Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

Schmidt, Udo; Riederer, Brigitte; Mörl, Mario; Schmelzer, Carlo; Ståhl, Ulf

doi:10.1002/j.1460-2075.1990.tb07400.x

Cited by 30 publications

(12 citation statements)

References 47 publications

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“…Oligonucleotide splints were used to delete individual domains of pJD20 according to the method of Kunkel (14) (using a Muta-gene kit from Bio-Rad); plasmids pJD2OAd2, pJD2OAd3, pJD2OAd4c, pJD2OAd5, and pJD2OAd6 encode precursor RNAs deleted cleanly for d2 to d6, respectively. We have previously characterized selfsplicing of an aISy substrate deleted for d4 transcribed from plasmid pJD2OA4 (10) 25 cycles. Amplified products were initially characterized on 2% agarose gels; the products were treated with T4 polynucleotide kinase and Klenow fragment (both from Promega) and then cloned into Bluescript(KS+) (Stratagene) that had been linearized by cleavage with SmaI.…”

Section: Methodsmentioning

confidence: 99%

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Group II introns deleted for multiple substructures retain self-splicing activity.

Koch¹,

Boulanger²,

Dib-Hajj³

et al. 1992

Mol. Cell. Biol.

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Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5'y of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.Group II introns are found in fungal mitochondrial DNAs and plant organelle genomes (17). Some of them self-splice in vitro (13,19,(24)(25)(26) by a pathway resembling that of mRNA introns of eukaryotes (6, 21). Group II intron primary sequences can be folded into secondary structures composed of six major substructures (domains 1 to 6 [dl to d6]) (see Fig. 1 for a specific example). It is likely that each domain participates in the splicing pathway in some way. It is already established that short sequences (exon binding sequences 1 and 2) in dl play essential roles in splicing by binding and presumably aligning the 5' exon for participation in both reaction steps (8,18). A recent study implicates another substructure of dl [the Cl(i) loop], capable of base pairing with a few nucleotides at the 5' end of the intron, as also playing a role in 5' splice site selection (9). d6 contains the branch site involved in the first transesterification reaction (23,24,26 In this study, each of the domains of aISy was tested for its possible involvement in d5-dependent reactions. Derivatives of the standard substrate deleted for various intron domains, singly and in combination, were made and characterized for self-splicing reactions in vitro as a means of locating the dS binding site. As expected, deletion of d5 blocks self-splicing, but all combinations of deletions of d2, d3, d4, and d6 tested, including the deletion of all four at once, retain at least the cleavage reaction at the 5' splice junction. Since 5' exon release requires d5 and still occurs even when all other substructures except dl are deleted, we conclude that the binding site of d5 is within dl. Our success in reducing the size of this intron...

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Section: Methodsmentioning

confidence: 99%

“…Some of them self-splice in vitro (13,19,(24)(25)(26) by a pathway resembling that of mRNA introns of eukaryotes (6, 21). Group II intron primary sequences can be folded into secondary structures composed of six major substructures (domains 1 to 6 [dl to d6]) (see Fig.…”

mentioning

confidence: 99%

Group II introns deleted for multiple substructures retain self-splicing activity.

Koch¹,

Boulanger²,

Dib-Hajj³

et al. 1992

Mol. Cell. Biol.

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“…More rarely, self-splicing can occur via the hydrolytic pathway, resulting in the excision of the intron as a linear molecule (Daniels et al 1996;Vogel and Börner 2002). A number of group II introns in yeast, algae, and bacteria have been shown, in fact, to catalyze their own removal from primary transcripts (Peebles et al 1986;Schmelzer and Schweyen 1986;van der Veen et al 1986;Schmidt et al 1990;Ferat and Michel 1993;Costa et al 1997;Robart and Zimmerly 2005).…”

Section: Introductionmentioning

confidence: 99%

A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

Mullineux¹,

Costa²,

Bassi³

et al. 2010

RNA

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A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37°C and at a concentration of MgCl 2 as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 39 OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.

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“…Several stop codons in the sequence of the intron result in a premature end to the translation of the transposase gene, rendering intron splicing essential for the mobility of the insertion sequence in the host genome (MartinezAbarca et al 1998). The location of the RmInt1 intron in a non-essential gene contrasts with the observed situation for mitochondrial group II introns, most of which are inserted into indispensable genes and require efficient splicing to ensure the survival of their hosts (Arnberg et al 1980;Schmidt et al 1990;Fontaine et al 1995;Simon et al 2008). With the lack of pressure for their efficient splicing, it has been suggested that RmInt1 and other bacterial group II introns behave essentially as retroelements rather than introns (Dai and Zimmerly 2002;Chillón et al 2011).…”

Section: Discussionmentioning

confidence: 96%

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element

Chillón

Molina-Sánchez

Fedorova

et al. 2014

RNA

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Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1 * sequence within the 3 ′ exon. We engineered an RmInt1 intron lacking the cryptic IBS1 * sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3 ′ splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3-IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.

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Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

Cited by 30 publications

References 47 publications

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns deleted for multiple substructures retain self-splicing activity.

A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element

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