Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA

Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a maturase protein, which promotes splicing through an unknown mechanism. The mechanism of splice site exchange within the RNA active site during catalysis also remains unclear. We determined two cryo-EM structures at 3.6-Å resolution of a group II intron reverse splicing into DNA. These structures reveal that the branch-site domain VI helix swings 90°, enabling substrate exchange during DNA integration. The maturase assists catalysis through a transient RNA-protein contact with domain VI that positions the branch-site adenosine for lariat formation during forward splicing. These findings provide the first direct evidence of the role the maturase plays during group II intron catalysis. The domain VI dynamics closely parallel spliceosomal branch-site helix movement and provide strong evidence for a retroelement origin of the spliceosome.

[1]  H. Stark,et al.  Cryo-EM structure of a human spliceosome activated for step 2 of splicing , 2017, Nature.

[2]  S. Dib-Hajj,et al.  Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron , 1995, Molecular and cellular biology.

[3]  S. Stevens,et al.  Spliceosomal intronogenesis , 2016, Proceedings of the National Academy of Sciences.

[4]  F. Michel,et al.  Frequent use of the same tertiary motif by self‐folding RNAs. , 1995, The EMBO journal.

[5]  Kai Zhang,et al.  Gctf: Real-time CTF determination and correction , 2015, bioRxiv.

[6]  E. Westhof,et al.  Crystal structures of a group II intron lariat primed for reverse splicing , 2016, Science.

[7]  P. Perlman,et al.  The stereochemical course of group II intron self-splicing. , 1994, Science.

[8]  C. Oubridge,et al.  A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation , 2019, Science.

[9]  Julie L. Fiore,et al.  An RNA folding motif: GNRA tetraloop–receptor interactions , 2013, Quarterly Reviews of Biophysics.

[10]  C. Taccioli,et al.  Transposable Elements Activity is Positively Related to Rate of Speciation in Mammals , 2018, Journal of Molecular Evolution.

[11]  T. Eickbush.  Mobile introns: Retrohoming by complete reverse splicing , 1999, Current Biology.

[12]  K. Rajashankar,et al.  Structural basis for the second step of group II intron splicing , 2018, Nature Communications.

[13]  M. Boudvillain,et al.  Defining functional groups, core structural features and inter‐domain tertiary contacts essential for group II intron self‐splicing: a NAIM analysis , 1998, The EMBO journal.

[14]  P. Perlman,et al.  Mutations of the two-nucleotide bulge of D5 of a group II intron block splicing in vitro and in vivo: phenotypes and suppressor mutations. , 1996, RNA.

[15]  G. Hausner,et al.  Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases. , 2001, RNA.

[16]  Anna Marie Pyle,et al.  Crystal Structure of a Self-Spliced Group II Intron , 2008, Science.

[17]  G. Mohr,et al.  Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns , 2010, PLoS biology.

[18]  Liskin Swint-Kruse,et al.  Resmap: automated representation of macromolecular interfaces as two-dimensional networks , 2005, Bioinform..

[19]  Anna Marie Pyle,et al.  RCrane: semi-automated RNA model building , 2012, Acta crystallographica. Section D, Biological crystallography.

[20]  F. Michel,et al.  Linking the branchpoint helix to a newly found receptor allows lariat formation by a group II intron , 2011, The EMBO journal.

[21]  Fred H. Gage,et al.  Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition , 2005, Nature.

[22]  F. Michel,et al.  Base-pairing interactions involving the 5' and 3'-terminal nucleotides of group II self-splicing introns. , 1990, Journal of molecular biology.

[23]  A. Lambowitz,et al.  Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications. , 2017, Molecular cell.

[24]  A. Lambowitz,et al.  A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA. , 1999, Molecular cell.

[25]  A. Lambowitz,et al.  A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility , 1995, Cell.

[26]  A. Lambowitz,et al.  Group II introns: mobile ribozymes that invade DNA. , 2011, Cold Spring Harbor perspectives in biology.

[27]  M. Batzer,et al.  Mammalian retroelements. , 2002, Genome research.

[28]  Chuangye Yan,et al.  Structural basis of pre-mRNA splicing , 2015, Science.

[29]  Soo-Chen Cheng,et al.  Both Catalytic Steps of Nuclear Pre-mRNA Splicing Are Reversible , 2008, Science.

[30]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[31]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[32]  E. Lindahl,et al.  Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2 , 2016, bioRxiv.

[33]  Randy J Read,et al.  Real-space refinement in PHENIX for cryo-EM and crystallography , 2018, bioRxiv.

[34]  Jonathan P. Staley,et al.  RNA catalyzes nuclear pre-mRNA splicing , 2013, Nature.

[35]  A. Pyle,et al.  Visualizing Group II Intron Catalysis through the Stages of Splicing , 2012, Cell.

[36]  K. Rajashankar,et al.  Crystal structure of a eukaryotic group II intron lariat , 2014, Nature.

[37]  C. Oubridge,et al.  CryoEM structure of the spliceosome immediately after branching , 2016, Nature.

[38]  A. Pyle,et al.  Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection. , 1995, RNA.

[39]  A. Pyle,et al.  The 2′-OH group at the group II intron terminus acts as a proton shuttle , 2009, Nature chemical biology.

[40]  F. Michel,et al.  Multiple exon-binding sites in class II self-splicing introns , 1987, Cell.

[41]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[42]  C. Oubridge,et al.  Crystal structure of Prp8 reveals active site cavity of the spliceosome , 2012, Nature.

[43]  Joseph H. Davis,et al.  Addressing preferred specimen orientation in single-particle cryo-EM through tilting , 2017, Nature Methods.

[44]  P. Perlman,et al.  Group II intron mobility occurs by target DNA-primed reverse transcription , 1995, Cell.

[45]  E. Westhof,et al.  A three‐dimensional perspective on exon binding by a group II self‐splicing intron , 2000, The EMBO journal.

[46]  G. Hausner,et al.  Phylogenetic relationships among group II intron ORFs. , 2001, Nucleic acids research.

[47]  Piotr Sliz,et al.  Collaboration gets the most out of software , 2013, eLife.

[48]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[49]  A conserved element that stabilizes the group II intron active site. , 2008, RNA.

[50]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[51]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[52]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.