Phylogenetic Genomewide Comparisons of the Pentatricopeptide Repeat Gene Family in indica and japonica Rice

More than 400 pentatricopeptide repeat (PPR) genes have been found in higher plants, but most of them have not been functionally analyzed and their origins are still obscure. In this study, we performed phylogenetic genomewide comparisons of the PPR gene family in indica and japonica rice to explore the expansion mechanisms of these genes in higher plants. The functions of PPR genes in plant CMS/Rf systems are also discussed. The results indicate that (1) unequal crossing over participated in the expansion of the newly evolved PPR genes in indica and japonica rice genomes, (2) CMS/Rf systems are different in monocots and dicots, (3) the BT-type CMS/Rf system exists in both indica and japonica rice, and (4) both the PPR gene family and the BT-type CMS/Rf system may have existed before the divergence of indica and japonica rice.     

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5′ and 3′ extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5′ processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria.Translation is the fundamental process decoding the genetic message present on mRNAs into proteins. In plant cells, mRNA translation occurs in the cytoplasm but also in two organelles, mitochondria and plastids. Because of their prokaryotic origin, the translation machineries operating in these two organelles share many characteristics with the bacterial translation apparatus (Bonen, 2004; Barkan, 2011). However, most of these bacteria-like features have been modified throughout evolution, and current organellar translation systems cooperate with numerous nucleus-encoded eukaryotic trans-factors. The divergence from bacteria is particularly obvious in plant mitochondria, notably because mitochondrial mRNAs lack the typical Shine and Dalgarno (SD) motif in their 5′ leaders and alternative start codons other than AUG are often used to initiate translation (Bonen, 2004). Proteomic and bioinformatic analyses allowed the identification of most proteins and RNA factors forming the core of the plant mitochondrial translation machinery, including translation initiation and elongation factors as well as ribosomal proteins (Bonen, 2004; Bonen and Calixte, 2006). However, the dynamics of this machinery remains largely obscure. In particular, nothing is known about the recruitment of mitochondrial ribosomes on 5′ untranslated regions in the absence of the SD motif and about the recognition of the correct translation initiation codon by the small ribosomal subunit. The high degree of sequence divergence among 5′ leaders of mitochondrial genes suggests a ribosome recruitment mechanism involving gene-specific cis-sequences and trans-factors (Hazle and Bonen, 2007; Choi et al., 2012). Up to now, only two proteins belonging to the Pentatricopeptide Repeat (PPR) family have been found to promote mitochondrial translation in higher plants (Uyttewaal et al., 2008b; Manavski et al., 2012). How they facilitate translation is still unclear, as for the few characterized PPR proteins shown to participate in plastid translation (Fisk et al., 1999; Schmitz-Linneweber et al., 2005; Cai et al., 2011; Zoschke et al., 2012, 2013). The plastid PENTATRICOPEPTIDE REPEAT PROTEIN10 (PPR10) protein of maize (Zea mays) is the only one for which the function has been elucidated at the molecular level. It was shown that, upon binding, PPR10 impedes the formation of a stem-loop structure in the 5′ leader of the ATP synthase subunit c (atpH) mRNA, permitting the recruitment of ribosomes through the liberation of an SD motif (Prikryl et al., 2011).PPR proteins represent a large family of RNA-binding proteins that has massively expanded in terrestrial plants (Barkan and Small, 2014). Most eukaryotes encode a handful of these proteins, whereas plant nuclear genomes express over 400 PPR proteins that are almost exclusively predicted to target mitochondria and/or plastids (Lurin et al., 2004; O’Toole et al., 2008). This family of proteins is characterized by the succession of tandem degenerate motifs of approximately 35 amino acids (Small and Peeters, 2000; Lurin et al., 2004). Based on the length of these repeats, the PPR family has been divided into two groups of roughly equal size in higher plants. P-type PPR proteins contain only successions of canonical 35-amino acid repeats (P), whereas PLS PPR proteins are composed of sequential repeats of P, short (S), and long (L) PPR motifs. P-type PPR proteins were shown to participate in various aspects of organellar RNA processing, whereas PLS PPR proteins have been almost exclusively associated with C-to-U RNA editing (for review, see Barkan and Small, 2014; Hammani and Giegé, 2014). Recent crystal structures showed that PPR motifs adopt an antiparallel helix-turn-helix fold whose repetition forms a solenoid-like structure (Ringel et al., 2011; Howard et al., 2012; Ban et al., 2013; Yin et al., 2013; Coquille et al., 2014; Gully et al., 2015). PPR tracks organize highly specific interaction domains that were shown to associate with single-stranded RNAs (Schmitz-Linneweber et al., 2005; Beick et al., 2008; Uyttewaal et al., 2008a; Williams-Carrier et al., 2008; Pfalz et al., 2009; Cai et al., 2011; Hammani et al., 2011; Prikryl et al., 2011; Khrouchtchova et al., 2012; Manavski et al., 2012; Zhelyazkova et al., 2012; Ke et al., 2013; Yin et al., 2013). The mechanism of sequence-specific RNA recognition by PPR proteins was recently uncovered, and combinations involving amino acid 6 of one motif and amino acid 1 of the subsequent motif correlate strongly with the identity of the RNA base to be bound (Barkan et al., 2012; Takenaka et al., 2013; Yagi et al., 2013).Besides those involved in RNA editing, few mitochondria-targeted PPR proteins have been characterized to date. Thus, our knowledge of the mechanisms governing the production and the expression of mitochondrial RNAs in higher plants is very limited. In this analysis, we describe the function of a novel mitochondria-targeted PPR protein of Arabidopsis (Arabidopsis thaliana) called MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1). Genetic and biochemical analyses indicate that MTL1 is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. Effectively, the Nad7 protein does not accumulate to detectable levels in mtl1 mutants, and this absence correlates with a lack of association of nad7 mature mRNA with mitochondrial polysomes. Interestingly, a partial but significant decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that the MTL1 protein is also involved in group II intron splicing. Since the decrease in splicing was only partial, this second function of MTL1 appears less essential for nad7 expression than its role in translation.     

