Identification of a novel pentatricopeptide repeat subfamily with a C-terminal domain of bacterial origin acquired via ancient horizontal gene transfer (original) (raw)
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Nucleic Acids Research, 2001
Bacterial tmRNA mediates a trans-translation reaction, which permits the recycling of stalled ribosomes and probably also contributes to the regulated expression of a subset of genes. Its action results in the addition of a small number of C-terminal amino acids to protein whose synthesis had stalled and these constitute a proteolytic recognition tag for the degradation of these incompletely synthesized proteins. Previous work has identified pseudoknots and stem-loops that are widely conserved in divergent bacteria. In the present work an alignment of tmRNA gene sequences within 13 β-proteobacteria reveals an additional sub-structure specific for this bacterial group. This sub-structure is in pseudoknot Pk2, and consists of one to two additional stem-loop(s) capped by stable GNRA tetraloop(s). Three-dimensional models of tmRNA pseudoknot 2 (Pk2) containing various topological versions of the additional substructure suggest that the sub-structures likely point away from the core of the RNA, containing both the tRNA and the mRNA domains. A putative tertiary interaction has also been identified.
Proceedings of the National Academy of Sciences, 2011
Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3′-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2′,3′-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3′-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn 2þ binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3′-P RNA ligase.
A chloroplastic RNA-binding protein is a new member of the PPR family
FEBS Letters, 2000
P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963^4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621^2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.
The natural history of transfer RNA and its interactions with the ribosome
Transfer RNA (tRNA) is undoubtedly the most central and one of the oldest molecules of the cell. Without it genetics and coded protein synthesis are impossible. The crucial specificities responsible for the genetic code and accurate translation are by far entrusted to interactions between tRNA and translation proteins, fundamentally aminoacyl-tRNA synthetase (aaRS) enzymes and elongation factor (EF) switches . Discrimination mediated by aaRSs and EFs against misincorporated tRNA and amino acids is at least 20 times more stringent than ribosomal recognition, editing, and other proofreading mechanisms . The fact that crucial genetic code specificities in highly selective interactions with protein enzymes do not involve the ribosomal ribonucleoprotein biosynthetic machinery challenges the "replicators first" origin of life scenario of an ancient RNA world (Caetano-Anollés and Seufferheld, 2013). It also highlights the central functional, mechanistic, and evolutionary roles of tRNA and its recognition determinants, which enable coevolution between nucleic acids and proteins. These coevolutionary relationships are compatible with a late origin of the ribosome in its mechanism and not in protein biosynthesis, which was inferred from the computational analysis of thousands of RNAs and proteomes (Harish and Caetano-Anollés, 2012). These analyses showed tight coevolution of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). While these relationships delimit molecular makeup when organisms use translation to negotiate growth and viability amidst environmental change, coevolution also constrains recruitment of the canonical L-shaped structure of the tRNA molecule into a multiplicity of modern functions. These new functions include the synthesis of antibiotics, bacterial cell wall peptidoglycans and tetrapyrroles, modification of bacterial membrane lipids, protein turnover, and the synthesis of other aminoacyl-tRNA molecules . Here we unfold coevolutionary relationships between tRNA substructures and translation proteins that embody crucial protein-nucleic acid interactions. We focus on a series of computational biology analyses of the structure and conformational diversity of tRNAs and their interacting proteins that provide information about the history of structural accretion of this "adaptor" molecule. Using this information, we place tRNA history within the framework of an evolutionary timeline of protein domain innovation, uncovering the natural history of tRNA within the context of the geological record.
