Metabolomics of Cassia Auriculata Plant Parts (Leaf, Flower, Bud) and Their Antidiabetic Medicinal Potentials.

Diabetes is a common chronic disease where therapeutics innovation is much needed. The search for novel antidiabetic molecules can be greatly facilitated by high throughput metabolomic characterization of herbal medicines. Cassia auriculata is a shrub used in Ayurvedic medicine and native to India and Sri Lanka. While C. auriculata has been used as a medicinal herb in diabetes, the molecular evidence for its antidiabetic medicinal potentials and components needs to be established. Moreover, the phytocomposition of the various plant parts is not fully known. We report a comprehensive metabolomic gas chromatography mass spectrometry study of the C. auriculata plant parts, including the leaf, flower, and bud. We identified a total of 102 primary and secondary metabolites in seven chemical groups, including amino acids (AA), carboxylic acids, nucleosides, fatty acids, among others. Interestingly, plant parts differed in their metabolomic signatures. While in the flowers and leaves nine and six AA were identified, respectively, no AA was detected in the buds. Some of the identified compounds have been previously noted for their antidiabetic, hypoglycemic, and hypolipidemic bioactivities. These findings offer a concrete metabolomic basis on the phytocomposition of individual C. auriculata plant parts. These omics data call for future research on the function of the identified compounds, and clinical studies to further evaluate their antidiabetic potentials and mechanisms of action in the clinic. Finally, we note that plant omics research offers an important avenue to inform, verify, and strengthen the evidentiary base and clinical testing of herbs with medicinal potentials.

Engineering and use of proteinoid polymers and nanocapsules containing agrochemicals.

To address global challenges such as population growth and climate change, introduction of new technologies and innovations in agriculture are paramount. Polymer-based formulations of agrochemicals have received much attention in recent years, and there is strong motivation to develop agrochemicals that are not harmful to the environment. Proteinoid polymers are produced by thermal step-growth polymerization of natural and unnatural amino acids. Under suitable gentle conditions, the proteinoid polymers may self-assemble to form nano-sized hollow proteinoid nanoparticles (NPs) of a relatively narrow size distribution. Agrochemical molecules may be encapsulated within these hollow proteinoid NPs, integrated in the crude proteinoid shell, or bound covalently/physically to the NP surface. In the present manuscript we prepared and characterized four model proteinoid polymers and NPs: P(KEf), P(KF), P(EWH-PLLA) and P(KWH-PLLA), where Ef denotes the unnatural herbicidal amino acid glufosinate. The NPs were fluorescently labeled and loaded with agrochemicals such as the plant hormone auxin. In addition, the NP surface was hydrophobized by covalent conjugation of dodecyl aldehyde via its surface primary amine groups. Following treatment of the plants with the different fluorescent-labeled NPs, fluorescent microscopic techniques enabled to localize the NPs and observe the accumulation in the plant's vascular system. Next, using genetically modified plants, which express fluorescent protein and are responsive to the level of auxin, we demonstrated the possibility to deliver encapsulated agrochemicals into cells. We also illustrated that the proteinoid NPs are non-toxic to human umbilical vein endothelial cells, and apart from P(KEf) also to lettuce plants.

Aminoacyl-tRNA synthetases and translational quality control in plant mitochondria.

Gene expression involves the transfer of information stored in the DNA to proteins by two sequential key steps: transcription and translation. Aminoacyl-tRNA synthetases (aaRSs), an ancient group of enzymes, are key to these processes as they catalyze the attachment of each of the 20 amino acids to their corresponding tRNA molecules. Yet, in addition to the 20 canonical amino acids, plants also produce numerous non-proteogenic amino acids (NPAAs), some of which are erroneously loaded into tRNAs, translated into non-functional or toxic proteins and may thereby disrupt essential cellular processes. While many studies have been focusing on plant organelle RNA metabolism, mitochondrial translation still lags behind its characterization in bacterial and eukaryotic systems. Notably, plant mitochondrial aaRSs generally have a dual location, residing also within the chloroplasts or cytosol. Currently, little is known about how mitochondrial aaRSs distinguish between amino acids and their closely related NPAAs. The organelle translation machineries in plants seem more susceptible to NPAAs due to protein oxidation by reactive oxygen species (ROS) and high rates of protein turnover. We speculate that plant organellar aaRSs have acquired high-affinities to their cognate amino acid substrates to reduce cytotoxic effects by NPAAs.

