tRNA-like Structure Research Articles

The tRNA‐like small noncoding RNA mascRNA promotes global protein translation

mascRNA is a small cytoplasmic RNA derived from the lncRNA MALAT1. After being processed by the tRNA processing enzymes RNase P and RNase Z, mascRNA undergoes CCA addition like tRNAs and folds into a tRNA-like cloverleaf structure. While MALAT1 functions in multiple cellular processes, the role of mascRNA was largely unknown. Here, we show that mascRNA binds directly to the multi-tRNA synthetase complex (MSC) component glutaminyl-tRNA synthetase (QARS). mascRNA promotes global protein translation and cell proliferation by positively regulating QARS protein levels. Our results uncover a role of mascRNA that is independent of MALAT1, but could be part of the molecular mechanism of MALAT1's function in cancer, and providea paradigm for understanding tRNA-like structures in mammalian cells.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3′ termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal. We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem–loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth

The RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA enzymes from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

Small RNA derived from Tobacco mosaic virus targets a host C2-domain abscisic acid-related (CAR) 7-like protein gene

Tobacco mosaic virus (TMV) is a positive-sense single-stranded RNA virus. The 3′ end of TMV genome is consisted of an upstream pseudoknot domain (UPD) and a tRNA-like structure (TLS), both of which are important RNA elements to enhance TMV replication and translation. Deep-sequencing analysis revealed that TMV-specific viral small interfering RNAs (vsiRNAs) were generated in TMV-infected Nicotiana benthamiana plants. A vsiRNA derived from the juxtaposition between UPD and TLS, named TMV-vsiRNA 22 nt (6285–6306), possessed high sequence complementarity to a host gene which encodes a C2-domain abscisic acid (ABA)-related (CAR) 7-like protein. CAR proteins play a critical role in ABA signaling pathway. The CAR protein-encoding gene was amplified from N. benthamiana leaves and termed as Nb-CAR7. In TMV-infected plants, accumulation of Nb-CAR7 transcripts was significantly decreased, as compared with that of mock-inoculated and TMV-43A-infected plants. TMV-43A is a mutant without the UPD sequence in its genome. Overexpression of Nb-CAR7 led to decreased TMV RNA accumulation in the TMV-inoculated leaves. Silencing of Nb-CAR7 enhanced TMV replication and resulted in a higher viral RNA accumulation. In addition, the expression level of Nb-CAR7 was positively correlated to that of a low-temperature-induced ABA responsive gene (LTI65). The effect of Nb-CAR7 on TMV RNA accumulation in host plants was linked to ABA signaling pathway. In conclusion, a vsiRNA derived from the juxtaposition between UPD and TLS at the 3′UTR of TMV targets a host CAR7 gene.

PlaMoM: a comprehensive database compiles plant mobile macromolecules.

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein-protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/.

Detection and in vitro studies of Cucurbita maxima phloem serpin-1 RNA-binding properties

Apart from being a conduit for photoassimilate transport in plants, the phloem serves as a pathway for transport of proteins and RNAs from sites of their synthesis to distant plant parts. As demonstrated for mRNAs and small RNAs such as miRNA and siRNA, their phloem transport is largely involved in responses to environmental cues including stresses and pathogen attacks. RNA molecules are believed to be transported in the phloem in the form of complexes with RNA-binding proteins; however, proteins forming such complexes are generally poorly studied. Here, we demonstrate that the Cucurbita maxima phloem serpin-1 (CmPS1), which has been previously described as a functional protease inhibitor capable of long-distance transport via the phloem, is able to bind RNA in vitro. Among different RNAs tested, CmPS1 exhibits a preference for imperfect RNA duplexes and the highest affinity to tRNA. A characteristic complex formed by CmPS1 with tRNA is not observed upon CmPS1 binding to tRNA-like structures of plant viruses. Mutational analysis demonstrates that the CmPS1 N-terminal region is not involved in RNA binding. Since antithrombin-III, the human protease inhibitor of serpin family most closely sequence-related to CmPS1, is found to be unable to bind RNA, one can suggest that, in its evolution, CmPS1 has gained the RNA binding capability as an additional function likely relevant to its specific activities in the plant phloem.

RNA mimicry in post-transcriptional regulation by aminoacyl tRNA synthetases.

Aminoacyl tRNA synthetases (aaRS) are well studied for their roles in tRNA charging with cognate amino acid. Nevertheless, numerous lines of evidence indicate that these proteins have roles other than tRNA charging. These include coordination of cellular signaling cascades, induction of cytokines outside the cell and transcription regulation. Herein, we focus on their roles in post-transcriptional regulation of mRNA expression. We describe functions that are related to antitermination of transcription, RNA splicing and mRNA translation. Cases were recognition of mRNA by the aaRS involves recognition of tRNA-like structures are described. Such recognition may be achieved by repurposing tRNA-binding domains or through domains added to the aaRS later in evolution. Furthermore, we describe cases in which binding by aaRS is implicated in autogenous regulation of expression. Overall, we propose RNA-mimicry as a common mode of interaction between aaRS and mRNA which allows efficient expression regulation. This article is categorized under: RNA Processing > tRNA Processing RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation.

