In Vivo Evolution of a Catalytic RNA Couples Trans-Splicing to Translation

Olson, Karen E.; Dolan, Gregory F.; Müller, Ulrich F.

doi:10.1371/journal.pone.0086473

Cited by 10 publications

(12 citation statements)

References 52 publications

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“…This efficiency is similar to the estimated 3-4% of CAT mRNA repaired in a similar bacterial system by a trans-splicing group I intron using only a single splice site [21], [36]. This suggests that the spliceozymes described in this study were similarly efficient as the previously used trans-splicing ribozymes.…”

Section: Discussionsupporting

confidence: 83%

Spliceozymes: Ribozymes that Remove Introns from Pre-mRNAs in Trans

2014

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Group I introns are pre-mRNA introns that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves and joining the flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby the ribozymes can recognize a splice site on a substrate RNA and replace the 5′- or 3′-portion of the substrate. Here we describe a new variant of the group I intron ribozyme from Tetrahymena that recognizes two splice sites on a substrate RNA, removes the intron sequences between the splice sites, and joins the flanking exons, analogous to the action of the spliceosome. This ‘group I spliceozyme’ functions in vitro and in vivo, and it is able to mediate a growth phenotype in E. coli cells. The intron sequences of the target pre-mRNAs are constrained near the splice sites but can carry a wide range of sequences in their interior. Because the splice site recognition sequences can be adjusted to different splice sites, the spliceozyme may have the potential for wide applications as tool in research and therapy.

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Section: Discussionsupporting

confidence: 83%

Spliceozymes: Ribozymes that Remove Introns from Pre-mRNAs in Trans

2014

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“…2A) using an evolution system established in earlier studies (Olson and Muller 2012;Olson et al 2014). As starting points for the evolution, two spliceozyme libraries were generated by mutagenic PCR, using as template either a single spliceozyme gene ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Increased efficiency of evolved group I intron spliceozymes by decreased side product formation

Amini

Müller

2015

RNA

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The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100-nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5 ′ -splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5 ′ -splice site but an increase in cleavage side products at the 3 ′ -splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5 ′ -splice site and increasing activity at the 3 ′ -splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to transsplicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed.

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“…Three studies describe the evolution of trans -splicing ribozymes over multiple rounds of evolution. The first evolution of trans -splicing group I intron ribozymes used the Tetrahymena ribozyme and proceeded over 21 rounds of evolution [ 66 ]. It used the same experimental setup as described in the selection above [ 50 ] but repeatedly introduced mutations over the entire length of the ribozyme ( Figure 4 A).…”

Section: Evolution Of Improved Trans -Splicing mentioning

confidence: 99%

“…Mutagenesis levels with 30 cycles of mutagenic PCR (corresponding to ~7.3 mutations per ribozyme) led to a collapse of the evolving population, while 10 or 20 cycles of mutagenic PCR, (~2.4 and 4.8 mutations per ribozyme, respectively) led to stable populations. The effect of recombination [ 67 ] was tested with ~1 recombination event per evolution round and ribozyme [ 66 ]. The most efficient, evolved motif did not benefit from recombination because it required four clustered mutations.…”

Section: Evolution Of Improved Trans -Splicing mentioning

confidence: 99%

“…For example, the group II intron shares a common ancestor with the spliceosome [ 79 ], and the evolution from a likely ribozyme precursor to a five-partite, multimegadalton, dynamic RNA-protein complex that is able to correctly recognize thousands of splice sites is far from being elucidated. The evolution of group I intron spliceozymes can serve as model for specific biochemical steps in the evolution of the spliceosome: Our previous evolution [ 68 ] showed how a ribozyme evolved for cis -splicing can adjust its mechanics to the trans -reaction, and an evolution with group I introns using a single splice site showed how an evolving RNA can recruit a cellular protein [ 66 ]. Further evolution studies may develop variants of group I introns that show more and more characteristics of spliceosomes.…”

Section: Possible Applications Of Evolved Trans mentioning

confidence: 99%

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Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

Müller

2017

Molecules

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Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

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In Vivo Evolution of a Catalytic RNA Couples Trans-Splicing to Translation

Cited by 10 publications

References 52 publications

Spliceozymes: Ribozymes that Remove Introns from Pre-mRNAs in Trans

Spliceozymes: Ribozymes that Remove Introns from Pre-mRNAs in Trans

Increased efficiency of evolved group I intron spliceozymes by decreased side product formation

Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

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