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

Abstract

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.

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

“…As starting points for the evolution, two spliceozyme libraries were generated by mutagenic PCR, using as template either a single spliceozyme gene ( Fig. 1; Amini et al 2014) or a pre-evolved library that already contained genetic diversity which, in some circumstances, can increase the rate at which new ribozyme characteristics are accessed by evolution (Hayden et al 2011). The spliceozyme libraries were cloned into the spliceozyme expression cassette of a library plasmid that also contained the expression cassette of the pre-mRNA of chloramphenicol acetyl transferase (CAT).…”

“…In vitro trans-splicing assays were completed essentially as described (Amini et al 2014). Spliceozyme and CAT pre-mRNA transcripts were generated by run-off transcription off PCR products and purified by denaturing PAGE.…”

“…Trans-splicing at both splice sites allows the removal of single nucleotides of a substrate RNA in cells, and up to 28 nt in vitro (Bell et al 2002;Dotson et al 2008). Using a different design principle for the substrate recognition at both splice sites, a Tetrahymena group I intron was recently shown to excise 100-nt-long introns from pre-mRNAs in vitro and in bacterial cells, efficiently enough to mediate antibiotic resistance (Amini et al 2014). These ribozymes were termed "spliceozymes" because their action is analogous to that of the spliceosome (Fig.…”

“…While the spliceosome likely evolved from a common ancestor with group II introns (Fica et al 2013), group I introns can be used as analogs to model comparable biochemical steps. The existing trans-splicing group I intron spliceozymes show that cissplicing ribozymes can make the evolutionary step to transsplicing ribozymes (Amini et al 2014), analogous to perhaps the first step in the evolution of the spliceosome from a cissplicing ancestor. Among the steps still required to obtain an analog of the spliceosome, the existing spliceozymes need (1) an increase in trans-splicing efficiency, (2) a reduction in side products and off-target effects, (3) the ability to recognize different splice sites, (4) the recruitment of proteins for structural and catalytic roles, and (5) fragmentation into multiple RNA-protein particles.…”

Crucial steps to life: From chemical reactions to code using agents

“…We think also of recombination tools that evolved out of repair abilities, not forgetting the several parts that diversified within cellular life such as group II introns that evolved into LTR regulatory tools (Lambowitz and Zimmerly, 2011;Zimmerly and Semper, 2015) or even into the important spliceosome that functions as a splicezyme (Matlin and Moore, 2007;Amini et al, 2014). A similar cooperative unit is the editosome in RNA editing (Witzany, 2011).…”