer and ladders for the primer extension assay; J.A. Liberman and S. Somarowthu for helpful discussions; and T.H. Dickey for reading the manuscript. The SEC-LS/UV/RI instrumentation is supported by NIH award no. 1S10RR023748-01. This work was supported by National Institutes of Health (NIH) grant RO1GM50313 (A.M.P.) and the Howard Hughes Medical Institute (A.M.P.). C.Z. is supported by a Yale University Fellowship and a Gruber Science Fellowship.

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Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Chen Zhao

  2. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Anna Marie Pyle

  3. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Anna Marie Pyle

  4. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Anna Marie Pyle

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  1. Chen Zhao
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Contributions

C.Z. and A.M.P. designed the project. C.Z. performed the experiments and solved the structure. C.Z. and A.M.P. wrote the manuscript.

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Correspondence to Anna Marie Pyle.

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Integrated supplementary information

Supplementary Figure 1 Domain construction, sequence, overall structure and active site of R.i. and E.r. maturases.

a. Organization of the maturase RT construct. All maturases contain an N-terminal RT domain (blue) and a C-terminal thumb/X domain (orange). b. Sequence alignment of the full-length maturase, comparing E.r. (Eu.re.I2) and R.i. (Ro.in.I1). The black boxes indicate identical residues, the grey boxes indicate similar residues, and the white box indicates residues with substantial differences. The red bar indicates the boundary for the RT domain used for crystallization. The blue arrow indicates the N-terminal RT domain and the orange arrow indicates the C-terminal X or thumb domain. c. R.i. and E.r. RT domain structures in cartoon representation. The R.i. RT is shown in green and the E.r. RT is shown in gray. d. Comparison of the E.r. RT active site and primer grip with an active telomerase (PDBID: 3KYL)49. The E.r. RT is shown in grey and telomerase is shown in cyan. For the residues shown as sticks, red color indicates oxygen and blue color indicates nitrogen atoms. The potassium in the R.i. RT active site is shown in purple, and the Mg2+ in the telomerase active site is shown in yellow. Metal ion coordination is indicated by orange dashed lines.

Supplementary Figure 2 Maturase RT forms a dimer.

a. Each panel shows the content of an ASU in different crystals, and the organism names and the space groups are indicated at the bottom of each panel. In the dimer, one monomer is colored in grey and the other is colored in green. The ASU of R.i. RT crystallized in P21 space group from condition A (see online Methods) contains 2 molecules. The ASU of R.i. RT crystallized in P212121 space group from condition B (see online methods) contains 4 molecules. The ASU of R.i. RT crystallized in P1 space group from condition C (see online methods) contains 4 molecules. The ASU of E.r. RT crystallized in P21 space group from condition D (see online methods) contains 8 molecules. b. The active site within the R.i. RT domain is partially obstructed by the dimerization interface. On the left panel, monomer A is represented as a cartoon diagram in grey, and monomer B is represented as a space-filling model in green. For monomer A, the active site loop is colored in red, the primer grip is colored in magenta and the insertion loop containing a short α-helix a10 is colored in yellow. The right panel is a close-up view of the interactions that stabilize the insertion loop shown in sticks. Oxygen atoms are colored in red, nitrogen atoms are colored in blue and hydrogen bonds are shown as orange dashed lines.

Supplementary Figure 3 A possible model for association of the maturase dimer with a group II intron RNA.

A cartoon diagram of oceanobacillus iheyensis group II intron (PDBID: 4FAQ)29 is shown on the left, where domain 1 (D1) is colored yellow and domain 4 (D4) is colored in cyan. All other domains are grey. The electrostatic surface of the E.r. RT dimer is shown at right, where blue indicates positive charge and red indicates negative charge. The sizes of the group II intron model and the maturase RT dimer model are not to scale. Based on the electrostatic surface potentials of the RT dimer, and previous results showing that maturase interacts with the intron as a dimer15-16 through a high-affinity binding site in intron D414 and additional auxiliary sites in D148, the potential surfaces involved in RNA-protein interactions are marked by arcs, and the potential interactions are indicated by dashed lines.

Supplementary Figure 4 E.r. RT binds RNA with high affinity and specificity.

a. Comparison of the secondary structures for D4 in the L1.LtrB14 and E.r. introns. The residues colored in red belong to the D4A region. The start codon is highlighted in green. The rest of the open reading frame (ORF) encoding the maturase is indicated by a circle. b. Electrophoretic mobility shift assay (EMSA) for E.r. RT binding to D4A or D2 (Fig. 3b and Fig. 3c). The protein concentrations (nM) for each lane are indicated at the top. c. Phosphate binding site on the R.i. RT domain. The R.i. RT is shown as a surface rendering in grey. The phosphorus atom is orange and the oxygens are red. The simulated annealing omit map for the phosphate ion is shown in blue at the 3σ level. The interactions stabilizing the phosphate are shown on the right panel. Carbon atoms are grey, oxygens are red and nitrogen are blue. Hydrogen bonds are shown as orange dashed lines. d. Molecular weight analysis of E.r. D4A RNA using SEC-MALS. The experiments for E.r. D4A RNA (shades of grey) were performed over two concentrations (in mg/mL) as indicated, and the MW at each concentration was plotted as triangles and circles respectively (upper right legend) on the right y-axis. For each concentration, the UV trace (curve) was plotted on the left y-axis with the elution volume indicated on the x-axis. The MW at elution peak is 23 kDa at 0.03 mg/mL and 24 kDa at 0.07 mg/mL.

Supplementary Figure 5 Primer extension assay for the maturase.

Lanes 1-3 are DNA sequencing ladders (corresponding to nucleotides A, G and T respectively) showing the sequence and the resolution of the primer extension products on the gel. Lane 4 is the control lane, which only contains the primer. Lane 5 is the primer extension product from the E.r. RT construct, and lane 6 is the primer extension product from the E.r. full-length maturase. In order to resolve DNA products of vastly different length on the same gel (enabling us to compare nonprocessive and processive activity by the RT domain and full-length maturase constructs, respectively), we used a long sequencing gel. The gel is slightly twisted, so each corresponding lane is also labeled at the bottom of the gel to avoid confusion.

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Zhao, C., Pyle, A. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nat Struct Mol Biol 23, 558–565 (2016). https://doi.org/10.1038/nsmb.3224

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