A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation

Costa, María; Fontaine, Jean-Marc; Goër, Susan Loiseaux‐de; Michel, François

doi:10.1006/jmbi.1997.1416

Cited by 75 publications

(98 citation statements)

References 33 publications

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“…Our results bear valuable information concerning the kinetics of group II intron self-splicing+ First, they show that binding of the 59 exon to the intron in the forward cis-splicing is rate-limiting in model precursor transcripts commonly used so far+ This conclusion is in accordance with the previously mentioned observation that the splicing efficiency of the pJD20 precursor transcript was improved by shortening its exon sequences (Perlman & Podar, 1996)+ However, even with the better model precursor transcript selected during this study, pEX93, the first step of forward splicing still appears to be limited by folding of the RNA rather than catalysis+ Indeed, the observed rate of branching in HS buffer and at 45 8C for this precursor transcript is k obs ' 0+67 min Ϫ1 , a value substantially lower than the rate of branching deduced from the reverse branching experiments at saturating 59 exon concentration (k br ' 3+5 6 1+0 min Ϫ1 )+ This difference is due to the fact that, in reverse branching experiments, preincubation of the lariat intron under splicing conditions before addition of the 59 exon avoids part of the folding process to interfere with catalysis+ This is most likely the reason why the value of 3+5 min Ϫ1 is the highest rate of branching determined so far for the aI5g intron+ This rate is similar to the highest rate of branching measured for the newly described Pl+LSU/2 intron from the brown alga Pylaiella littoralis (on the order of 5 min Ϫ1 ), which provides a new lower bound for the rate of chemistry for the first splicing step catalyzed by group II ribozymes (Costa et al+, 1997b)+…”

Section: Consequences On Group II Intron Self-splicing Kineticsmentioning

confidence: 64%

Influence of substrate structure on in vitro ribozyme activity of a group II intron

Nolte

Chanfreau

Jacquier

1998

RNA

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Substrate sequences surrounding catalytic RNAs but not involved in specific, conserved interactions can severely interfere with in vitro ribozyme activity. Using model group II intron precursor transcripts with truncated or randomized exon sequences, we show that unspecific sequences within the 59 exon are particularly prone to inhibit both the forward and the reverse first splicing step (branching). Using in vitro selection, we selected efficient 59 exons for the reverse branching reaction. Precursor RNAs carrying these selected 59 exons reacted more homogeneously and faster than usual model precursor transcripts. This suggests that unfavorable structures induced by the 59 exon can introduce a folding step that limits the rate of in vitro self-splicing. These results stress how critical is the choice of the sequences retained or discarded when isolating folding domains from their natural sequence environments. Moreover, they suggest that exon sequences not involved in specific interactions are more evolutionarily constrained with respect to splicing than previously envisioned.

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Section: Consequences On Group II Intron Self-splicing Kineticsmentioning

confidence: 64%

Influence of substrate structure on in vitro ribozyme activity of a group II intron

Nolte

Chanfreau

Jacquier

1998

RNA

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“…We also tried combining the mutations in the distal stems of the two best-performing variants, DV14 and DV20, but found that the combined variant had lower retrohoming efficiency (2.2-fold wild type) than either single variant (16-and 22-fold wild type for DV14 and DV20, respectively). Finally, we substituted the DV distal stem from the Pylaiella littoralis LSU/2, which selfsplices at unusually low magnesium concentrations (32), and found the retrohoming efficiency to be only 1.3-fold that of the wild-type intron (Fig. 3F).…”

Section: Characteristics Of Variants With Increased Retrohoming Efficmentioning

confidence: 98%

Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

Truong

Sidote

Russell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg 2+ concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg 2+ transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg 2+ concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg 2+ concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg 2+ binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg 2+ -dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg 2+ concentrations, with implications for evolution and potential applications in gene targeting. directed evolution | Mg 2+ transport | RNA structure M obile group II introns are retrotransposons that are found in prokaryotes and the mitochondrial and chloroplast DNAs of some eukaryotes and are thought to be evolutionary ancestors of spliceosomal introns, the spliceosome, retrotransposons, and retroviruses in higher organisms (1). They consist of two components-an autocatalytic intron RNA ("ribozyme") and an intron-encoded protein (IEP) with reverse transcriptase activity-that function together in a ribonucleoprotein (RNP) complex to promote RNA splicing and site-specific integration of the intron into new DNA sites in a process called retrohoming (1). Like spliceosomal introns, group II introns splice via two sequential transesterification reactions that yield an excised intron lariat RNA (2). For group II introns, the splicing reactions are catalyzed by the intron RNA with the assistance of the IEP, which binds specifically to the intron RNA and stabilizes the catalytically active RNA structure. The IEP then remains bound to the excised intron lariat RNA in an RNP that promotes retrohoming via reverse splicing of the intron RNA directly into DNA sites followed by reverse transcription by the IEP. The resulting intron cDNA is integrated into the genome by host enzymes (3, 4). The ribozyme-based, site-specific DNA integration mechanism used by g...