Positive Darwinian Selection Promotes Heterogeneity Among Members of the Antifreeze Protein Multigene Family

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption–inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function. Received: 27 February 2001 / Accepted: 12 September 2001     

神经富亮氨酸重复蛋白家族研究进展

神经富亮氨酸重复(neural leucine-rich repeat,NLRR)蛋白家族是一类进化上保守、功能多样的Ⅰ型跨膜蛋白.NLRR主要表达于神经组织,肺部、心脏、肝、肾也有微量表达.自1996年Taguchi在发育的小鼠神经系统中发现了新的LRR蛋白质mNLRR-1和mNLRR-2后,一系列含有类似结构的NLRR蛋白质在小鼠、大鼠、爪蟾、斑马鱼和人类等种属中被发现.NLRR蛋白家族成员同源性高,主要含有一些共同的蛋白质结构域:LRR(leucine-rich repeat)、AFR(amino-flanking region)、CFR(carboxy-flanking region)、IgC2(immunoglobulin-like C2 type domain)和FNⅢ(fibronectin type Ⅲ-like domain)等.迄今为止,已有实验证明NLRR作为细胞黏附分子或配体受体分子在神经发育与再生过程中发挥着重要作用.主要对NLRR的时空表达模式、结构及主要生物学功能等进行了综述.     

Structure of a PLS-class Pentatricopeptide Repeat Protein Provides Insights into Mechanism of RNA Recognition

Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA-binding proteins that form a pervasive family of proteins conserved in yeast, plants, and humans. The plant PPR proteins are grouped mainly into the P and PLS classes. Here, we report the crystal structure of a PLS-class PPR protein from Arabidopsis thaliana called THA8L (THA8-like) at 2.0 Å. THA8L resembles THA8 (thylakoid assembly 8), a protein that is required for the splicing of specific group II introns of genes involved in biogenesis of chloroplast thylakoid membranes. The THA8L structure contains three P-type PPR motifs flanked by one L-type motif and one S-type motif. We identified several putative THA8L-binding sites, enriched with purine sequences, in the group II introns. Importantly, THA8L has strong binding preference for single-stranded RNA over single-stranded DNA or double-stranded RNA. Structural analysis revealed that THA8L contains two extensive patches of positively charged residues next to the residues that are proposed to comprise the RNA-binding codes. Mutations in these two positively charged patches greatly reduced THA8L RNA-binding activity. On the basis of these data, we constructed a model of THA8L-RNA binding that is dependent on two forces: one is the interaction between nucleotide bases and specific amino acids in the PPR motifs (codes), and the other is the interaction between the negatively charged RNA backbone and positively charged residues of PPR motifs. Together, these results further our understanding of the mechanism of PPR protein-RNA interactions.     

