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Structural basis for exon recognition by a group II intron.

ABSTRACT: Free group II introns are infectious retroelements that can bind and insert themselves into RNA and DNA molecules via reverse splicing. Here we report the 3.4-A crystal structure of a complex between an oligonucleotide target substrate and a group IIC intron, as well as the refined free intron structure. The structure of the complex reveals the conformation of motifs involved in exon recognition by group II introns.

SUBMITTER: Toor N

PROVIDER: S-EPMC2735234 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Structural basis for exon recognition by a group II intron.

Toor Navtej N Rajashankar Kanagalaghatta K Keating Kevin S KS Pyle Anna Marie AM

Nature structural & molecular biology 20081026 11

Free group II introns are infectious retroelements that can bind and insert themselves into RNA and DNA molecules via reverse splicing. Here we report the 3.4-A crystal structure of a complex between an oligonucleotide target substrate and a group IIC intron, as well as the refined free intron structure. The structure of the complex reveals the conformation of motifs involved in exon recognition by group II introns. ...[more]

PMID: 18953333

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Cation-induced kinetic heterogeneity of the intron-exon recognition in single group II introns.

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Structural Insights into the Mechanism of Group II Intron Splicing.

Project description:While the major architectural features and active-site components of group II introns have been known for almost a decade, information on the individual stages of splicing has been lacking. Recent advances in crystallography and cryo-electron microscopy (cryo-EM) have provided major new insights into the structure of intact lariat introns. Conformational changes that mediate the steps of splicing and retrotransposition are being elucidated, revealing the dynamic, highly coordinated motions that are required for group II intron activity. Finally, these ribozymes can now be viewed in their larger, more natural context as components of holoenzymes that include encoded maturase proteins. These studies expand our understanding of group II intron structural diversity and evolution, while setting the stage for rigorous mechanistic analysis of RNA splicing machines.

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Structural accommodations accompanying splicing of a group II intron RNP.

Project description:Group II introns, the putative progenitors of spliceosomal introns and retrotransposons, are ribozymes that are capable of self-splicing and DNA invasion. In the cell, group II introns form ribonucleoprotein (RNP) complexes with an intron-encoded protein, which is essential to folding, splicing and retromobility of the intron. To understand the structural accommodations underlying splicing, in preparation for retromobility, we probed the endogenously expressed Lactococcus lactis Ll.LtrB group II intron RNP using SHAPE. The results, which are consistent in vivo and in vitro, provide insights into the dynamics of the intron RNP as well as RNA-RNA and RNA-protein interactions. By comparing the excised intron RNP with mutant RNPs in the precursor state, confined SHAPE profile differences were observed, indicative of rearrangements at the active site as well as disengagement at the functional RNA-protein interface in transition between the two states. The exon-binding sequences in the intron RNA, which interact with the 5' exon and the target DNA, show increased flexibility after splicing. In contrast, stability of major tertiary and protein interactions maintains the scaffold of the RNA through the splicing transition, while the active site is realigned in preparation for retromobility.

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Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1.

Project description:BackgroundGroup II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(?-?' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The ? nucleotide is involved in base pairing with another intron residue (?') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.ResultsIn this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and ?-?' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical ?-?' pairing and its importance for the intron excision in vivo.ConclusionsThe excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.

| S-EPMC3123198 | biostudies-literature

A three-dimensional perspective on exon binding by a group II self-splicing intron.

Project description:We have used chemical footprinting, kinetic dissection of reactions and comparative sequence analysis to show that in self-splicing introns belonging to subgroup IIB, the sites that bind the 5' and 3' exons are connected to one another by tertiary interactions. This unanticipated arrangement, which contrasts with the direct covalent linkage that prevails in the other major subdivision of group II (subgroup IIA), results in a unique three-dimensional architecture for the complex between the exons, their binding sites and intron domain V. A key feature of the modeled complex is the presence of several close contacts between domain V and one of the intron-exon pairings. These contacts, whose existence is supported by hydroxyl radical footprinting, provide a structural framework for the known role of domain V in catalysis and its recently demonstrated involvement in binding of the 5' exon.

| S-EPMC314214 | biostudies-literature

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