The Phytotoxicity of Meta-Tyrosine Is Associated With Altered Phenylalanine Metabolism and Misincorporation of This Non-Proteinogenic Phe-Analog to the Plant's Proteome.

Plants produce a myriad of specialized (secondary) metabolites that are highly diverse chemically, and exhibit distinct biological functions. Here, we focus on meta-tyrosine (m-tyrosine), a non-proteinogenic byproduct that is often formed by a direct oxidation of phenylalanine (Phe). Some plant species (e.g., Euphorbia myrsinites and Festuca rubra) produce and accumulate high levels of m-tyrosine in their root-tips via enzymatic pathways. Upon its release to soil, the Phe-analog, m-tyrosine, affects early post-germination development (i.e., altered root development, cotyledon or leaf chlorosis, and retarded growth) of nearby plant life. However, the molecular basis of m-tyrosine-mediated (phyto)toxicity remains, to date, insufficiently understood and are still awaiting their functional characterization. It is anticipated that upon its uptake, m-tyrosine impairs key metabolic processes, or affects essential cellular activities in the plant. Here, we provide evidences that the phytotoxic effects of m-tyrosine involve two distinct molecular pathways. These include reduced steady state levels of several amino acids, and in particularly altered biosynthesis of the phenylalanine (Phe), an essential α-amino acid, which is also required for the folding and activities of proteins. In addition, proteomic studies indicate that m-tyrosine is misincorporated in place of Phe, mainly into the plant organellar proteomes. These data are supported by analyses of adt mutants, which are affected in Phe-metabolism, as well as of var2 mutants, which lack FtsH2, a major component of the chloroplast FtsH proteolytic machinery, which show higher sensitivity to m-tyrosine. Plants treated with m-tyrosine show organellar biogenesis defects, reduced respiration and photosynthetic activities and growth and developmental defect phenotypes.

Excessive nitrogen impairs hydraulics, limits photosynthesis, and alters the metabolic composition of almond trees.

Horticulture nitrogen (N) runoffs are major environmental and health concerns, but current farming practices cannot detect ineffective N applications. Hence, we set to recognize high N conditions and characterize their effects on the physiology of almond trees grown in drainage lysimeters. Water and nutrients mass balances exhibited that N benefitted almond trees in a limited range (below 60 mg N L-1 in irrigation), while higher N conditions (over a 100 mg N L-1) reduced evapotranspiration (ET) by 50% and inherently constrained N uptake. Respectively, whole-tree hydraulic conductance reduced by 37%, and photosynthesis by 17%, which implied that high N concentrations could damage trees. Through gas-chromatography, we realized that high N conditions also affected components of the citric acid cycle (TCA) and carbohydrates availability. Such changes in the metabolic composition of roots and leaves probably interfered with N assimilation and respiration. It also determined the proportions between N and starch in almond leaves, which formed a new index (N:ST) that starts at 0.4 in N deficiency and reaches 0.6-0.8 in optimal N conditions. Importantly, this index continues to increase in higher N conditions (as starch reduces) and essentially indicates to excessive N applications when it exceeds 1.1.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

Structure of subcomplex Iß of mammalian respiratory complex I leads to new supernumerary subunit assignments.

Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved "core" subunits and 31 "supernumerary" subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iß, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iß and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX9C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein-ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation.

Universal pathway for posttransfer editing reactions: insights from the crystal structure of TtPheRS with puromycin.

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA(Phe) to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2' and 3' isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA(Phe) where deacylation occurs from either the 2'- or 3'-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.

The mechanistic and evolutionary aspects of the 2'- and 3'-OH paradigm in biosynthetic machinery.

