A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing.
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.
Downing
Maureen E
ME
Center for RNA Molecular Biology, Case Western Reserve University, School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4960, USA.
Brady
Kristina L
KL
Caprara
Mark G
MG
D001233:Aspergillus nidulans / Q000201:enzymology*; D001665:Binding Sites; D007438:Introns; D010446:Peptide Fragments; D011485:Protein Binding; D017510:Protein Folding; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D012322:RNA Precursors; D012331:RNA, Fungal; D012194:RNA-Directed DNA Polymerase; C454879:maturase I-AniI