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A Jacquier

Publications and source records attributed to A Jacquier.

At least 37 records · Page 2Linked to original sources
PubMed1997

Multiple tertiary interactions involving domain II of group II self-splicing introns.

The ribozyme core of group II introns is organized into six domains of secondary structure. Of these, domain II was long thought to be relatively unimportant for group II self-splicing. However, we now demonstrate the existence, in both major subdivisions of the group II family, of essential tertiary interactions involving domain II. theta-theta' is a novel tertiary interaction between the terminal loop of the IC1 stem of domain I and the basal stem of domain II. The theta-theta' interaction appears to stabilize the group II ribozyme core: it is essential for efficient self-splicing at elevated temperatures but, as shown by the use of a bimolecular reaction system, molecules with a defective theta-theta' contact are not affected in catalysis. An interaction, eta-eta', between domains II and VI of subgroup IIB introns was recently reported to mediate a conformational rearrangement between the two steps of the self-splicing reaction. We now show that domains II and VI of subgroup IIA introns also contact each other, although in a somewhat different way. Reinforcement of the eta-eta' interaction of a subgroup IIA intron prevents the use of a specific 2'-hydroxyl group in domain VI to initiate splicing by transesterification at the 5' splice site; the 5' intron-exon junction is hydrolyzed instead. Since disruption of eta-eta' has exactly opposite effects, and promotes reversal of the first transesterification step, it is concluded that formation of eta-eta' mediates a conformational change in subgroup IIA introns as well. Just like the eta-eta' interaction of subgroup IIB introns, the eta-eta' interaction of subgroup IIA introns (and the theta-theta' interaction) involves terminal loops of the GNRA family and their RNA receptors. Therefore, these motifs are used by nature not only to stabilize three-dimensional RNA architectures, but also in situations that require dynamic interactions.

Base Sequence↗
PubMed1996

An RNA conformational change between the two chemical steps of group II self-splicing.

As for nuclear pre-mRNA introns, the splicing pathway of group II self-splicing introns proceeds by two successive transesterifications involving substrates with different chemical configurations. These two reactions have been proposed to be catalysed by two active sites, or alternatively by a single active site rearranging its components to accommodate the successive substrates. Here we show that the structural elements specific for the second splicing step are clustered in peripheral structures of domains II and VI. We show that these structures are not required for catalysis of the second chemical step but, instead, take part in a conformational change that occurs between the two catalytic steps. This rearrangement involves the formation of a tertiary contact between part of domain II and a GNRA tetraloop at the tip of domain VI. The fact that domain VI, which carries the branched structure, is involved in this structural rearrangement and the fact that modifications affecting the structures involved have almost no effect when splicing proceeds without branch formation, suggest that the conformational change results in the displacement of the first-step product out of the active site. These observations give further support to the existence of a single active site in group II introns.

Base Sequence↗
PubMed1996

Group II introns: elaborate ribozymes.

Group II introns are found in organelle genomes of plants, fungi and algae as well as in some bacteria. Some group II introns have been shown to self-splice in vitro and thus constitute examples of ribozymes. Their splicing pathway is analogous to the splicing pathway of nuclear pre-mRNA introns. They thus constitute simple models to analyze RNA catalysis of this type of splicing reactions. In this review article, I will summarize our current state of understanding of the ribozyme activity of group II introns and show that their large size correlates with their ability to perform complex tasks. After discussing the similarities found between group II and nuclear pre-mRNA introns, I will briefly evoke how the ribozyme activity of group II introns might be involved in their transposition at the DNA level.

Binding Sites↗
PubMed1994

Catalytic site components common to both splicing steps of a group II intron.

The splicing of group II introns occurs in two steps involving substrates with different chemical configurations. The question of whether these two steps are catalyzed by a single or two separate active sites is a matter of debate. Here, certain bases and phosphate oxygen atoms at conserved positions in domain V of a group II self-splicing intron are shown to be required for catalysis of both splicing steps. These results show that the active sites catalyzing the two steps must, at least, share common components, ruling out the existence of two completely distinct active sites in group II introns.

Base Sequence↗
PubMed1994

Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae.

The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities. Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns. We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/. Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide. These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection.

