Research Articles

Crystal structures of a group II intron lariat primed for reverse splicing

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Science  02 Dec 2016:
Vol. 354, Issue 6316, aaf9258
DOI: 10.1126/science.aaf9258

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Tie me up, cut me down

Group II in trons are mobile genetic elements found in all domains of life. They are large ribozymes that can excise themselves from host RNA. Costa et al. determined the structure of an excised group II intron in its branched conformation. This conformation is comparable to the branched “lariat” seen during the splicing of nuclear RNA transcripts. The lariat conformation helps assemble the group II active site for the reverse splicing reaction. The lariat in spliceosomal splicing may also have a similar role in the second step of messenger RNA intron removal.

Science, this issue p. 10.1126/science.aaf9258

Structured Abstract


Self-splicing group II introns are catalytic RNAs (ribozymes) that can excise by themselves from precursor RNA molecules. These ribozymes are widespread in the bacterial world and can also be found in the bacterial-derived organelles (mitochondria and chloroplasts) of some higher organisms. Group II self-splicing is believed to have evolved into nuclear pre-mRNA splicing, a fundamental step in the expression of eukaryotic genes during which a large ribonucleoprotein machinery (the spliceosome) catalyzes the removal of introns from nascent premessenger transcripts. Both group II and pre-mRNA splicing proceed via two sequential phosphoryl transfer reactions. First, a 2′-5′ phosphodiester bond is created between a conserved intron adenosine and the first intron nucleotide. The resulting splicing intermediate with a branched conformation is called a “lariat.” In a second step, completion of splicing leads to the ligation of the flanking 5′ and 3′ exons and the release of the intron lariat. Bacterial group II introns are composite elements that, in addition to their ribozyme core, carry an open reading frame encoding a reverse transcriptase (RT) enzyme. In association with their RT, freed group II intron lariats behave as mobile genetic elements that colonize genomes through retrotransposition. The group II mobility pathway is initiated by “reverse splicing” of the intron lariat into a DNA target, followed by synthesis of a DNA copy of the integrated intron by the RT enzyme.


Eukaryotic pre-mRNA splicing relies entirely on formation of the 2′-5′ branch structure. In group II introns, the same branch structure is required for efficient and faithful catalysis of the second step of splicing and for complete, accurate reverse splicing of the intron into DNA targets during intron mobility. Moreover, in both splicing systems, the branched nucleotides must translocate within the active site between the two steps of splicing. To understand the molecular mechanisms at play, we crystallized the lariat form of a group II intron either alone or bound to a nonreactive analog of the 5′ exon.


Our crystal structures at 3.4 and 3.5 Å resolution reveal that the 2′-5′ branched nucleotides are part of a network of hydrogen bonds and stacking interactions that involve highly conserved nucleotides at the intron core and boundaries. The resulting architecture organizes the second-step active site by juxtaposing the intron 5′ and 3′ ends and promotes positioning of the last intron nucleotide into the catalytic center. After the ligated exons have been released, the terminal ribose of the lariat intron remains docked in the reaction center, with its 3′-hydroxyl group activated by a highly coordinated metal ion and poised for catalysis of the reverse-splicing reaction. Stable docking of the 2′-5′ branch structure into the active site is promoted by a rearrangement of the base-pairing pattern within the helix that contains the adenosine branchpoint. This rearrangement operates between the two steps of splicing and is essential for recognition of the proper 3′ splice site. Comparison of lariat structures in the presence and absence of the 5′ exon reveals that substrate binding results in an induced fit that extends into the catalytic center and contributes to coordination of a second catalytic metal ion. This “exon-sensing” device could ensure that catalysis of reverse splicing is dependent on the accuracy of intron-exon pairings.


The present crystal structures bring to light the crucial role of the 2′-5′ branch in organizing the lariat intron catalytic site for efficient and accurate ligation of the flanking exons during the last stage of splicing. Making use of the branch structure to build the second-step active site results in coupling the two steps of splicing and contributes decisively to the fidelity of the overall process. Moreover, the presence of the 2′-5′ branch locks the active site into a near-transition-state configuration for catalysis of reverse splicing, which must have contributed to selection of the lariat conformation during the evolution of mobile group II introns. As all nucleotides involved in the catalytic center have potential homologs in the spliceosomal system, a group II-based model in which the 2′-5′ branch fulfills the same organizational role is proposed for the spliceosome second-step active site. As in group II introns, the postulated architecture implies a notable conformational rearrangement of the spliceosome active center between the two steps of pre-mRNA splicing. Our findings rationalize the extreme conservation of the branched conformation both during the diversification of group II introns and along the evolutionary path that gave rise to the nuclear pre-mRNA splicing apparatus of eukaryotes.

Structural basis for reverse splicing by group II introns.

Self-splicing group II introns and their evolutionary descendants, the spliceosomal introns of eukaryotes, are excised as branched molecules (lariats) with a 2′-5′ phosphodiester bond. The 2′-5′ branch organizes the lariat active site, priming it for catalysis of splicing and reverse splicing. Reverse splicing into DNA is used to initiate group II intron mobility in bacteria.


The 2′-5′ branch of nuclear premessenger introns is believed to have been inherited from self-splicing group II introns, which are retrotransposons of bacterial origin. Our crystal structures at 3.4 and 3.5 angstrom of an excised group II intron in branched (“lariat”) form show that the 2′-5′ branch organizes a network of active-site tertiary interactions that position the intron terminal 3′-hydroxyl group into a configuration poised to initiate reverse splicing, the first step in retrotransposition. Moreover, the branchpoint and flanking helices must undergo a base-pairing switch after branch formation. A group II–based model of the active site of the nuclear splicing machinery (the spliceosome) is proposed. The crucial role of the lariat conformation in active-site assembly and catalysis explains its prevalence in modern splicing.

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