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

Abstract

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.

Structural Insights into the Mechanism of Group II Intron Splicing

“…These structures capture the lariat after it has completed the first step of reverse-splicing, which may represent the same intron structure as the pre-second step during forward splicing. These structures and supporting mutational data indicate that formation of the second step active site is coupled to a striking rearrangement in the D6 secondary structure (Figure 4A) [32]. During the first step of splicing, D6 forms a one-nucleotide bulge that only contains the nucleophile adenosine (Figure 4A), as described in previous studies [32].…”

“…- A.v. , solved to 3.5Å resolution) (Figure 3B) [32, 33]. Together, these structures have provided a wealth of information on the structural states that stimulate splicing by group II introns.…”

“…- A.v. chimera provide important insights into the configuration of the second-step active site [32]. These structures capture the lariat after it has completed the first step of reverse-splicing, which may represent the same intron structure as the pre-second step during forward splicing.…”

Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications

“…The crystal structure enabled us to model the binding of the full-length GsI-IIC RT to a group IIC intron lariat RNA (Costa et al, 2016) by using positioning information from the cryo-EM structures of Ll.LtrB RT bound to a group IIA intron RNA and spliceosomal protein Prp8 bound to snRNAs. In order to mimic the configuration of the complex just prior to reverse splicing of the intron lariat RNA into a DNA target site during retrohoming, a DNA strand containing the exon junction (EJ) and 5′-exon hairpin recognized by group IIC introns was added to the model based on the position of an RNA bound at the exon-binding site in a group IIC intron RNA structure (PDB:3IGI) (Fig.…”

Mobile Group II Introns as Ancestral Eukaryotic Elements

“…First, both group II and spliceosomal introns undergo two identical transesterification reactions resulting in a lariat form of the intron and ligated exons (Figure 2A). Second, structural and functional parallels between group II intron RNAs and spliceosomal snRNAs are striking [2, 4, 5, 7, 77]. The most frequently invoked feature is the branch-site motif with the bulged A involved in the very first step of splicing.…”