A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing

Abstract

Comparative genomics has provided evidence for numerous conserved protein domains whose functions remain unknown. We identified a protein harboring ''domain of unknown function 860'' (DUF860) as a component of group II intron ribonucleoprotein particles in maize chloroplasts. This protein, assigned the name WTF1 (''what's this factor?''), coimmunoprecipitates from chloroplast extract with group II intron RNAs, is required for the splicing of the introns with which it associates, and promotes splicing in the context of a heterodimer with the RNase III-domain protein RNC1. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. DUF860 is found only in plants, where it is represented in a protein family comprising 14 orthologous groups in angiosperms. Most members of the DUF860 family are predicted to localize to chloroplasts or mitochondria, suggesting that proteins with this domain have multiple roles in RNA metabolism in both organelles. These findings add to emerging evidence that the coevolution of nuclear and organellar genomes spurred the evolution of diverse noncanonical RNA-binding motifs that perform organelle-specific functions.DUF860 ͉ mitochondria ͉ plastid T he evolution of mitochondria and chloroplasts from bacterial endosymbionts was accompanied by large scale transfer of genes to the nucleus (1). Accordingly, many organellar proteins are encoded by nuclear genes of bacterial ancestry that retain their ancestral function. However, during the long coevolution of mitochondria and chloroplasts with their host cell, both organelles acquired features that are not typical of their bacterial ancestors. The origin of the genes that confer such traits is only beginning to be elucidated.The complex RNA metabolism characteristic of plant mitochondria and chloroplasts provides striking examples of acquired, nonprocaryotic traits. For example, both organellar genomes are rich in introns, RNAs in both organelles are modified by RNA editing, and posttranscriptional events have the predominant role in determining gene product abundance (2, 3-5). Many proteins that participate in such processes were not derived from the endosymbiont, but rather emerged in the context of nuclear-organellar coevolution. For example, the pentatricopeptide repeat (PPR) protein family is found only in eucaryotes, where it has been implicated in RNA-related processes in mitochondria and chloroplasts (6). Current data argue that PPR proteins generally function as RNA-interaction platforms, but they appear to be derived from the tetratricopeptide repeat (TPR) motif, a more ancient motif that binds protein ligands.Here, we present evidence that a previously uncharacterized protein family defined by ''domain of unknown function 860'' (DUF860) fits this general paradigm. We show that the DUF860 protein WTF1 (''what's this factor?'') is required for the splicing of group II introns in chloroplasts, that it associates in vivo with its genetically-defined RNA ligands, and tha...

APO1 Promotes the Splicing of Chloroplast Group II Introns and Harbors a Plant-Specific Zinc-Dependent RNA Binding Domain

“…Reverse-genetic and RNA coimmunoprecipitation analyses then confirmed the roles of these proteins in splicing Prikryl et al, 2008;Kroeger et al, 2009). The maize ortholog of APO1, Zm-APO1, was detected in a coimmunoprecipitate with the splicing factor CAF1 (see Supplemental Figure 1 online), prompting us to consider a role for APO1 in chloroplast group II intron splicing.…”

“…There is now strong evidence for the involvement of one chloroplast-encoded and 13 nucleus-encoded proteins in chloroplast intron splicing (reviewed in Kroeger et al, 2009;Stern et al, 2010). Unlike the nuclear spliceosome, whose core has a uniform composition, chloroplast splicing factors function in a combinatorial manner, with each intron requiring distinct but overlapping protein sets.…”

“…The material used for RNA binding assays is shown in lane 2 in the panel to the right. MBP expressed from the empty vector and purified by successive amylose-affinity and gel filtration chromatography has been shown previously not to bind RNA (Kroeger et al, 2009). M, marker.…”

“…This approach has revealed that many such genes were not derived from the chloroplast's cyanobacterial ancestor, but rather emerged after endosymbiosis. For example, genetic analyses identified 12 nucleus-encoded proteins that are required for the splicing of one or more of the ;18 group II introns in angiosperm chloroplasts, and most of these belong to RNA binding protein families that evolved in eukaryotes (summarized in Kroeger et al, 2009). These proteins function combinatorially to promote the splicing of different intron subsets and are found in large ribonucleoprotein particles that include their cognate intron RNAs (reviewed in Kroeger et al, 2009;Stern et al, 2010).…”

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

“…The petB sequence is not edited and serves as a negative control. MBP has been shown to lack RNA binding activity under the conditions used here (66). B, bound RNA; U, unbound RNA.…”