BACKGROUND: The translation machinery underlies a multitude of biological processes within the cell. The design and implementation of the modern translation apparatus on even the simplest course of action is extremely complex, and involves different RNA and protein factors. According to the "RNA world" idea, the critical link in the translation machinery may be assigned to an adaptor tRNA molecule. Its exceptional functional and structural characteristics are of primary importance in understanding the evolutionary relationships among all these macromolecular components. PRESENTATION OF THE HYPOTHESIS: The 2'-3' hydroxyls of the tRNA A76 constitute chemical groups of critical functional importance, as they are implicated in almost all phases of protein biosynthesis. They contribute to: a) each step of the tRNA aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (aaRSs); b) the isomerase activity of EF-Tu, involving a mixture of the 2'(3')- aminoacyl tRNA isomers as substrates, thereby producing the required combination of amino acid and tRNA; and c) peptide bond formation at the peptidyl transferase center (PTC) of the ribosome. We hypothesize that specific functions assigned to the 2'-3' hydroxyls during peptide bond formation co-evolved, together with two modes of attack on the aminoacyl-adenylate carbonyl typical for two classes of aaRSs, and alongside the isomerase activity of EF-Tu. Protein components of the translational apparatus are universally recognized as being of ancient origin, possibly replacing RNA-based enzymes that may have existed before the last universal common ancestor (LUCA). We believe that a remnant of these processes is still imprinted on the organization of modern-day translation. TESTING AND IMPLICATIONS OF THE HYPOTHESIS: Earlier publications indicate that it is possible to select ribozymes capable of attaching the aa-AMP moiety to RNA molecules. The scenario described herein would gain general acceptance, if a ribozyme able to activate the amino acid and transfer it onto the terminal ribose of the tRNA, would be found in any life form, or generated in vitro. Interestingly, recent studies have demonstrated the plausibility of using metals, likely abandoned under primordial conditions, as biomimetic catalysts of the aminoacylation reaction.

Anticodon G recognition by tRNA synthetases mimics the tRNA core.

Ancient mechanisms for nucleotide base recognition in the RNA world are candidates for mimicking by early proteins like tRNA synthetases. In the core of the tRNA, conserved G22 interacts with two internal bases in a complex further stabilized by stacking interactions. This particular tRNA format for G recognition is shown here to be adapted by nine different and even nonhomologous anticodon binding domains (ABDs) of tRNA synthetases, in which amino acid side chains mimic all of the tRNA G22 base interactions. We offer the possibility that mimicking this RNA-based mechanism for guanine recognition is perhaps one of the selective pressures for choosing amino acids for the genetic code.

Crystal structure of human mitochondrial PheRS complexed with tRNA(Phe) in the active "open" state.

Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.0 Å. Unlike bacterial PheRSs, the hmPheRS recognizes C74, the G1-C72 base pair, and the "discriminator" base A73, proposed to contribute to tRNA(Phe) identity in the yeast mitochondrial enzyme. An interaction of the tRNA acceptor stem with the signature motif 2 residues of hmPheRS is of critical importance for the stabilization of the CCA-extended conformation and its correct placement in the synthetic site of the enzyme. The crystal structure of hmPheRS-tRNA(Phe) provides direct evidence that the formation of the complex with tRNA requires a significant rearrangement of the anticodon-binding domain from the "closed" to the productive "open" state. Global repositioning of the domain is tRNA modulated and governed by long-range electrostatic interactions.

Bacterial and eukaryotic phenylalanyl-tRNA synthetases catalyze misaminoacylation of tRNA(Phe) with 3,4-dihydroxy-L-phenylalanine.

Aminoacyl-tRNA synthetases exert control over the accuracy of translation by selective pairing the correct amino acids with their cognate tRNAs, and proofreading the misacylated products. Here we show that three existing, structurally different phenylalanyl-tRNA synthetases-human mitochondrial (HsmtPheRS), human cytoplasmic (HsctPheRS), and eubacterial from Thermus thermophilus (TtPheRS), catalyze mischarging of tRNA(Phe) with an oxidized analog of tyrosine-L-dopa. The lowest level of L-dopa discrimination over the cognate amino acid, exhibited by HsmtPheRS, is comparable to that of tyrosyl-tRNA synthetase. HsmtPheRS and TtPheRS complexes with L-dopa revealed in the active sites an electron density shaping this ligand. HsctPheRS and TtPheRS possessing editing activity are capable of hydrolyzing the exogenous L-dopa-tRNA(Phe) as efficiently as Tyr-tRNA(Phe). However, editing activity of PheRS does not guarantee reduction of the aminoacylation error rate to escape misincorporation of L-dopa into polypeptide chains.