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“…Products were identified based on (1) reverse transcription of gel-extracted molecules (see Fig. 4) and (2) their electrophoretic mobility, compared to that of known splicing products of a P. littoralis LSU1787 (Table 1; Costa et al 1997b) precursor transcript (MW lane: band 1, 640 nt, lariat; band 2, 872 nt, precursor; band 3, 640 nt, linear intron; band 4, 232 nt, ligated exons). (B) Time course of self-splicing reactions of a Grifola SSU788 precursor RNA at 42°C in 40 mM Tris-Cl (pH 7.5 at 25°C), 20 mM MgCl 2 , and 1 M NH 4 Cl (circles and solid curve, generated by a biphasic exponential fit with k 1 = 0.9 6 0.2 min À1 and k 2 = 0.03 min À1 ) (see Materials and Methods) or 1 M KCl (squares and dashed curve, lariat intron; lozenges and dotted curve, linear intron; both from single exponential fits).…”

Section: Distribution Of 59-terminal Inserts In Mitochondrial Subgroumentioning

confidence: 99%

“…RNA synthesis and purification were carried out as described in Costa et al (1997b), except that the transcription mixture contained 10% DMSO so as to avoid premature transcription stops and a 1.55 molar concentration ratio of magnesium over nucleotides was used to prevent premature intron splicing.…”

Section: Sequence Analyses Of Laglidadg Proteinsmentioning

confidence: 99%

“…Purification of splicing products from preparative denaturing polyacrylamide gels and their reverse transcription with 32 P-labeled, gel-purified oligonucleotides were performed essentially as described by Costa et al (1997b). The following oligonucleotides (see Supplemental Table S1) were used for reverse transcription: BMS103B, to sequence ligated exons and determine the branchpoint of the G. frondosa intron-39exon lariat; Gr-R2, to determine the 59 splice of the G. frondosa intron lariat; Py-R2, to determine the 59 extremity of P. fulgens linear intron molecules.…”

Section: Reverse Transcription Of Splicing Productsmentioning

confidence: 99%

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Recurrent insertion of 5′-terminal nucleotides and loss of the branchpoint motif in lineages of group II introns inserted in mitochondrial preribosomal RNAs

Costa²,

Bassi³

et al. 2011

RNA

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A survey of sequence databases revealed 10 instances of subgroup IIB1 mitochondrial ribosomal introns with 1 to 33 additional nucleotides inserted between the 59 exon and the consensus sequence at the intron 59 end. These 10 introns depart further from the IIB1 consensus in their predicted domain VI structure: In contrast to its basal helix and distal GNRA terminal loop, the middle part of domain VI is highly variable and lacks the bulging A that serves as the branchpoint in lariat formation. In vitro experiments using two closely related IIB1 members inserted at the same ribosomal RNA site in the basidiomycete fungi Grifola frondosa and Pycnoporellus fulgens revealed that both ribozymes are capable of efficient self-splicing. However, whereas the Grifola intron was excised predominantly as a lariat, the Pycnoporellus intron, which possesses six additional nucleotides at the 59 end, yielded only linear products, consistent with its predicted domain VI structure. Strikingly, all of the introns with 59 terminal insertions lack the EBS2 exon-binding site. Moreover, several of them are part of the small subset of group II introns that encode potentially functional homing endonucleases of the LAGLIDADG family rather than reverse transcriptases. Such coincidences suggest causal relationships between the shift to DNA-based mobility, the loss of one of the two ribozyme sites for binding the 59 exon, and the exclusive use of hydrolysis to initiate splicing.

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A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation

Cited by 75 publications

References 33 publications

Influence of substrate structure on in vitro ribozyme activity of a group II intron

Influence of substrate structure on in vitro ribozyme activity of a group II intron

Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

Recurrent insertion of 5′-terminal nucleotides and loss of the branchpoint motif in lineages of group II introns inserted in mitochondrial preribosomal RNAs

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