tRNA Research Articles

TRNA modification reprogramming contributes to artemisinin resistance in Plasmodium falciparum

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

Mimicking bacterial infection in male mice changes sperm small RNA profiles and multigenerationally alters offspring behavior and physiology

Paternal pre-conceptual exposures, including stress, diet, substance abuse, parasite infection, and viral immune activation via Poly I:C, have been reported to influence the brains and behavior of offspring through sperm epigenetic changes. However, the effects of paternal (F0) pre-conceptual exposure to bacterial-induced immune activation on the behavior and physiology of F1 and F2 generations remain unexplored. We examined this using C57BL/6J mice. Eight-week-old males (F0) received a single intraperitoneal injection of the bacterial mimetic lipopolysaccharide (LPS: 5 mg/kg) or 0.9 % saline (vehicle control) before mating with naïve females at four weeks post-injection. Comprehensive behavioral assessments were conducted to investigate anxiety, social behaviors, depressive-like behaviors and cognition in both the F1 and F2 generations within the age range of 8 to 14 weeks. Results demonstrated that only female offspring of LPS-exposed fathers exhibited reduced anxiety levels in the light/dark box, large open field, and novelty-suppressed feeding test. These F1 female offspring also exhibited heightened sociability in the 3-chambered social interaction test and a reduced preference for saccharin in the saccharin preference test. Additionally, the F1 male offspring of LPS-challenged males demonstrated an increased total distance traveled in the light/dark box and a longer distance covered in the light zone. They also exhibited diminished preference for social novelty in the 3-chambered social interaction test and an elevated novel arm preference index in the Y-maze. In the F2 generation, male descendants of LPS-treated fathers showed reduced latency to feed in the novelty-suppressed feeding test. Additionally, the F2 generation of LPS-challenged fathers, but not the F1 generation, displayed enhanced immune response in both sexes after an acute LPS immune challenge (5 mg/kg). Analysis of sperm small noncoding RNA profiles from LPS-treated F0 mice revealed significant changes at 4 weeks after administration of LPS. These changes included three microRNAs, eight PIWI-interacting RNAs, and two transfer RNAs, exhibiting significant upregulation (mmu-miR-146a-5p, mmu-piR-27082 and mmu-piR-29102) or downregulation (mmu-miR-5110, mmu-miR-467e-3p, mmu-piR-22583, mmu-piR-23548, mmu-piR-36341, mmu-piR-50293, mmu-piR-16583, mmu-piR-36507, Mus_musculus_tRNA-Ile-AAT-2-1 and Mus_musculus_tRNA-Tyr-GTA-1-1). Additionally, we detected 52 upregulated small noncoding RNAs (including 9 miRNAs, 41 piRNAs, and 2 tRNAs) and 7 downregulated small noncoding RNAs (3 miRNAs, 3 piRNAs, and 1 tRNA) in the sperm of F1 offspring from LPS-treated males. These findings provide compelling evidence for the involvement of epigenetic mechanisms in the modulation of brain function and immunity, and associated behavioral and immunological traits, across generations, in response to bacterial infection.

Activity reconstitution of Kre33 and Tan1 reveals a molecular ruler mechanism in eukaryotic tRNA acetylation.