Examination of the Dimerization States of the Single-stranded RNA Recognition Protein Pentatricopeptide Repeat 10 (PPR10)

Pentatricopeptide repeat (PPR) proteins, particularly abundant in plastids and mitochrondria of angiosperms, include a large number of sequence-specific RNA binding proteins that are involved in diverse aspects of organelle RNA metabolisms. PPR proteins contain multiple tandom repeats, and each repeat can specifically recognize a RNA base through residues 2, 5, and 35 in a modular fashion. The crystal structure of PPR10 from maize chloroplast exhibits dimeric existence both in the absence and presence of the 18-nucleotide psaJ RNA element. However, previous biochemical analysis suggested a monomeric shift of PPR10 upon RNA binding. In this report, we show that the amino-terminal segments of PPR10 determine the dimerization state of PPR10. A single amino acid alteration of cysteine to serine within repeat 10 of PPR10 further drives dimerization of PPR10. The biochemical elucidation of the determinants for PPR10 dimerization may provide an important foundation to understand the working mechanisms of PPR proteins underlying their diverse physiological functions.     

Regulation of Inflorescence Branch Development in Rice Through a Novel Pathway Involving the Pentatricopeptide Repeat Protein sped1-D

Panicle type has a direct bearing on rice yield. Here, we characterized a rice clustered-spikelet mutant, sped1-D, with shortened pedicels and/or secondary branches, which exhibits decreased pollen fertility. We cloned sped1-D and found that it encodes a pentatricopeptide repeat protein. We investigated the global expression profiles of wild-type, 9311, and sped1-D plants using Illumina RNA sequencing. The expression of several GID1L2 family members was downregulated in the sped1-D mutant, suggesting that the gibberellin (GA) pathway is involved in the elongation of pedicels and/or secondary branches. When we overexpressed one GID1L2, {"type":"entrez-nucleotide","attrs":{"text":"AK070299","term_id":"32980323","term_text":"AK070299"}}AK070299, in sped1-D plants, the panicle phenotype was restored to varying degrees. In addition, we analyzed the expression of genes that function in floral meristems and found that RFL and WOX3 were severely downregulated in sped1-D. These results suggest that sped1-D may prompt the shortening of pedicels and secondary branches by blocking the action of GID1L2, RFL, and Wox3. Moreover, overexpression of sped1-D in Arabidopsis resulted in the shortening of pedicels and clusters of siliques, which indicates that the function of sped1-D is highly conserved in monocotyledonous and dicotyledonous plants.     

Structural and Energetic Characterization of the Ankyrin Repeat Protein Family

Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily amenable for design, with promising biotechnological and biomedical applications. Studying repeat protein families presents technical challenges due to the high sequence divergence among the repeating units. We developed and applied a systematic method to consistently identify and annotate the structural repetitions over the members of the complete Ankyrin Repeat Protein Family, with increased sensitivity over previous studies. We statistically characterized the number of repeats, the folding of the repeat-arrays, their structural variations, insertions and deletions. An energetic analysis of the local frustration patterns reveal the basic features underlying fold stability and its relation to the functional binding regions. We found a strong linear correlation between the conservation of the energetic features in the repeat arrays and their sequence variations, and discuss new insights into the organization and function of these ubiquitous proteins.     

Selection at work in self-incompatible Arabidopsis lyrata: mating patterns in a natural population

Identification and characterization of the self-incompatibility genes in Brassicaceae species now allow typing of self-incompatibility haplotypes in natural populations. In this study we sampled and mapped all 88 individuals in a small population of Arabidopsis lyrata from Iceland. The self-incompatibility haplotypes at the SRK gene were typed for all the plants and some of their progeny and used to investigate the realized mating patterns in the population. The observed frequencies of haplotypes were found to change considerably from the parent generation to the offspring generation around their deterministic equilibria as determined from the known dominance relations among haplotypes. We provide direct evidence that the incompatibility system discriminates against matings among adjacent individuals. Multiple paternity is very common, causing mate availability among progeny of a single mother to be much larger than expected for single paternity.     

Expression,Purification, and Secondary Structure Prediction of Pentatricopeptide Repeat Protein RF1A from Rice

Pentatricopeptide repeat protein (PPR) proteins are putative RNA-binding proteins which are particularly prevalent in terrestrial plants. Previous research has reported the great difficulty in purifying soluble PPR proteins in Escherichia coli, therefore hindering further study of their functions. In this paper, we report the use of the pMAL ^™ prokaryotic expression system to acquire a soluble expression of a PPR protein, RF1A from rice (Oryza sativa L.). After purification, we identified RF1A by ESI-TOF-MS/MS. We also made an estimation of its secondary structure using the circular dichroism spectroscopy. These results supported the bioinformatic prediction of helical-hairpin model about PPR proteins.