A Plant-Specific RNA-Binding Domain Revealed Through Analysis of Chloroplast Group II Intron Splicing

A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing Tiffany S. Kroegera,1, Kenneth P. Watkinsa,1, Giulia Frisob, Klaas J. van Wijkb, and Alice Barkana,2 aInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403; and bDepartment of Plant Biology, Cornell University, Ithaca, NY 14853 Edited by Alan M. Lambowitz, University of Texas, Austin, TX, and approved January 7, 2009 (received for review December 8, 2008) Comparative genomics has provided evidence for numerous con- members of the DUF860 family are predicted to localize to served protein domains whose functions remain unknown. We chloroplasts or mitochondria, suggesting that proteins with this identified a protein harboring ‘‘domain of unknown function 860’’ domain have multiple roles in gene expression in both organelles. (DUF860) as a component of group II intron ribonucleoprotein Although DUF860 is found only in land plants, it is distantly particles in maize chloroplasts. This protein, assigned the name related to a class of ubiquitin hydrolases (UBH) found through- WTF1 (‘‘what’s this factor?’’), coimmunoprecipitates from chloro- out the eucaryotes. However, structural modeling suggests that plast extract with group II intron RNAs, is required for the splicing DUF860 adopts a structure that differs from UBH enzymes, and of the introns with which it associates, and promotes splicing in the that has a surface that is reminiscent of helical repeat RNA- context of a heterodimer with the RNase III-domain protein RNC1. binding motifs such as the PPR and PUM-HD motifs. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. Results DUF860 is found only in plants, where it is represented in a protein WTF1 Is Found in Group II Intron Ribonucleoprotein Particles (RNPs). family comprising 14 orthologous groups in angiosperms. Most WTF1 came to our attention during our analysis of the machin- members of the DUF860 family are predicted to localize to chlo- ery that promotes the splicing of group II introns in chloroplasts. roplasts or mitochondria, suggesting that proteins with this do- Nine nucleus-encoded chloroplast splicing factors have been main have multiple roles in RNA metabolism in both organelles. identified in land plants, each involved in the splicing of distinct These findings add to emerging evidence that the coevolution of intron subsets (7–14). In an effort to identify additional factors, nuclear and organellar genomes spurred the evolution of diverse we used antibodies to the splicing factors CRS1, CAF1, and noncanonical RNA-binding motifs that perform organelle-specific CAF2 to immunopurify intron RNPs from chloroplast extract, functions. and we identified coimmunoprecipitating proteins by mass spec- trometry. Two proteins identified in this manner were shown ͉ ͉ DUF860 mitochondria plastid previously to be authentic components of intron RNPs (7, 15). A predicted protein containing DUF860 (IPR008578) was he evolution of mitochondria and chloroplasts from bacterial identified in both the CAF1 and CAF2 coimmunoprecipitates Tendosymbionts was accompanied by large scale transfer of [supporting information (SI) Table S1 and Fig. S1]. As no genes to the nucleus (1). Accordingly, many organellar proteins functional information was available for this or any DUF860 are encoded by nuclear genes of bacterial ancestry that retain protein, we named it WTF1. The rice and Arabidopsis pro- their ancestral function. However, during the long coevolution of teomes include 17 and 15 DUF860 proteins, respectively, which mitochondria and chloroplasts with their host cell, both or- comprise 14 orthologous groups (Fig. S1 and Fig. S2). WTF1 ganelles acquired features that are not typical of their bacterial and its orthologs are predicted to localize to chloroplasts (Fig. ancestors. The origin of the genes that confer such traits is only S2), consistent with the isolation of WTF1 from a chloroplast beginning to be elucidated. preparation. The complex RNA metabolism characteristic of plant mitochondria and chloroplasts provides striking examples of ac- Recovery of wtf1 Insertion Mutants. To gain insight into the quired, nonprocaryotic traits. For example, both organellar function of WTF1, we screened our collection of transposon- genomes are rich in introns, RNAs in both organelles are induced nonphotosynthetic maize mutants for insertions in the modified by RNA editing, and posttranscriptional events have wtf1 gene. The mutant alleles used for subsequent experiments the predominant role in determining gene product abundance are shown in Fig. 1. The wtf1-1 insertion cosegregates with a PLANT BIOLOGY (2, 