Base Sequence↗
PubMed1994

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Base Sequence↗
PubMed1993

Interaction of intronic boundaries is required for the second splicing step efficiency of a group II intron.

Group II and nuclear pre-mRNAs introns share a common splicing pathway involving a lariat intermediate, as well as some primary sequence similarities at the splice junctions. In this work, we analyze the role of the conserved nucleotides at the first and penultimate positions (G1 and A886) of a group II self-splicing intron. We show that the G1 nucleotide is essential for the efficiency of both the first and the second splicing steps, while substitutions at the penultimate nucleotide affect mostly the efficiency of the second step. A reciprocal suppression of the second splicing step defect can be observed in some double mutants. This result is best explained by a non-Watson-Crick interaction between the first and the penultimate nucleotides of the intron, which occurs after lariat formation. The finding that an interaction between intron boundaries is required for the second splicing step in both group II and nuclear pre-mRNA introns strengthens the idea that both systems employ similar mechanisms, albeit with differences in the details of the nucleotide interactions.

Base Sequence↗
PubMed1992

Sequence of a 10.7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases.

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 10.7 kb. The sequence was determined using a double-strand sequencing strategy adapted from the random-clone strategy. The segment contains seven non-overlapping long open reading frames, YKL500, 505, 510, 513, 516, 518 and 520 and one tRNA gene. YKL505 and YKL513 are two already sequenced genes, the BAF1/ABF1 and the APN1 genes, respectively. YKL510 exhibits a strong homology to the RAD2 protein and YKL516 is presumably a protein kinase.

Amino Acid Sequence↗
PubMed1991

Splice site selection and role of the lariat in a group II intron.

The structural elements involved in 5' and 3' splice site (SS) selection in a group II intron were analyzed. While 5' SS selection appears to be defined by only one element, the EBS1-IBS1 pairing, four distinct structural components contribute to 3' SS selection, one of which being analogous to the "internal guide sequence" described for group I introns. Moreover, some of the mutants analyzed during this study induce efficient 5' SS hydrolysis and suggest how 5' SS transesterification is selected against hydrolysis. Finally, the lariat structure was found to accelerate both steps of splicing, suggesting that it "locks" the ribozyme in an active configuration.

Base Sequence↗
PubMed1990

Base-pairing interactions involving the 5' and 3'-terminal nucleotides of group II self-splicing introns.

By combining comparative sequence analyses and nucleotide replacements, we show that formation of the active center of group II introns rests in part on two novel long-range base-pairing interactions. (1) The last nucleotide of group II introns forms a solitary Watson-Crick base-pair with one of the nucleotides in the short sequence stretch connecting domains II and III. Formation of this base-pair is rate-limiting for the 3' cleavage and ligation step. (2) Nucleotides 3 and 4 form base-pairs with two consecutive nucleotides in a well-conserved internal loop of domain I. This interaction is involved in both the 5' and 3' cleavage steps. Possible relationships between group II and nuclear pre-mRNA introns are reassessed by taking into account these new pieces of information.

Bacteriophages↗
PubMed1990

Self-splicing group II and nuclear pre-mRNA introns: how similar are they?

The splicing pathway of pre-mRNA introns bears similarities to that of the group II introns, some members of which undergo self-splicing. The snRNAs may provide the pre-mRNA introns with RNA structures in trans comparable to those available in cis in group II introns. This article examines the available evidence for the hypothesis that the catalysis of these two splicing pathways is fundamentally equivalent.

Animals↗
PubMed1987

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

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.

Base Sequence↗
PubMed1986

Efficient trans-splicing of a yeast mitochondrial RNA group II intron implicates a strong 5' exon-intron interaction.

The reaction mechanism for self-splicing introns requires the existence of a 5' exon binding site on the intron. Experimental evidence is now presented consistent with the existence of such a binding site by demonstrating efficient and accurate trans-self-splicing of a yeast mitochondrial group II intron. Partial and complete trans-splicing reactions take place in the absence of branch formation, part of the usual pathway of nuclear splicing and group II self-splicing. In addition to indicating the existence of a 5' exon binding site on the intron, the results have mechanistic implications for group II self-splicing and perhaps for nuclear splicing as well.

Exons↗
PubMed1986

Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease.

