Molecular Recognition in a Trans Excision-Splicing Ribozyme:  Non-Watson−Crick Base Pairs at the 5‘ Splice Site and ωG at the 3‘ Splice Site Can Play a Role in Determining the Binding Register of Reaction Substrates

Abstract

Trans excision-splicing (TES) ribozymes, derived from a Pneumocystis carinii group I intron, can catalyze the excision of targeted sequences from within RNAs. In this report, the sequence requirements of the splice sites are analyzed. These conserved sequences include a u-G wobble pair at the 5' splice site and a guanosine in the omega position at the 3' splice site (in the substrate). We report that 7 out of 16 base pair combinations at the 5' splice site produce appreciable TES product. This promiscuity is in contrast to results reported for analogous self-splicing reactions using a Tetrahymena ribozyme. At long reaction times TES products dissociate and rebind free ribozyme, at which point product degradation occurs via the 5' cleavage reaction. Unexpectedly, only in cases where Watson-Crick base pairs form at the 5'splice site do we see degradation of TES products at cryptic sites, suggesting that non-Watson-Crick base pairs at the 5' splice site are acting in concert with other factors to precisely determine the binding register of TES reaction substrates within the ribozyme. Moreover, cryptic site degradation does not occur with the corresponding reaction substrates, which additionally contain omegaG, suggesting that omegaG can play a similar role. We report that omegaG cannot be replaced by any other base, so TES substrates require a guanosine as the last (or only) base to be excised. Additionally, we demonstrate that P9.0 and P10 are expendable for TES reactions, suggesting that omegaG is sufficient as a 3' molecular recognition element.

Tetrahymena thermophila and Candida albicans Group I intron-derived ribozymes can catalyze the trans-excision-splicing reaction

“…First, the initial ribozyme constructs lacked the nucleotides required to form the P10 helix (RE3), and so an RE3 region (native to the ribozyme) was added. As seen for the Pneumocystis ribozyme, P10 helix formation has been shown to aid in binding the 3′-exon region of TES substrates (9,10). Second, the 3′-end of each ribozyme sequence was modified to include the addition of a terminal guanosine, (which is found naturally) as it is required as the intramolecular nucleophile in the substrate-cleavage reaction (4).…”

“…This was done because the formation of the P10 helix interaction is important for the second reaction step of the TES reaction (9,10). Note that the RE3 sequence added was native to each respective intron.…”

Engineering a family of synthetic splicing ribozymes

“…Despite the large number of splicing ribozymes determined by alignment, only several have ever been experimentally characterized and may not function as a self-splicing ribozyme in a bacterial host. Other ribozymes that have been studied include the ribozymes from Azoarcus (9,10), Pneumocystis (11,12), Didymium iridis (DiGIR2) (13,14), and Fuligo (Fse.L569 and Fse.L1898) (13). We tested two uncharacterized sequences found in the alignment for their ability to function as self-splicing ribozymes, but neither ribozyme showed cis -splicing activity (Supplementary Data).…”

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme

“…Other considerations for ribozyme targeting include reconstituting au-G wobble pair at the 5 0 splice site and aguanosine at the 3 0 splice site as the last (or only) base of the sequence targeted for excision (Baum et al 2005). Also note that previous work with this ribozyme demonstrated that segments larger than a single nucleotide can be excised in vitro (Bell et al 2002), so we anticipate that larger regions could be excised in vivo.…”