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The group II intron ribonucleoprotein precursor is a large, loosely packed structure.

ABSTRACT: Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.

SUBMITTER: Huang T

PROVIDER: S-EPMC3074136 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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The group II intron ribonucleoprotein precursor is a large, loosely packed structure.

Huang Tao T Shaikh Tanvir R TR Gupta Kushol K Contreras-Martin Lydia M LM Grassucci Robert A RA Van Duyne Gregory D GD Frank Joachim J Belfort Marlene M

Nucleic acids research 20101203 7

Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this ...[more]

PMID: 21131279

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Project description:BACKGROUND: The Streptophyta comprises all land plants and six groups of charophycean green algae. The scaly biflagellate Mesostigma viride (Mesostigmatales) and the sarcinoid Chlorokybus atmophyticus (Chlorokybales) represent the earliest diverging lineages of this phylum. In trees based on chloroplast genome data, these two charophycean green algae are nested in the same clade. To validate this relationship and gain insight into the ancestral state of the mitochondrial genome in the Charophyceae, we sequenced the mitochondrial DNA (mtDNA) of Chlorokybus and compared this genome sequence with those of three other charophycean green algae and the bryophytes Marchantia polymorpha and Physcomitrella patens. RESULTS: The Chlorokybus genome differs radically from its 42,424-bp Mesostigma counterpart in size, gene order, intron content and density of repeated elements. At 201,763-bp, it is the largest mtDNA yet reported for a green alga. The 70 conserved genes represent 41.4% of the genome sequence and include nad10 and trnL(gag), two genes reported for the first time in a streptophyte mtDNA. At the gene order level, the Chlorokybus genome shares with its Chara, Chaetosphaeridium and bryophyte homologues eight to ten gene clusters including about 20 genes. Notably, some of these clusters exhibit gene linkages not previously found outside the Streptophyta, suggesting that they originated early during streptophyte evolution. In addition to six group I and 14 group II introns, short repeated sequences accounting for 7.5% of the genome were identified. Mitochondrial trees were unable to resolve the correct position of Mesostigma, due to analytical problems arising from accelerated sequence evolution in this lineage. CONCLUSION: The Chlorokybus and Mesostigma mtDNAs exemplify the marked fluidity of the mitochondrial genome in charophycean green algae. The notion that the mitochondrial genome was constrained to remain compact during charophycean evolution is no longer tenable. Our data raise the possibility that the emergence of land plants was not associated with a substantial gain of intergenic sequences by the mitochondrial genome.

Project description:Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

Dataset Information

The group II intron ribonucleoprotein precursor is a large, loosely packed structure.

Publications

The group II intron ribonucleoprotein precursor is a large, loosely packed structure.

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