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Protein-facilitated folding of group II intron ribozymes.

ABSTRACT: Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5gamma group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5gamma intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although ATP is required for the protein turnover. As Mss 116 binds RNA nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5gamma ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. Dimethyl sulfate probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.

SUBMITTER: Fedorova O

PROVIDER: S-EPMC2912160 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Protein-facilitated folding of group II intron ribozymes.

Fedorova Olga O Solem Amanda A Pyle Anna Marie AM

Journal of molecular biology 20100206 3

Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5gamma group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5gamma intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. ...[more]

PMID: 20138894

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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

Protein-facilitated folding of group II intron ribozymes.

Publications

Protein-facilitated folding of group II intron ribozymes.

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