A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Abstract

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N-and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

An allosteric-feedback mechanism for protein-assisted group I intron splicing

“…COB intron does not affect protein-dependent splicing (Geese and Waring 2001). These observations are consistent with the idea that the mechanisms of maturase recognition and facilitation of RNA folding can differ significantly even among closely related maturases (Downing et al 2005). This most likely is a reflection of the different evolutionary paths that each intron/maturase pair follows during the process of adaptation to protein-assisted splicing.…”

“…2). Previously, the P7 and P3 helices were judged to be unfolded by their susceptibility to cleavage by the single-stranded specific Ribonuclease 1 (Downing et al 2005). Thus, the four central helices that define the group I intron core are unstable in A.n.…”

“…Transcription and purification of 32 P-internally labeled and end-labeled RNAs were carried out as described by Solem et al (2002) and Downing et al (2005). For iodine structure mapping experiments, RNAs were prepared by adding NTPaS to the transcription reaction.…”

“…Dissociation kinetics were performed and analyzed as described by Solem et al (2002) and Downing et al (2005). The association kinetic experiments were performed by preincubating 50 pM 32 Plabeled RNA in 50 mM Tris-HCl, pH-7.5, 100 mM NaCl, 5 mM Mg 2+ , 10 mM DTT, 10% glycerol, and 0.1 mg/mL BSA at 37°C for 20 min.…”

A DExH/D-box Protein Coordinates the Two Steps of Splicing in a Group I Intron

“…Unlabeled RNAs were transcribed as described 31. End-labeled RNAs were transcribed and 32 P-labeled at the 5’ end and purified as described 53. High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α- 32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP.…”