LRP130, a single-stranded DNA/RNA-binding protein, localizes at the outer nuclear and endoplasmic reticulum membrane, and interacts with mRNA in vivo

LRP130 (also known as a LRPPRC) is an RNA and single-stranded DNA-binding protein, and recently identified as a candidate gene responsible for the Leigh syndrome, a French-Canadian type cytochrome c oxidase deficiency. However, the biological function of LRP130 still remains largely unresolved. In the present study, we found that the C-terminal half of the mouse LRP130 located within a 120 amino acid sequence (a.a. 845-964) binds to synthetic RNA homopolymers, poly(G), poly(U), and poly(C), as well as r(CUGCC)(6). Assessment of the subcellular localization indicated both nuclear/endoplasmic reticulum (ER) and mitochondrial fractions to be positive. To further analyze the subcellular localization of LRP130, a nuclear/ER fraction was fractionated into the nucleoplasm (NP) and nuclear envelope (NE)/ER, and the latter was further separated into outer nuclear membrane (ONM)/ER and inner nuclear membrane (INM) by treatment with Triton X-100. LRP130 was detectable in all three fractions, and the distribution pattern was in good accordance with that known for ONM/ER proteins. Interestingly, immunostaining of HeLa cells demonstrated nuclear rim staining of LRP130, specifically at the outside of the NE and also at ER, and association of LRP130 with poly(A)(+) RNA was restricted only to the ONM/ER fraction. Overexpression of full-length mouse LRP130 fused with EGFP resulted in nuclear accumulation of poly(A)(+) RNA in HeLa cells. Taking all these results together, it is suggested that LRP130, a novel type of RNA-binding protein, associates with mRNA/mRNP complexes at the outside of NE and ER, and plays a role in control of mRNA metabolisms.     

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.     

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.     

The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial COX1 mRNA in yeast

Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein.     

Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

PPR蛋白影响植物生长发育的研究进展

植物生长发育是一个极其复杂的生理生化过程,受内外因素共同作用。PPR蛋白是核基因编码的具有重复PPR基序的蛋白,分布广泛,在高等植物中数量巨大。PPR蛋白的靶标一般是线粒体和叶绿体中转录的RNA前体,多数可与MORF互作,参与线粒体和叶绿体基因的RNA编辑。PPR蛋白缺失的突变体植株多数呈现异常表型,影响植物的正常生长发育。本文就近年来发现的PPR蛋白结构、分布,与RNA编辑的关系,及其对植物生长发育的影响进行了综述。     

Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L (long) and L2 (long2) and the S2 (short2) motifs. We now find that inclusion of these motifs improves the prediction of RNA editing target sites. Previously overlooked RNA editing target sites are suggested from the PPR motif structures of known E-class PPR proteins and are experimentally verified. RNA editing target sites are assigned for the novel PPR protein MEF32 (mitochondrial editing factor 32) and are confirmed in the cDNA.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Mitochondrial–nuclear co‐evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins

Mitochondrial–nuclear incompatibility has a major role in reproductive isolation between species. However, the underlying mechanism and driving force of mitochondrial–nuclear incompatibility remain elusive. Here, we report a pentatricopeptide repeat‐containing (PPR) protein, Ccm1, and its interacting partner, 15S rRNA, to be involved in hybrid incompatibility between two yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus. S. bayanus‐Ccm1 has reduced binding affinity for S. cerevisiae‐15S rRNA, leading to respiratory defects in hybrid cells. This incompatibility can be rescued by single mutations on several individual PPR motifs, demonstrating the highly evolvable nature of PPR proteins. When we examined other PPR proteins in the closely related Saccharomyces sensu stricto yeasts, about two‐thirds of them showed detectable incompatibility. Our results suggest that fast co‐evolution between flexible PPR proteins and their mitochondrial RNA substrates may be a common driving force in the development of mitochondrial–nuclear hybrid incompatibility.     

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.