The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein is a G-tract Specific RNA Binding Protein

Pakoyo F Kamba,Jennifer L Ekstrom,Neil A White,Charles G Hoogstraten,David A Dickson,Donna J Koslowsky

doi:10.1038/s41598-018-34377-9

Pakoyo F Kamba, Jennifer L Ekstrom + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-34377-9

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Abstract

Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite’s single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T. brucei PPR protein (KRIPP11) using electrophoretic mobility shift assays, fluorescence anisotropy, circular dichroism spectroscopy, and in vitro selection. We found KRIPP11 to be an RNA binding protein with specificity for sequences of four or more consecutive guanosine residues (G-tracts). Such G-tracts are dramatically enriched in T. brucei mitochondrial transcripts that are destined for extensive uridine insertion/deletion editing but are not present in mRNAs following editing. We further found that the quadruplex oligoguanosine RNA conformation is preferentially recognized by KRIPP11 over other conformational forms, and is bound without disruption of the quadruplex structure. In combination with prior data demonstrating association of KRIPP11 with the small ribosomal subunit, these results suggest possible roles for KRIPP11 in bridging mRNA maturation and translation or in facilitating translation of unusual dual-coded open reading frames.

Highlights

RNA editing and other post-transcriptional RNA processing events in trypanosome mitochondria are executed by a variety of RNA binding proteins whose roles and mechanisms of action are only being www.nature.com/scientificreports/
First discovered in Arabidopsis thaliana in 2000, pentatricopeptide repeat (PPR) proteins are characterized by tandem repeats of 35 amino acids, with each motif folding into a pair of antiparallel alpha-helices similar to the peptide-binding tetratricopeptide (34 amino acid) repeat (TPR) motif[18,19]
In the initial work reported here, we focused on the 27 kDa T. brucei PPR protein KRIPP11 (TriTrypDB Tb927.8.6040; genbank XM_842341; previously PPR27), which is one of the smallest and simplest members of the protein family

Summary

Introduction

RNA editing and other post-transcriptional RNA processing events in trypanosome mitochondria are executed by a variety of RNA binding proteins whose roles and mechanisms of action are only being www.nature.com/scientificreports/. Maize PPR10 protects and defines both the 5′ and 3′ termini of transcripts from two different loci (atpH and psaJ) by binding to a highly conserved RNA motif located in the intergenic regions downstream of each gene[34,35] Another PPR protein, CRR4 from Arabidopsis thaliana, specifies the site of RNA editing in the chloroplast ndhD gene by recognizing a specific motif surrounding the editing site[24,36]. Crystal structures of two plant-derived PPR proteins in complex with RNA targets (maize PPR10 and Arabidopsis thaliana THA8) confirmed the recognition of individual nucleotides of single-stranded RNA by individual PPR repeats and elucidated the basis for many aspects of the emerging recognition code[44,45,46]. These correspondences have been more finely mapped by a set of four crystal structures of designed PPR proteins (dPPRs) in complex with their predicted RNA targets[47]

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