Complete mitochondrial genome sequence of Bekko Tombo Libellula angelina Selys, 1883 (Odonata: Libellulidae)

The dragonfly Libellula angelina Selys, 1883 (Odonata: Libellulidae) has been listed as a critically endangered species by the International Union for Conservation of Nature (IUCN) and is also an endangered insect in South Korea. We sequenced the whole genome (15,233 bp) of L. angelina species, which included a set of typical genes and one major non-coding AT-rich region with an arrangement identical to that observed in most insect genomes. The A + T-rich region harbored one identical repeat composed of 65 bp and two tRNA-like structures (trnF and trnK-like sequences) with proper anticodon and clover-leaf structures. Phylogenetic reconstruction using the concatenated sequences of 13 protein-coding genes (PCGs) and two rRNAs of the representative odonate mitogenomes utilizing both Bayesian inference and maximum-likelihood methods revealed a strong support for the monophyletic Zygoptera and a moderate to high support for the monophyletic Anisoptera suborders. Unlike that in conventional phylogenetic analysis, a relatively strong sister relationship was revealed between the suborders of Anisozygoptera and Zygoptera.

RNA phloem transport mediated by pre-miRNA and viral tRNA-like structures

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3′-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.

Analyzing and Predicting Phloem Mobility of Macromolecules with an Online Database.

Phloem, a specialized plant tissue, serves as a superhighway for macromolecular exchanges between different organs or tissues in plants. These mobile macromolecules may function as signaling molecules to sense intrinsic developmental cues or environmental inputs. Among these mobile molecules, RNAs generally need non-cell-autonomous pathway proteins (NCAPPs) to bind to and help them move along the symplasmic passage (through plasmodesmata) into the phloem stream. Grafting experiments combined with next-generation sequencing discovered that around 11.4% of identified Arabidopsis mobile mRNAs have a tRNA-like structure (TLS) motif. Adding an artificial tRNA-like structure at the 5' end of cell-autonomous RNAs (e.g., GUS transcript) can trigger its mobility and movement across a grafting junction to distant organs. Based on the accumulated data and the role of the TLS motif in RNA mobility, we built a web server in our database PLaMoM (a database for plant mobile macromolecules) to enable plant biologists to predict and analyze the transcripts they are interested in. In this chapter, we describe how to use our built-in web server to investigate RNA mobility.

Synergistic SHAPE/Single-Molecule Deconvolution of RNA Conformation under Physiological Conditions

Structural RNA domains are widely involved in the regulation of biological functions, such as gene expression, gene modification, and gene repair. Activity of these dynamic regions depends sensitively on the global fold of the RNA, in particular, on the binding affinity of individual conformations to effector molecules in solution. Consequently, both the 1) structure and 2) conformational dynamics of noncoding RNAs prove to be essential in understanding the coupling that results in biological function. Toward this end, we recently reported observation of three conformational states in the metal-induced folding pathway of the tRNA-like structure domain of Brome Mosaic Virus, via single-molecule fluorescence resonance energy transfer studies. We report herein selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-directed structure predictions as a function of metal ion concentrations ([Mn+]) to confirm the three-state folding model, as well as test 2° structure models from the literature. Specifically, SHAPE reactivity data mapped onto literature models agrees well with the secondary structures observed at 0–10 mM [Mg2+], with only minor discrepancies in the E hairpin domain at low [Mg2+]. SHAPE probing and SHAPE-directed structure predictions further confirm the stepwise unfolding pathway previously observed in our single-molecule studies. Of special relevance, this means that reduction in the metal-ion concentration unfolds the 3′ pseudoknot interaction before unfolding the long-range stem interaction. This work highlights the synergistic power of combining 1) single-molecule Förster resonance energy transfer and 2) SHAPE-directed structure-probing studies for detailed analysis of multiple RNA conformational states. In particular, single-molecule guided deconvolution of the SHAPE reactivities permits 2° structure predictions of isolated RNA conformations, thereby substantially improving on traditional limitations associated with current structure prediction algorithms.

Viral tRNA Mimicry from a Biocommunicative Perspective.