The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (r1 intron) possesses a 235 codon long internal open reading frame (r1 ORF) whose translation product determines the duplicative transposition of that intron during crosses between intron-plus strains (omega+) and intron-minus ones (omega-). Using site-directed mutagenesis, we have constructed a universal code equivalent of the r1 ORF that, under appropriate promoter control, allows the overexpression in E. coli of a protein identical to the mitochondrial intron encoded "transposase". This protein exhibits a double strand endonuclease activity specific for the omega- site. This finding demonstrates, for the first time, the enzymatic activity of an intron encoded protein whose function is to promote the spreading of that intron by generating double strand breaks at a specific sequence within a gene.

Amino Acid Sequence↗
PubMed1986

Mitochondrial introns as mobile genetic elements: the role of intron-encoded proteins.

Introns of organelle genes share distinctive RNA secondary structures that allow their classification into two known families. These structures are believed to play an essential role in splicing, and members of both structural classes have recently been shown to perform self-splicing reactions in vitro. In lower eukaryotes, many structured introns also contain long internal open reading frames (ORFs), which are able to code for hydrophilic proteins. Several properties of self-splicing structured introns suggest that they resemble mobile genetic elements, even though no actual transposition event involving these introns has yet been found. We report here on the characterization of two intron-encoded proteins that strongly support this attractive idea. First, we show that the class I intron of the 21S ribosomal RNA (rRNA) gene of Saccharomyces cerevisiae omega+ strains (rl intron) encodes a specific transposase. This protein has been partially purified from Escherichia coli cells that overexpress it from an artificial universal code equivalent to the rl intronic ORF. The omega transposase shows a double-strand endonuclease activity in vitro. This activity creates a 4-bp staggered cut with 3' OH overhangs within a specific sequence of the 21S rRNA gene of omega- strains. It is precisely within this sequence that the rl intron inserts by a duplicative transposition. Second, we report on the synthesis, in E. coli, of a putative reverse transcriptase encoded by the class II intron of the cytochrome b gene of Schizosaccharomyces pombe. This synthesis was obtained from E. coli expression vectors, using the class II intronic ORF linked to an artificial initiator sequence. As further support of the idea that structured introns are mobile, we show, from a systematic screening of introns in various yeast species, that the rl intron has transposed into the ATPase subunit 9 gene of Kluyveromyces fragilis. Structural features observed at the new intron homing site may be relevant to the transposition event.

Adenosine Triphosphatases↗
PubMed1986

RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point.

Two mutant genes, both of which contain an A----C transversion at the absolutely conserved branch point of the yeast "TACTAAC box" (TACTAAC----TACTACC), were constructed and introduced into yeast cells. Splicing and gene expression are almost completely eliminated by this mutation, but a low level (approximately equal to 0.1%) of proper splicing is detectable. Branch point mapping indicates that the mutant branch is formed at the normal location--i.e., to cytidine rather than adenosine. The mutant branch is also a very poor substrate for the HeLa cell debranching enzyme. Although splicing of the mutant transcripts is very poor, the cells contain a high level of mutant intron because these excised introns are remarkably stable. The results imply that the normal branch point is important not only for branch formation and splicing but also for intron turnover.

Base Sequence↗
PubMed1985

A quantitative analysis of the effects of 5' junction and TACTAAC box mutants and mutant combinations on yeast mRNA splicing.

We have introduced four point mutations into the 5' junction (GTATGT) and TACTAAC box of a yeast intron-containing gene coding for beta-galactosidase. To analyze quantitatively mutant combinations, we deliberately avoided nucleotides adjacent to the sites of cleavage (GTATGT) and lariat formation (TACTAAC) and chose positions with expected weak effects on splicing. All four mutants do not affect, or only marginally affect, gene expression. The levels of precursor RNA and intermediates indicate, however, that splicing efficiency is affected in all cases; the first stage of splicing, 5' cleavage and lariat formation, is reduced two to 15-fold in these four mutants. The mutants were combined to generate compensatory and noncompensatory double mutant combinations. No evidence for a specific base-pairing interaction between the 5' junction and TACTAAC box could be obtained. The results suggest that all conserved positions in the 5' junction and TACTAAC box play a role in splicing efficiency.

Base Sequence↗