Structure of the Trypanosoma brucei p22 Protein, a Cytochrome Oxidase Subunit II-specific RNA-editing Accessory Factor

Abstract

Kinetoplastid RNA (k-RNA) editing is a complex process in the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, that involves the guide RNA-directed insertion and deletion of uridines from precursor-mRNAs to produce mature, translatable mRNAs. k-RNA editing is performed by multiprotein complexes called editosomes. Additional non-editosome components termed k-RNA-editing accessory factors affect the extent of editing of specific RNAs or classes of RNAs. The T. brucei p22 protein was identified as one such accessory factor. Here we show that p22 contributes to cell growth in the procyclic form of T. brucei and functions as a cytochrome oxidase subunit II-specific k-RNA-editing accessory factor. To gain insight into its functions, we solved the crystal structure of the T. brucei p22 protein to 2.0-Å resolution. The p22 structure consists of a six-stranded, antiparallel β-sheet flanked by five α-helices. Three p22 subunits combine to form a tight trimer that is primarily stabilized by interactions between helical residues. One side of the trimer is strikingly acidic, while the opposite face is more neutral. Database searches show p22 is structurally similar to human p32, which has a number of functions, including regulation of RNA splicing. p32 interacts with a number of target proteins via its α1 N-terminal helix, which is among the most conserved regions between p22 and p32. Co-immunoprecipitation studies showed that p22 interacts with the editosome and the k-RNA accessory protein, TbRGG2, and α1 of p22 was shown to be important for the p22-TbRGG2 interaction. Thus, these combined studies suggest that p22 mediates its role in k-RNA editing by acting as an adaptor protein.

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

“…The p22 protein with the z64-kDa MBP-tag migrates as a band just below the BSA band, which served as a negative control because it does not bind RNA (Fig. 2, lanes 4,6; Sprehe et al 2010). TbRGG2-GST acted as a positive control for RNA binding ).…”

Association of UBP1 to ribonucleoprotein complexes is regulated by interaction with the trypanosome ortholog of the human multifunctional P32 protein

“…The P22–RBP16 interaction seems to be unstable or occurring with only a limited amount of P22 molecules, as it cannot be obtained by in vivo immunoprecipitation (Sprehe et al ., ). However, TbRGG2, an RNA‐editing factor from T. brucei , can be indeed immunoprecipitated (Sprehe et al ., ), suggesting P22 interacts with many different factors in vivo . In T. brucei , these interactions seem to be important for k‐RNA editing, primarily as a COII‐specific editing factor (Sprehe et al ., ).…”

“…2B). Mam33 family proteins have been identified in several organisms, which include T. brucei P22 and human P32 (Jiang et al, 1999;Hayman et al, 2001;Sprehe et al, 2010). All these proteins share structural topology and have thus been designated as orthologous proteins (Sprehe et al, 2010).…”

An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

“…Additionally, simultaneous depletion of RBP16 and MRP1/2 revealed that these proteins have a redundant function in the maintenance of edited A6 (ATPase subunit 6) and cytochrome oxidase subunit III (COIII) RNAs (14). Depletion of the p22 protein resulted in a specific defect in editing of the COII RNA (15). The MERS1 (mitochondrial edited mRNA stability) Nudix hydrolase and TbRGG1 exhibit more global effects.…”

“…The enzymes that catalyze editing, including endonucleolytic cleavage, uridine (U) addition or U removal, and the final resealing by ligation, are found in a stable multiprotein structure termed the RNA editing core complex (RECC) or 20S editosome (8-11). In addition, several noneditosome proteins facilitate the editing of one or more mRNAs in vivo (12)(13)(14)(15)(16). More recently, a large, dynamic macromolecular complex, termed the mitochondrial RNA binding 1 (MRB1) complex, otherwise known as RESC (RNA editing substrate binding complex [17]), has emerged as essential for mitochondrial RNA editing (4).…”