RNA viruses have very small genomes which limits the functions they can encode. One of the strategies employed by these viruses is to mimic key factors of the host cell so they can take advantage of the interactions and activities these factors typically participate in. The viral RNA genome itself was first observed to mimic cellular tRNA over 40 years ago. Since then researchers have confirmed that distinct families of RNA viruses are accessible to a battery of cellular factors involved in tRNA-related activities. Recently, potential tRNA-like structures have been detected within the sequences of a 100 mRNAs taken from human cells, one of these being the host defense interferon-alpha mRNA; these are then additional to the examples found in bacterial and yeast mRNAs. The mimetic relationship between tRNA, cellular mRNA, and viral RNA is the central focus of two considerations described below. These are subsequently used as a preface for a final hypothesis drawing on concepts relating to mimicry from the social sciences and humanities, such as power relations and creativity. Firstly, the presence of tRNA-like structures in mRNAs indicates that the viral tRNA-like signal could be mimicking tRNA-like elements that are contextualized by the specific carrier mRNAs, rather than, or in addition to, the tRNA itself, which would significantly increase the number of potential semiotic relations mediated by the viral signals. Secondly, and in particular, mimicking a host defense mRNA could be considered a potential new viral strategy for survival. Finally, we propose that mRNA’s mimicry of tRNA could be indicative of an ancestral intracellular conflict in which species of mRNAs invaded the cell, but from within. As the meaning of the mimetic signal depends on the context, in this case, the conflict that arises when the viral signal enters the cell can change the meaning of the mRNAs’ internal tRNA-like signals, from their current significance to that they had in the distant past.

Sequencing of the complete mitochondrial genome of a fish-parasitic flatworm Paratetraonchoides inermis (Platyhelminthes: Monogenea): tRNA gene arrangement reshuffling and implications for phylogeny

BackgroundParatetraonchoides inermis (Monogenea: Tetraonchoididae) is a flatworm parasitising the gills of uranoscopid fishes. Its morphological characteristics are ambiguous, and molecular data have never been used to study its phylogenetic relationships, which makes its taxonomic classification controversial. Also, several decades of unsuccessful attempts to resolve the relationships within the Monogenea present a strong indication that morphological datasets may not be robust enough to be used to infer evolutionary histories. As the use of molecular data is currently severely limited by their scarcity, we have sequenced and characterized the complete mitochondrial (mt) genome of P. inermis. To investigate its phylogenetic position, we performed phylogenetic analyses using Bayesian inference and maximum likelihood approaches using concatenated amino acid sequences of all 12 protein-coding genes on a dataset containing all available monogenean mt genomes.ResultsThe circular mt genome of P. inermis (14,654 bp) contains the standard 36 genes: 22 tRNAs, two rRNAs, 12 protein-encoding genes (PCGs; Atp8 is missing) and a major non-coding region (mNCR). All genes are transcribed from the same strand. The A + T content of the whole genome (82.6%), as well as its elements, is the highest reported among the monogeneans thus far. Three tRNA-like cloverleaf structures were found in mNCR. Several results of the phylogenomic analysis are in disagreement with previously proposed relationships: instead of being closely related to the Gyrodactylidea, Tetraonchidea exhibit a phylogenetic affinity with the Dactylogyridea + Capsalidea clade; and the order Capsalidea is neither basal within the subclass Monopisthocotylea, nor groups with the Gyrodactylidea, but instead forms a sister clade with the Dactylogyridea. The mt genome of P. inermis exhibits a unique gene order, with an extensive reorganization of tRNAs. Monogenea exhibit exceptional gene order plasticity within the Neodermata.ConclusionsThis study shows that gene order within monopisthocotylid mt genomes is evolving at uneven rates, which creates misleading evolutionary signals. Furthermore, our results indicate that all previous attempts to resolve the evolutionary history of the Monogenea may have produced at least partially erroneous relationships. This further corroborates the necessity to generate more molecular data for this group of parasitic animals.

Conservation of tRNA mimicry in the 5'-untranslated region of distinct HIV-1 subtypes.

Human tRNALys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1) and anneals to the complementary primer binding site (PBS) in the genome. All tRNALys isoacceptors interact with human lysyl-tRNA synthetase (hLysRS) and are selectively packaged into virions. tRNALys3 must be released from hLysRS in order to anneal to the PBS, and this process is proposed to be facilitated by the interaction of hLysRS with a tRNA-like element (TLE) first identified in the HIV-1 5′-untranslated region (5′-UTR) of the subtype B NL4-3 virus. However, a significant subset of HIV-1 strains represented by the MAL isolate possess a different secondary structure in this region of the genome. Thus, to establish the conservation of this mechanism for primer targeting and release, we investigated the subtype A-like 5′-UTR of the MAL isolate. hLysRS bound to a 229-nt MAL RNA containing the PBS domain with high affinity (Kd = 47 nM), and to a 98-nt truncated construct with ∼10-fold reduced affinity. These results resemble previous studies using analogous NL4-3-derived RNAs. However, in contrast to studies with NL4-3, no binding was observed to smaller stem–loop elements within the MAL PBS domain. The tertiary structure of the 98-nt construct was analyzed using small-angle X-ray scattering, revealing remarkable global structural similarity to the corresponding NL4-3 PBS/TLE region. These results suggest that the tRNA-like structure within the 5′-UTR is conserved across distinct HIV-1 subtypes and that hLysRS recognition of the MAL isolate is likely not conferred by specific sequence elements but by 3D structure.

