Multiple exon-binding sites in class II self-splicing introns

doi:10.1016/0092-8674(87)90658-1

Cell

Volume 50, Issue 1, 3 July 1987, Pages 17-29

https://doi.org/10.1016/0092-8674(87)90658-1 Get rights and content

Abstract

Partial deletion of the exon 5′ to S. cerevisiae intron a5, a self-splicing mitochondrial class II intron, reveals the existence of several sites of intron-exon interaction. We have identified two of the corresponding exon-binding sites in intron a5 by comparative sequence analysis and RNAase H digestion of the intron complexed to a DNA version of its 5′ exon. Introduction of mutations in either the intronic sites or the complementary exonic sequences affects splicing in vitro, whereas double mutants in which intron-exon pairings have been restored show normal activity. Some of the mutants accumulate a product that was shown to be the intron-3′ exon lariat, a postulated splicing intermediate. The possible role of one of the intronic sites in aligning exons for the ligation step is discussed.

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Citation Excerpt :
The RNA component of a group II intron is a ribozyme that has a conserved secondary structure with six domains (Lambowitz and Zimmerly, 2011; Figure S2). Domain I (DI) contains EBS1–EBS3 (Costa et al., 2000; Jacquier and Michel, 1987), used to target the group II intron to a specific DNA sequence. Domain IV (DIV) contains the open reading frame (ORF) for the maturase protein and provides the main binding site for the RT domain to form the fully assembled group II intron ribonucleoprotein (RNP) complex (Wank et al., 1999).
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.
The role of Mg(II) in DNA cleavage site recognition in group II intron ribozymes: Solution structure and metal ion binding sites of the RNA·DNA complex
2014, Journal of Biological Chemistry
Citation Excerpt :
The EBS1·IBS1 interaction confers high specificity to the site of reinsertion of the intron, thus preventing insertion into sites from which the intron cannot splice again. However, it has been shown that EBS sequences are not conserved within different group II introns (17, 20, 21). For this reason, any desired sequence can be bound and cleaved by the intron in trans as long as the EBS and IBS sequences are complementary (22–25).
Group II intron ribozymes catalyze the cleavage of (and their reinsertion into) DNA and RNA targets using a Mg²⁺-dependent reaction. The target is cleaved 3′ to the last nucleotide of intron binding site 1 (IBS1), one of three regions that form base pairs with the intron's exon binding sites (EBS1 to -3). We solved the NMR solution structure of the d3′ hairpin of the Sc.ai5γ intron containing EBS1 in its 11-nucleotide loop in complex with the dIBS1 DNA 7-mer and compare it with the analogous RNA·RNA contact. The EBS1·dIBS1 helix is slightly flexible and non-symmetric. NMR data reveal two major groove binding sites for divalent metal ions at the EBS1·dIBS1 helix, and surface plasmon resonance experiments show that low concentrations of Mg²⁺ considerably enhance the affinity of dIBS1 for EBS1. Our results indicate that identification of both RNA and DNA IBS1 targets, presentation of the scissile bond, and stabilization of the structure by metal ions are governed by the overall structure of EBS1·dIBS1 and the surrounding loop nucleotides but are irrespective of different EBS1·(d)IBS1 geometries and interstrand affinities.
A 971-bp insertion in the rns gene is associated with mitochondrial hypovirulence in a strain of Cryphonectria parasitica isolated from nature
2011, Fungal Genetics and Biology
Citation Excerpt :
A BLASTn search using the putative domain V sequence as a query also recovered several group II intron domain V sequences from fungi and plant mtDNA group II introns. Next to domain V, a sequence was detected that folds into domain VI, a domain that harbors the bulging adenine that provides a 2′ OH group for initiating a series of transesterification reactions that results in the splicing of the flanking exon sequences and the release of the intron RNA as a lariat (Costa et al., 2000; Jacquier and Michel, 1987; Kück et al., 1990; Lambowitz and Zimmerly, 2004; Michel et al., 1989, 2009; Schmelzer and Müller, 1987; Toor et al., 2001; van der Veen et al., 1986). The 5′ end of the intron starts with the characteristic GUGYG sequence (reviewed in Bonen and Vogel, 2001) and features such as the IBS1 and 2 and the corresponding EBS1 and 2 motifs (Bonen and Vogel, 2001; Michel et al., 1989) were also detected (Fig. 2).
In the chestnut-blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes frequently are elicited by double-stranded RNA (dsRNA) virus infections. However, some strains manifest cytoplasmically transmissible hypovirulence traits without containing any mycovirus. In this study, we describe an altered form of mtDNA that is associated with hypovirulence and senescence in a virus-free strain of C. parasitica, KFC9, which was obtained from nature and has an elevated level of cyanide-resistant respiration. In this strain, a 971-bp DNA element, named InC9, has been inserted into the first exon of the mitochondrial small-subunit ribosomal RNA (rns) gene. Sequence analysis indicates that InC9 is a type A1 group II intron that lacks a maturase-encoding ORF. RT-PCR analyses showed that the InC9 sequence is spliced inefficiently from the rRNA precursor. The KFC9 strain had very low amounts of mitochondrial ribosomes relative to virulent strains, thus most likely is deficient in mitochondrial protein synthesis and lacks at least some of the components of the cyanide-sensitive, cytochrome-mediated respiratory pathway. The attenuated-virulence trait and the splicing-defective intron are transferred asexually and concordantly by hyphal contact from hypovirulent donor strains to virulent recipients, confirming that InC9 causes hypovirulence.
The mechanism of splicing as told by group II introns: Ancestors of the spliceosome
2019, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
Citation Excerpt :
Group II introns have conserved 5′ GUGYG sequences and 3′ AY end sequences, resembling the 5′ GU and 3′ AG sequences of spliceosomal introns [46]. Domain I (DI) is the largest domain at the 5′ end of the intron and contains sequences that base pair to exon sequences in order to align exon junctions at the active site for splicing and reverse splicing reactions [23,41,47]. These sequences consist of exon binding sequences (EBS) that recognize and bind intron binding sequences (IBS) in the exons and the δ nucleotide that binds the δ' nucleotide in DI (Fig. 2) [23,41,46,47].
Spliceosomal introns and self-splicing group II introns share a common mechanism of intron splicing where two sequential transesterification reactions remove intron lariats and ligate exons. The recent revolution in cryo-electron microscopy (cryo-EM) has allowed visualization of the spliceosome's ribozyme core. Comparison of these cryo-EM structures to recent group II intron crystal structures presents an opportunity to draw parallels between the RNA active site, substrate positioning, and product formation in these two model systems of intron splicing. In addition to shared RNA architectural features, structural similarity between group II intron encoded proteins (IEPs) and the integral spliceosomal protein Prp8 further support a shared catalytic core. These mechanistic and structural similarities support the long-held assertion that group II introns and the eukaryotic spliceosome have a common evolutionary origin. In this review, we discuss how recent structural insights into group II introns and the spliceosome facilitate the chemistry of splicing, highlight similarities between the two systems, and discuss their likely evolutionary connections. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.
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Cell

ArticleMultiple exon-binding sites in class II self-splicing introns

Abstract

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Article
Multiple exon-binding sites in class II self-splicing introns