The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

Abstract

The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors. Interaction modules are crucial for the molecular and cellular function of proteins, and a wide repertoire of diverse domains and motifs mediates a variety of regulatory mechanisms. Proteins that exhibit a repeating structural unit with an ␣-helical architecture are classified as members of the ␣-solenoid superfamily (1). This large family is widely distributed in eukaryotes and includes, among others, the transcription activator-like (TAL) effectors, the tetratricopeptide repeat (TPR) proteins, and the pentatricopeptide repeat (PPR) proteins (2-4). The ␣-helical structure of diverse members mediates binding to either proteins or nucleic acids.A well-characterized example of RNA binding proteins is the PPR protein family (5). The degenerate PPR motif is described as two antiparallel, ␣-helical tracts of 35 amino acids, whose repeats are often arranged in tandem arrays. Recently, two approaches provided insights into the predictable RNA binding code of this motif (6, 7). Structural analyses indicate that RNA binding is mediated by helical tracts forming a superhelical groove with exposed residues. Despite the low sequence conservation of the PPR motif being restricted to several amino acids, PPR proteins specifically recognize single-stranded RNA sequences through a combinatorial amino acid code (2, 6, 7). Mod...

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns

“…In C. reinhardtii, 43 OPR proteins were predicted that participate in RNA metabolism-related processes (31). This includes the psaA mRNA splicing factors Raa1, Rat2, and Raa8 (18,21,31,32). In most land plants, the genomes encode only a single OPR gene (33).…”

“…For C. reinhardtii, seven splicing factors, specific for group II introns, have been described at the molecular level (17)(18)(19)(20)(21)(22)(23). Whereas splicing factor Raa1 (RNA maturation of psaA RNA) is involved in splicing of both reactions, Raa3, Raa4, Raa8, and Rat2 (RNA maturation of psaA tscA RNA) are psaA-i1-specific.…”

CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid Cytochrome c Assembly in the Green Alga Chlamydomonas reinhardtii

“…Such motifs, named OPR for o ctatrico p eptide r epeats were first defined in TBC2 ( t ranslation factor for chloroplast ps bC mRNA), a nuclear encoded protein required for the translation of psbC RNA in the chloroplast ( Auchincloss et al, 2002 ). OPRs are also recognized in other factors controlling translation (TDA1, TAB1), maturation (RAT2, RAA1, RAA8, RAP), or stability (MCG1,MBI1) of chloroplast transcripts ( Balczun et al, 2005 ; Merendino et al, 2006 ; Eberhard et al, 2011 ; Rahire et al, 2012 ; Kleinknecht et al, 2014 ; Marx et al, 2015 ; Wang et al, 2015 ).…”

“…The OPR family is further characterized by the presence of low complexity regions, which are regions containing little diversity in their amino acid composition ( Balczun et al, 2005 ; Merendino et al, 2006 ; Eberhard et al, 2011 ; Rahire et al, 2012 ; Marx et al, 2015 ; Wang et al, 2015 ). Indeed, we noted the occurrence of several stretches of three or more alanine, glycine, serine, or glutamine repeats in CCS2 ( Figure 4A ).…”

A pioneer protein is part of a large complex involved in trans‐splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii

“…Raa2 is involved in splicing of intron 2 (Perron et al ., ), and together with Raa1 is part of a 500 kDa multiprotein complex (Perron et al ., ). Raa3, and the recently discovered Raa8, are necessary for the splicing of intron 1 (Rivier et al ., ; Marx et al ., ) and are part of a large 1700 kDa complex that includes the tscA RNA and the precursor of psaA exon 1. Raa4 is also necessary for the splicing of intron 1, and was shown to bind tscA RNA in vitro .…”