3–5). Many proteins that participate in such processes were recessive mutation conferring a pale green phenotype, whereas not derived from the endosymbiont, but rather emerged in the the wtf1-3 and wtf1-4 insertions cosegregate with recessive context of nuclear-organellar coevolution. For example, the mutations conferring an albino phenotype (Fig. 1B). The inser- pentatricopeptide repeat (PPR) protein family is found only in tions in wtf1-3 and wtf1-4 disrupt the ORF (Fig. 1A), and are 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 plat- Author contributions: T.S.K., K.P.W., and A.B. designed research; T.S.K., K.P.W., and G.F. forms, but they appear to be derived from the tetratricopeptide performed research; T.S.K., K.P.W., G.F., K.J.v.W., and A.B. analyzed data; and A.B. wrote repeat (TPR) motif, a more ancient motif that binds protein the paper. ligands. The authors declare no conflict of interest. Here, we present evidence that a previously uncharacterized This article is a PNAS Direct Submission. protein family defined by ‘‘domain of unknown function 860’’ Data deposition: The sequence reported in this paper has been deposited in the GenBank (DUF860) fits this general paradigm. We show that the DUF860 database (accession no. FJ264201). protein WTF1 (‘‘what’s this factor?’’) is required for the splicing 1T.S.K. and K.P.W. contributed equally to this work. of group II introns in chloroplasts, that it associates in vivo with 2To whom correspondence should be addressed. E-mail: [email protected]. its genetically-defined RNA ligands, and that both WTF1 and its This article contains supporting information online at www.pnas.org/cgi/content/full/ resident DUF860 exhibit RNA-binding activity in vitro. Most 0812503106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812503106 PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4537–4542 Downloaded by guest on September 30, 2021 the WTF1 antibody (Fig. 2A). WTF1 is enriched in isolated chloroplasts with respect to its concentration in leaf, and was detected solely in the stromal fraction. Antibodies to WTF1 coimmunoprecipitated both CAF1 and CAF2 from stroma (Fig. 2B), confirming that these ‘‘bait’’ proteins, with which WTF1 was initially identified, are associated with WTF1. Furthermore, WTF1 coimmunoprecipitated with several other chloroplast splicing factors (Fig. 2B). Coimmunoprecipitation with RNC1 was particularly strong, whereas weak but reciprocal coimmunoprecipitation was detected with CFM3. WTF1 antibody also coimmunoprecipitated CRS1. CAF1, CAF2, CFM3, RNC1, and CRS1 reside in stromal particles that include group II intron RNAs (7, 10, 12). When stroma was fractionated by sedimen- tation through sucrose gradients, WTF1 was found in particles spanning the same size range as these group II intron RNPs (Ϸ600–800 kDa; see Fig. 2C). Fig. 1. Mutant alleles of wtf1. (A) Mu transposon insertions in the wtf1 gene. The wtf1 ORF lacks introns and is indicated by a rectangle. (B) Phenotypes of wtf1 mutants. Plants indicated by 2 alleles are the heteroallelic progeny of WTF1 Is Associated with Group II Intron RNAs in Chloroplast Extract. complementation crosses. (C) Loss of WTF1 protein in wtf1 mutant chloro- The coimmunoprecipitation of WTF1 with group II intron plasts. Chloroplasts purified from seedlings of the indicated genotypes were splicing factors and its cosedimentation with intron RNPs sug- analyzed on immunoblots with anti-WTF1 antiserum. Cpn60 was used as a gested that WTF1 is itself associated with group II introns. To loading control. Strong wtf1 alleles could not be analyzed in this manner identify RNAs that associate with WTF1, we used a genome- because plastids cannot be purified in sufficient quantity from albino plants. wide RNA coimmunoprecipitation assay (‘‘RIP-chip’’) as an initial screen. WTF1 was immunoprecipitated from stromal extract; RNAs purified separately from the pellet and superna- anticipated to be null alleles; the wtf1-1 insertion is upstream of tant were labeled with red- or green-fluorescing dye, respec- the ORF, consistent with the weaker phenotype observed. The tively, combined and hybridized to a tiling microarray of the F1 progeny of crosses between plants heterozygous for each maize chloroplast genome. Two replicate experiments were allele segregated chlorophyll-deficient, seedling lethal mutants, performed with WTF1 antiserum; the results were compared demonstrating that these phenotypes result from the disruption with those obtained by using an antibody to OE16, which does of wtf1. The chlorophyll deficiency of wtf1-1/wtf1-4 plants is not bind RNA. The data are summarized in Table S2 and Fig. intermediate between that conditioned by the

Load more