Archaeal Guide RNAs Function in rRNA Modification in the Eukaryotic Nucleus
Current Biology, 2002
C and Box D elements in at least six archaeal species [11, 12] suggested the remarkable possibility that the essential elements of the Box C/D RNAs had not diverged in Archaea and Eukarya over a vast period of Summary independent evolution. However, the minimal features required for assembly of a functional Box C/D RNP in In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short comple-eukaryotes have not yet been defined. The archaeal Box C/D RNAs do not possess significant sequence mentary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2-O-homology with eukaryotic Box C/D RNAs outside of the Box C, CЈ, D, and DЈ sequence elements [11, 12]. methylation [1-3]. Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known Moreover, the identified archaeal Box C/D RNAs are more compact than eukaryotic Box C/D RNAs (e.g., on proteins [4-10]. Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by average 56 nucleotides in P. furiosus versus 104 in Saccharomyces cerevisiae) [11, 22]. biochemical analysis and computational genomic screens [11, 12] and have likely evolved independently Analysis of the sequences of archaeal homologs of the eukaryotic Box C/D RNA-associated proteins suggests in Archaea and Eukarya for more than 2000 million years [13-15]. We have microinjected Box C/D RNAs that the archaeal Box C/D RNAs interact with a simpler set of proteins. For example, archaeal fibrillarin homo-from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog logs lack the glycine arginine-rich (GAR) domain that is characteristic of fibrillarin in eukaryotes [18, 23, 24]. Xenopus laevis. Our results show that Box C/D RNAs derived from this prokaryote are retained in the nu-Moreover, five of the eukaryotic proteins, Nop56, Nop58, p50, p55, and p15.5 kDa, appear to be products of gene cleus, localize to nucleoli, and interact with the X. laevis Box C/D RNA binding proteins fibrillarin, Nop56, duplications that have occurred since the divergence of Archaea and Eukarya [7, 20, 23]. For example, Nop56, and Nop58. Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific Nop58, and the spliceosomal protein Prp31 are related eukaryotic proteins all represented by a single archaeal 2-O-methylation of ribosomal RNA. Our studies have revealed the remarkable ability of archaeal Box C/D homolog [23]. Thus, the six eukaryotic Box C/D RNAassociated proteins are represented by just four homol-RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus. ogous proteins in archaea [20]. All six of the eukaryotic Box C/D RNA-associated proteins are essential [6, 10, 25, 26], indicating that the duplicated genes (paralogs) Results and Discussion have evolved to encode proteins with distinct and critical functions in eukaryotes. We were interested to know The numerous Box C/D RNAs that guide modification whether the eukaryotic proteins would recognize archof rRNA in eukaryotic cells are characterized by two aeal Box C/D RNAs. very short conserved sequence elements, Box C In this work, we have tested the consequences of the (UGAUGA) and Box D (CUGA), found near the 5Ј and 3Ј evolutionary distance and extant differences between termini of the RNAs, respectively. Two additional elethe Box C/D RNAs and associated proteins in Archaea ments, Box CЈ (UGAUGA) and Box DЈ (CUGA), are also and Eukarya. We cloned the coding sequence of four present within Box C/D guide RNAs but are usually less (of 52) Box C/D RNAs predicted to exist in P. furiosus: highly conserved [16]. Box C/D RNAs base pair with sR2, sR3, sR29, and sR42 ( [11] and equivalent to sR10, rRNA via complementary guide sequences (10 to 21 25, 35, and 30, respectively, in [12]). We also confirmed nucleotides in length) found immediately upstream of by RT-PCR analysis that the computationally predicted Box D (or Box DЈ). Interaction of a Box C/D RNA with P. furiosus sR2, sR3, sR29, and sR42 are expressed RNAs (data not shown). In eukaryotes, Box C/D RNAs 4 Correspondence: mterns@bmb.uga.edu [M.P.T.], rterns@bmb.uga. edu [R.M.T.] are actively retained in the nucleus and function within
Small RNA profiling in Chlamydomonas: insights into chloroplast RNA metabolism
Nucleic Acids Research
In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded transacting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long-and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5 end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5 end by the cognate factor. Enzymatic removal of 5-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32-34 nt ribosome-protected fragments. Using this 'in vivo ribosome footprinting', we identified the function and molecular target of two candidate transacting factors.
Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein
Proceedings of the National Academy of Sciences, 2011
Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI-atpH intergenic region (i) blocks both 5′→3′ and 3′→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5′-terminus in conjunction with a generic 5′→3′ exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10's ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins. mitochondria | plastid | RNA binding protein | RNA processing P entatricopeptide repeat (PPR) proteins are a recently recognized class of RNA-binding proteins that profoundly affect gene expression in mitochondria and chloroplasts (reviewed in 1). PPR proteins are defined by tandem arrays of a degenerate 35-aa repeat that is predicted to adopt a helical hairpin structure (2). A wealth of genetic data documents the importance of PPR proteins for organellar RNA metabolism: Mutations in PPRencoding genes cause defects in the splicing, editing, stabilization, processing, or translation of subsets of organellar RNAs, with downstream defects in respiration, photosynthesis, and organismal development.
We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5Ј untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of c ytochrome b 6 f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation-known as the CES process-in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process.