Mechanistic insight into pentatricopeptide repeat proteins as sequence-specific RNA-binding proteins for organellar RNAs in plants

The pentatricopeptide repeat (PPR) protein family is highly expanded in terrestrial plants. Arabidopsis contains 450 PPR genes, which represents 2% of the total protein-coding genes. PPR proteins are eukaryote-specific RNA-binding proteins implicated in multiple aspects of RNA metabolism of organellar genes. Most PPR proteins affect a single or small subset of gene(s), acting in a gene-specific manner. Studies over the last 10 years have revealed the significance of this protein family in coordinated gene expression in different compartments: the nucleus, chloroplast and mitochondrion. Here, we summarize recent studies addressing the mechanistic aspect of PPR proteins.     

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.     

LRP130, a pentatricopeptide motif protein with a noncanonical RNA-binding domain,is bound in vivo to mitochondrial and nuclear RNAs

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.     

A combinatorial amino Acid code for RNA recognition by pentatricopeptide repeat proteins

The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA-binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA-protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.     

番茄PPR基因家族的鉴定与生物信息学分析

PPR(Pentatricopeptide repeats)基因家族在植物中广泛存在, 其在植物生长发育过程中至关重要。文章采用生物信息学方法, 利用Pfam已鉴定的PPR保守结构域序列检索番茄(Solanum lycopersicum L.)基因组计划注释的蛋白序列, 最终确定了番茄中可能存在的471个PPR编码基因; 根据拟南芥(Arabidopsis thaliana L.)中鉴定的各个结构域的特点对其进行了蛋白结构分析、分类和保守序列分析, 并对番茄PPR基因家族进行了系统进化树构建、染色体定位、亚细胞定位预测、表达和GO分析等。结果表明：番茄PPR基因家族分为P和PLS两个亚家族, 各占序列数目的一半, PLS亚家族又分为PLS、E、E+和DYW四类, 且在进化树中形成不同的分支; 各个结构域在植物中非常保守; PPR基因家族分布在番茄12条染色体上, 且多数无内含子结构; 大部分PPR蛋白具有线粒体或叶绿体定位序列, GO分析表明PPR蛋白参与RNA相关的生物学过程     

The roles of S1 RNA-binding domains in Rrp5's interactions with pre-rRNA

RNA-binding proteins mediate the function of all RNAs. Since few distinct RNA-binding domains (RBDs) exist, with most RBDs contacting only a few nucleotides, RNA-binding proteins often combine multiple RNA-binding motifs to achieve a higher affinity and selectivity for their targets. Rrp5, a ribosome assembly factor essential for both 40S and 60S ribosome maturation, is an extreme example as it contains 12 tandem S1 RNA-binding domains. In this study, we use a combination of RNA binding and DMS probing experiments to probe interactions of Rrp5 with pre-rRNA mimics. Our data localize Rrp5's binding site to three distinct regions within internal transcribed spacer 1 (ITS1), the sequence between 18S and 5.8S rRNAs. One of these regions is directly adjacent to a recently uncovered helical structure, which prevents premature cleavage at the 3'-end of 18S rRNA. This finding, together with previous results, suggests a role for Rrp5 in regulating the above-mentioned helical element. Furthermore, we have produced two truncated forms of the protein, Rrp5N and Rrp5C, which together encompass the entire protein and fully restore growth. Quantitative analysis of the RNA affinity of these Rrp5 fragments indicates that the first nine S1 motifs contribute much of Rrp5's RNA affinity, while the last three domains alone provide its specificity for the pre-rRNA. This surprising division of labor is unique, as it suggests that S1 domains can bind RNA both specifically as well as nonspecifically with high affinity; this has important implications for the molecular details of the Rrp5?pre-rRNA complex.