Idiosyncrasy and identity in the prokaryotic Phe-system: crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP.

The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αß)2 heterotetramer: the αß heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N-terminus, A1 and A2 belong to the α-subunit and B1-B8 domains to the ß subunit. The structure of EcPheRS revealed that architecture of four helix-bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil-short helix structural fragments. The N-terminal domain of the α-subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N-terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA(Phe) binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ∼5 Å is required for C-terminal domain B8, and of ∼6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.

Structure of human cytosolic phenylalanyl-tRNA synthetase: evidence for kingdom-specific design of the active sites and tRNA binding patterns.

The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs. We report herein the crystal structure of hcPheRS in complex with phenylalanine at 3.3 A resolution. A novel structural module has been revealed at the N terminus of the alpha subunit. It stretches out into the solvent of approximately 80 A and is made up of three structural domains (DBDs) possessing DNA-binding fold. The dramatic reduction of aminoacylation activity for truncated N terminus variants coupled with structural data and tRNA-docking model testify that DBDs play crucial role in hcPheRS activity.

Structural Aspects of Phenylalanylation and Quality Control in Three Major Forms of Phenylalanyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS)-bacterial (αß)(2), eukaryotic/archaeal cytosolic (αß)(2), and mitochondrial α-is a prominent example of structural diversity within the aaRSs family. Although archaeal/eukaryotic and bacterial PheRSs share common topology of the core domains and the B3/B4 interface, where editing activity of heterotetrameric PheRSs is localized, the detailed investigation of the three-dimensional structures from three kingdoms revealed significant variations in the local design of their synthetic and editing sites. Moreover, as might be expected from structural data eubacterial, Thermus thermophilus and human cytoplasmic PheRSs acquire different patterns of tRNA(Phe) anticodon recognition.

Large-scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl-tRNA synthetase.

Structural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a "closed" form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl-tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.

Intra-protein compensatory mutations analysis highlights the tRNA recognition regions in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases (aaRSs) covalently attach amino acids to their corresponding nucleic acid adapter molecules, tRNAs. The interactions in the tRNA-aaRSs complexes are mostly non-specific, and largely electrostatic. Tracing a way of aaRS-tRNA mutual adaptation throughout evolution offers a clearer view of understanding how aaRS-tRNA systems preserve patterns of tRNA recognition and binding. In this study, we used the compensatory mutations analysis to explore adaptation of aaRSs in respond to random mutations that can occur in the tRNA-recognition area. We showed that the frequency of compensatory mutations among residues that belong to the recognition region is 1.75-fold higher than that of the exposed residues. The highest frequencies of compensatory mutations are observed for pairs of charged residues, wherein one residue is located within the tRNA-recognition area, while the second is placed outside of the area, and contributes to the formation of the aaRS electrostatic landscape. Given charged residues are compensated by buried charge residues in more than 60% of the analyzed mutations. The cytoplasmatic and mitochondrial aaRSs preserve similar patterns of compensatory mutations in the tRNA recognition areas. Moreover, we found that mitochondrial aaRSs demonstrate a significant increase in the frequency of compensatory mutations in the area. Our findings shed light on the physical nature of compensatory mutations in aaRSs, thereby keeping unchanged tRNA-recognition patterns.

Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta-tyrosine.

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA synthetases in this process has been hypothesized, the specific pathway by which ROS-damaged amino acids are incorporated into proteins remains unclear. We provide herein evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases (HsmtPheRS and HsctPheRS, respectively) catalyze direct attachment of m-Tyr to tRNA(Phe), thereby opening the way for delivery of the misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into eukaryotic proteins. Crystal complexes of mitochondrial and bacterial PheRSs with m-Tyr reveal the net of highly specific interactions within the synthetic and editing sites.

The tRNA-induced conformational activation of human mitochondrial phenylalanyl-tRNA synthetase.

All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 A resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the alpha and anticodon binding domain (ABD) of the bacterial beta subunit of (alphabeta)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through approximately 160 degrees) of the ABD upon tRNA binding.

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.