Direct selection for ribozyme cleavage activity in cells

Chen, Xi; Denison, L.; Levy, Matthew; Ellington, Andrew D.

doi:10.1261/rna.1635209

Cited by 22 publications

(22 citation statements)

References 41 publications

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“…The hammerhead derivatives were specifically developed in vivo and showed different cleavage efficiencies in comparison to the original hammerhead, with 1401 6 228-, 225 6 70-, and 53 6 13-fold increase for N117, N79, and N93, respectively (Yen et al 2004). In addition, transcripts containing the N79 ribozyme were recently shown to be completely cleaved in vivo in transfected cells (Chen et al 2009). The Rzs or their corresponding inactive variants were inserted between the EDA exon and the DRE, and the resulting minigenes were analyzed for the EDA splicing pattern and nascent pre-mRNA.…”

Section: Resultsmentioning

confidence: 99%

Intron cleavage affects processing of alternatively spliced transcripts

Pastor

Mas²,

Talotti³

et al. 2011

RNA

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We previously showed that the insertion of a hammerhead ribozyme (Rz) in a critical intronic position between the EDA exon and a downstream regulatory element affects alternative splicing. Here we evaluate the effect of other intronic cotranscriptional cleavage events on alternative pre-mRNA processing using different ribozymes (Rz) and Microprocessor target sequences (MTSs). In the context of the fibronectin EDA minigene, intronic MTSs were cleaved very inefficiently and did not affect alternative splicing or the level of mature transcripts. On the contrary, all hammerhead Rz derivatives and hepatitis d Rz were completely cleaved before a splicing decision and able to affect alternative splicing. Despite the very efficient Rz-mediated cleavage, the levels of mature mRNA were only reduced to~40%. We show that this effect on mature transcripts occurs regardless of the type and intronic position of Rzs, or changes in alternative splicing and exon definition. Thus, we suggest that intron integrity is not strictly required for splicing but is necessary for efficient pre-mRNA biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Intron cleavage affects processing of alternatively spliced transcripts

Pastor

Mas²,

Talotti³

et al. 2011

RNA

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“…The latter values are well below the optimal Mg 2+ concentrations for protein-assisted group II intron RNA splicing and reverse splicing in vitro (5-10 mM) (21). For some ribozymes, it has been possible to select new variants that function at lower Mg 2+ concentrations or use different metal ions (22)(23)(24). However, the extent to which group II introns can be similarly evolved or engineered to decrease their Mg 2+ dependence is unknown.…”

Section: Significancementioning

confidence: 99%

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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“…Naturally occurring hammerheads as well as artificially selected motifs have rather variable sequences in the loop and bulge regions with specific combinations particular to different ribozyme sources. 67–69 One exception to this is the widely conserved adenosine in the sixth position of Loop II of type I and III ribozymes. In the full-length ribozyme tertiary structure, this base is involved in a noncanonical interaction with the conserved uridine from the substrate strand.…”

Section: Hammerhead Ribozymes In Mammalian Gene Regulationmentioning

confidence: 99%

“…This interaction exists in identical orientation in all but one natural hammerhead ribozyme analyzed to date and in most artificially selected species. 67 Preferential CG base pairing emphasizes the need for a reinforced helix at this position possibly due to the nature of the adjacent loop–bulge interaction. However underappreciated, this interaction may play an important role in fine-tuning ribozyme function.…”

Section: Hammerhead Ribozymes In Mammalian Gene Regulationmentioning

confidence: 99%

The Hammerhead Ribozyme

Scott¹,

Horan²,

Martick³

2013

Progress in Molecular Biology and Translational Science

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The hammerhead ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand hammerhead ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the ribozyme’s active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length hammerhead ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2′-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the hammerhead more recently has been found embedded in the 3′-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation.

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Direct selection for ribozyme cleavage activity in cells

Cited by 22 publications

References 41 publications

Intron cleavage affects processing of alternatively spliced transcripts

Intron cleavage affects processing of alternatively spliced transcripts

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

The Hammerhead Ribozyme

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