An in Vitro Peptide Complementation Assay for CYT-18-Dependent Group I Intron Splicing Reveals a New Role for the N‑Terminus

Abstract

The mitochondrial tyrosyl tRNA synthetase from <i>Neurospora crassa</i> (CYT-18 protein) is a bifunctional group I intron splicing cofactor. CYT-18 is capable of splicing multiple group I introns from a wide variety of sources by stabilizing the catalytically active intron structures. CYT-18 and mt TyrRSs from related fungal species have evolved to assist in group I intron splicing in part by the accumulation of three N-terminal domain insertions. Biochemical and structural analysis indicate that the N-terminal insertions serve primarily to create a structure-stabilizing scaffold for critical tertiary interactions between the two major RNA domains of group I introns. Previous studies concluded that the primarily α-helical N-terminal insertion, H0, contributes to protein stability and is necessary for splicing the <i>N. crassa</i> ND1 intron but is dispensable for splicing the <i>N. crassa</i> mitochondrial LSU intron. Here, we show that CYT-18 with a complete H0 deletion retains residual ND1 intron splicing activity and that addition of the missing N-terminus <i>in trans</i> is capable of restoring a significant portion of its splicing activity. The development of this peptide complementation assay has allowed us to explore important characteristics of the CYT-18/group I intron interaction including the stoichiometry of H0 in intron splicing and the importance of specific H0 residues. Evaluation of truncated H0 peptides in this assay and a re-examination of the CYT-18 crystal structure suggest a previously unknown structural role of the first five N-terminal residues of CYT-18. These residues interact directly with another splicing insertion, making H0 a central structural element responsible for connecting all three N-terminal splicing insertions