ICTV Virus Taxonomy Profile: Virgaviridae.

The family Virgaviridae is a family of plant viruses with rod-shaped virions, a ssRNA genome with a 3′-terminal tRNA-like structure and a replication protein typical of alpha-like viruses. Differences in the number of genome components, genome organization and the mode of transmission provide the basis for genus demarcation. Tobacco mosaic virus (genus Tobamovirus) was the first virus to be discovered (in 1886); it is present in high concentrations in infected plants, is extremely stable and has been extensively studied. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Virgaviridae, which is available at www.ictv.global/report/virgaviridae.

First Mitochondrial Genome from Nemouridae (Plecoptera) Reveals Novel Features of the Elongated Control Region and Phylogenetic Implications.

The complete mitochondrial genome (mitogenome) of Nemoura nankinensis (Plecoptera: Nemouridae) was sequenced as the first reported mitogenome from the family Nemouridae. The N. nankinensis mitogenome was the longest (16,602 bp) among reported plecopteran mitogenomes, and it contains 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Most PCGs used standard ATN as start codons, and TAN as termination codons. All tRNA genes of N. nankinensis could fold into the cloverleaf secondary structures except for trnSer (AGN), whose dihydrouridine (DHU) arm was reduced to a small loop. There was also a large non-coding region (control region, CR) in the N. nankinensis mitogenome. The 1751 bp CR was the longest and had the highest A+T content (81.8%) among stoneflies. A large tandem repeat region, five potential stem-loop (SL) structures, four tRNA-like structures and four conserved sequence blocks (CSBs) were detected in the elongated CR. The presence of these tRNA-like structures in the CR has never been reported in other plecopteran mitogenomes. These novel features of the elongated CR in N. nankinensis may have functions associated with the process of replication and transcription. Finally, phylogenetic reconstruction suggested that Nemouridae was the sister-group of Capniidae.

Identification and Characterization of a Class of MALAT1-like Genomic Loci

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript (MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant long noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Thus, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.

PlaMoM: a comprehensive database compiles plant mobile macromolecules.

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein–protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/.

TRNA-Related Sequences Trigger Systemic mRNA Transport in Plants.

In plants, protein-coding mRNAs can move via the phloem vasculature to distant tissues, where they may act as non-cell-autonomous signals. Emerging work has identified many phloem-mobile mRNAs, but little is known regarding RNA motifs triggering mobility, the extent of mRNA transport, and the potential of transported mRNAs to be translated into functional proteins after transport. To address these aspects, we produced reporter transcripts harboring tRNA-like structures (TLSs) that were found to be enriched in the phloem stream and in mRNAs moving over chimeric graft junctions. Phenotypic and enzymatic assays on grafted plants indicated that mRNAs harboring a distinctive TLS can move from transgenic roots into wild-type leaves and from transgenic leaves into wild-type flowers or roots; these mRNAs can also be translated into proteins after transport. In addition, we provide evidence that dicistronic mRNA:tRNA transcripts are frequently produced in Arabidopsis thaliana and are enriched in the population of graft-mobile mRNAs. Our results suggest that tRNA-derived sequences with predicted stem-bulge-stem-loop structures are sufficient to mediate mRNA transport and seem to be necessary for the mobility of a large number of endogenous transcripts that can move through graft junctions.

The Restorer-of-fertility-like 2 pentatricopeptide repeat protein and RNase P are required for the processing of mitochondrial orf291 RNA in Arabidopsis.

Eukaryotes harbor mitochondria obtained via ancient symbiosis events. The successful evolution of energy production in mitochondria has been dependent on the control of mitochondrial gene expression by the nucleus. In flowering plants, the nuclear-encoded pentatricopeptide repeat (PPR) superfamily proteins are widely involved in mitochondrial RNA metabolism. Here, we show that an Arabidopsis nuclear-encoded RNA-binding protein, Restorer-of-fertility-like PPR protein 2 (RFL2), is required for RNA degradation of the mitochondrial orf291 transcript via endonucleolytic cleavage of the transcript in the middle of its reading frame. Both invivo and invitro, this RNA cleavage requires the activity of mitochondrial proteinaceous RNase P, which is possibly recruited to the site by RFL2. The site of RNase P cleavage likely forms a tRNA-like structure in the orf291 transcript. This study presents an example of functional collaboration between a PPR protein and an endonuclease in RNA cleavage. Furthermore, we show that the RFL2-binding region within the orf291 gene is hypervariable in the family Brassicaceae, possibly correlated with the rapid evolution of the RNA-recognition interfaces of the RFL proteins.