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RNA Binding and RNA Remodeling Activities of the Half-A-Tetratricopeptide (HAT) Protein HCF107 Underlie Its Effects on Gene Expression

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RNA Binding and RNA Remodeling Activities of the Half-A-Tetratricopeptide (HAT) Protein HCF107 Underlie Its Effects on Gene Expression RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression Kamel Hammania, William B. Cookb, and Alice Barkana,1 aInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403; and bDepartment of Biology, Midwestern State University, Wichita Falls, TX 76308 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved February 29, 2012 (received for review January 6, 2012) The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat findings obtained with HCF107 are likely to elucidate general motif found in proteins that influence various aspects of RNA mechanisms through which HAT domains act. metabolism, including rRNA biogenesis, RNA splicing, and poly- HCF107 is a nucleus-encoded protein that localizes to the adenylation. This functional association with RNA suggested that chloroplasts of land plants. Mutations in Arabidopsis HCF107 HAT repeat tracts might bind RNA. However, RNA binding activity cause defects in the translation and stabilization of mRNAs psbH has not been reported for any HAT repeat tract, and recent derived from the chloroplast gene (6, 15). Our results show that HCF107 binds ssRNA with specificity for sequences at the 5′ literature has emphasized a protein binding role. In this study, psbH we show that a chloroplast-localized HAT protein, HCF107, is end of the processed mRNA isoforms that require HCF107 fi for their accumulation. In addition, we show that bound HCF107 a sequence-speci c RNA binding protein. HCF107 consists of 11 i ′ → ′ tandem HAT repeats and short flanking regions that are also ( ) blocks a 5 3 exonuclease in vitro, accounting for its ability to stabilize psbH RNA in vivo, and (ii) remodels local RNA predicted to form helical hairpins. The minimal HCF107 binding site structure in a manner that can account for its ability to enhance spans ∼11 nt, consistent with the possibility that HAT repeats bind psbH translation. We suggest that the RNA binding, RNA pro- RNA through a modular one repeat–1 nt mechanism. Binding of psbH ′ tection, and RNA remodeling activities observed for HCF107 HCF107 to its native RNA ligand in the 5 UTR remodels local are likely to be general features of long HAT repeat tracts, and RNA structure and protects the adjacent RNA from exonucleases that these properties are likely to contribute to the functions in vitro. These activities can account for the RNA stabilizing, RNA of HAT domains found in other ribonucleoprotein complexes processing, and translational activation functions attributed to throughout the cell. HCF107 based on genetic data. We suggest that analogous activ- ities contribute to the functions of HAT domains found in ribonu- Results – cleoprotein complexes in the nuclear cytosolic compartment. HCF107 Is a Sequence-Specific RNA Binding Protein. HCF107 is tar- geted to chloroplasts by an N-terminal transit peptide (6). Ma- helical repeat protein | plastid | pentatricopeptide repeat ture HCF107 (i.e., lacking the cleaved transit peptide) has 11 consecutive HAT motifs flanked by ∼100 aa on each side (Fig. he half-a-tetratricopeptide (HAT) motif is a helical repeat S1A). However, a structural model generated by I-TASSER (16) Tmotif that has been functionally linked to RNA because of its predicts that virtually the entire mature portion of HCF107 presence exclusively in complexes that influence RNA metabolism consists of helical hairpins (Fig. S1B), with approximately two (1). Examples of HAT domain proteins include Utp6, which is additional helical hairpin units flanking each side of the canon- involved in nuclear pre-rRNA processing, Prp6, which is involved ical HAT repeats. in nuclear pre-mRNA splicing, and CstF-77, which is involved in Mature HCF107 was expressed as a fusion to maltose binding protein in Escherichia coli and purified by successive amylose pre-mRNA cleavage and polyadenylation. The HAT motif is re- fi fi lated to the tetratricopeptide repeat (TPR), a degenerate 34-aa af nity and gel ltration chromatography (Fig. S2). Recombi- motif that forms a pair of antiparallel α-helices (reviewed in ref. nant HCF107 (rHCF107) has a monomeric molecular mass of 67 kDa but eluted from a size exclusion column at a position cor- 2). TPR motifs are typically found in tandem arrays, which stack to responding to a globular protein of ∼150 kDa, indicating that it is form a broad surface that binds protein ligands. Crystal structures fi an elongated monomer or forms homodimers. of CstF-77 con rmed that HAT repeat tracts adopt a TPR-like To test whether rHCF107 has the capacity to bind non- structure (3, 4). The possibility that HAT repeat tracts might bind fi – speci cally to nucleic acids, gel mobility shift assays were per- RNA has been suggested (1, 5 7), but the notion that HAT repeat formed under low stringency binding conditions (i.e., in the tracts serve as a scaffold for binding other proteins dominates absence of competitor RNA or heparin) using oligonucleotides recent literature (3, 8–10).Thisviewisreflected by the annotation of the same sequence but in the form of either ssRNA, dsRNA, of the HAT motif at InterPro, which states only that “the repeats ssDNA, or dsDNA (Fig. 1A). rHCF107 bound to the ssRNA but may be involved in protein–protein interactions” (http://www.ebi. not the other nucleic acid forms. ac.uk/interpro/IEntry?ac=IPR003107). HCF107 influences the metabolism of the polycistronic RNA Although TPR, HEAT, and several other helical repeat motifs derived from the chloroplast psbB-psbT-psbH-petB-petD gene form protein binding surfaces, similar structures have been cluster (15). Mutations in HCF107 cause the specific loss of shown to bind nucleic acids. Puf and pentatricopeptide repeat processed RNAs with a 5′ end 44 nt upstream of the psbH start (PPR) motifs have been shown to bind RNA (reviewed in refs. codon (Figs. 2B and 3A), and also decrease psbH translational 11 and 12), and mTERF, ALK, and TAL repeat motifs have been shown to bind DNA (reviewed in refs. 13 and 14). Thus, it seemed quite plausible that the presence of HAT domains ex- Author contributions: K.H., W.B.C., and A.B. designed research; K.H. and W.B.C. per- clusively in ribonucleoprotein complexes reflects the fact that formed research; K.H. and A.B. analyzed data; and K.H. and A.B. wrote the paper. HAT repeat tracts interact directly with RNA. In this study, we The authors declare no conflict of interest. show that the HAT domain protein HCF107 does, in fact, bind This article is a PNAS Direct Submission. RNA and that it does so with high affinity and specificity for its 1To whom correspondence should be addressed. E-mail: [email protected]. fi BIOCHEMISTRY genetically de ned site of action. HCF107 consists almost en- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tirely of HAT motifs and lacks other functional domains. Thus, 1073/pnas.1200318109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1200318109 PNAS | April 10, 2012 | vol. 109 | no. 15 | 5651–5656 Downloaded by guest on September 28, 2021 [rHCF107]: Zm-hcf107 A A ATG (+1) STOP (+1834) +1527 B TCATCAAGAAGAGGCTMuGAAGAGGCTGGCCCTC B C kb WT Zm-hcf107 crp1 WT U WT Zm-hcf107 WT crp1 6 34 nt psbH 5’ 4 ssDNA dsDNA ssRNA dsRNA 3 2 34 nt petB-petD B 1.5 [rHCF107]: 0.7 1 0.6 0.5 EtBr 0.5 B 0.4 psbH 0.3 methylene atpF/A 0.2 blue fraction bound 0.1 0 Fig. 2. Amaizehcf107 mutant (Zm-hcf107) lacks an sRNA corresponding to U 20 40 60 80 100 120 140 the HCF107-dependent psbH 5′ end. (A) Position of a Mu1-transposon insertion psbH atpF/A [rHCF107] in Zm-hcf107. The diagram shows its position within the spliced ORF. The se- quence flanking the insertion is shown below, with the target site duplication underlined. (B) RNA gel blot showing loss of processed psbH mRNAs in the Zm- 0.5 C hcf107 mutant. The missing transcripts match those transcripts that are missing 0.4 [rHCF107]: in Arabidopsis hcf107 mutants (15). (C) RNA gel blot showing chloroplast sRNAs from two intergenic regions. Total leaf RNA (15 μg) from a Zm-hcf107 0.3 B psbH mutant, a crp1 mutant, and a phenotypically normal sibling of each (WT) was 0.2 atpF/A U fractionated in a polyacrylamide gel and electrophoretically transferred to nylon membrane. Replicate membranes were probed with oligodeoxynucleo- 0.1 psbH atpF/A fraction bound tides complementary to the sRNA in the psbH 5′ UTR (the putative HCF107 0 footprint) or the sRNA in the petB–petD intergenic region (the putative CRP1 0 50 100 150 200 250 footprint) (20). The position of a 34-nt RNA size marker is shown. [rHCF107] Fig. 1. Gel mobility shift assays showing the RNA binding capacity of rHCF107. (A) Synthetic 31-mers of the same sequence but in ssRNA, dsRNA, rice, barley, and maize (19, 20). The binding sites of several PPR ssDNA, or dsDNA form were incubated with rHCF107 (0, 50, 100, and 200 proteins accumulate as sRNAs in vivo, and there is evidence that nM) and resolved on a native polyacrylamide gel. Bound (B) and unbound these sRNAs are in vivo protein footprints that accumulate be- (U) RNAs/DNAs are marked. (B) RNAs derived from the 5′ UTR of Arabidopsis cause they are protected by the cognate protein from endoge- psbH (26 nt) or the maize atpF/atpA intergenic region (27 nt) were incubated nous ribonucleases (17, 19, 20). To evaluate whether the sRNA with rHCF107 (0, 31, 62, and 125 nM) under stringent conditions (150 mM mapping to the 5′ end of processed psbH RNA is the HCF107 NaCl, 0.5 mg/mL heparin).
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