RNA acetylation is a universal post-transcriptional modification that occurs in various RNAs. Transfer RNA (tRNA) acetylation is found at position 34 (ac4C34) in bacterial tRNAMet and position 12 (ac4C12) in eukaryotic tRNASer and tRNALeu. The biochemical mechanism, structural basis and functional significance of ac4C34 are well understood; however, despite being discovered in the 1960s and identification of Kre33/NAT10 and Tan1/THUMPD1 as modifying apparatuses, ac4C12 modification activity has never been reconstituted for nearly six decades. Here, we successfully reconstituted the ac4C12 modification activity of yeast Kre33 and Tan1. Biogenesis of ac4C12 is primarily dependent on a minimal set of elements, including a canonical acceptor stem, the presence of the 11CCG13 motif and correct D-arm orientation, indicating a molecular ruler mechanism. A single A13G mutation conferred ac4C12 modification to multiple non-substrate tRNAs. Moreover, we were able to introduce ac4C modifications into small RNAs. ac4C12 modification contributed little to tRNA melting temperature and aminoacylation in vitro and in vivo. Collectively, our results realize in vitro activity reconstitution, delineate tRNA substrate selection mechanism for ac4C12 biogenesis and develop a valuable system for preparing acetylated tRNAs as well as non-tRNA RNA species, which will advance the functional interpretation of the acetylation in RNA structures and functions.

An Integrative Study of Scrophularia takesimensis Nakai in an Ovalbumin-Induced Murine Model of Asthma: The Effect on T Helper 2 Cell Activation.

Scrophularia have traditionally been used as herbal medicines to treat neuritis, sore throats, and laryngitis. In particular, S. takesimensis, a Korean endemic species with restricted distribution on Ulleung Island, holds significant resource and genetic value. However, its pharmacological properties have not been thoroughly evaluated. Thus, we provide detailed morphological characteristics and genomic information for S. takesimensis in this study. Moreover, its pharmacological activity was evaluated in an ovalbumin-induced asthma rat model, using extracts of S. takesimensis roots (100 or 200 mg/kg). The distinguishing features of S. takesimensis from related species include the presence or absence of stem wings, leaf shape, and habitat. The chloroplast (cp) genome of this species is 152,420 bp long and exhibits a conserved quadripartite structure. A total of 114 genes were identified, which included 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The gene order, content, and orientation of the S. takesimensis cp genome was highly conserved and consistent with the general structure observed in S. buergeriana and S. ningpoensis cp genomes. Confirming the anti-inflammatory effects of S. takesimensis extract (STE) using an established mouse model of ovalbumin-induced asthma, we observed reduced asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and suppression of T helper 2 (Th2) cell. Furthermore, STE treatment reduced Th2 cell activation and differentiation. This study underscores the medicinal value of S. takesimensis. The importance of preserving S. takesimensis was revealed and crucial insights were provided for further research on its utilization as a medicinal resource.

Altered expression of transfer RNAs and their possible roles in brain white matter injury.

Transfer RNAs (tRNAs) can regulate cell behavior and are associated with neurological disorders. Here, we aimed to investigate the expression levels of tRNAs in oligodendrocyte precursor cells (OPCs) and their possible roles in the regulation of brain white matter injury (WMI). Newborn Sprague-Dawley rats (postnatal day 5) were used to establish a model that mimicked neonatal brain WMI. RNA-array analysis was performed to examine the expression of tRNAs in OPCs. psRNAtarget software was used to predict target mRNAs of significantly altered tRNAs. Gene ontology (GO) and KEGG were used to analyze the pathways for target mRNAs. Eighty-nine tRNAs were changed after WMI (fold change absolute ≥1.5, P < 0.01), with 31 downregulated and 58 upregulated. Among them, three significantly changed tRNAs were identified, with two being significantly increased (chr10.trna1314-ProTGG and chr2.trna2771-ProAGG) and one significantly decreased (chr10.trna11264-GlyTCC). Further, target mRNA prediction and GO/KEGG pathway analysis indicated that the target mRNAs of these tRNAs are mainly involved in G-protein coupled receptor signaling pathways and beta-alanine metabolism, which are both related to myelin formation. In summary, the expression of tRNAs in OPCs was significantly altered after brain WMI, suggesting that tRNAs may play important roles in regulating WMI. This improves the knowledge about WMI pathophysiology and may provide novel treatment targets for WMI.

Abstract LB171: First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer

Abstract METTL1 is an RNA methyltransferase that catalyzes N7-methylation on guanine at position 46 (m7G46) in a subset of transfer RNAs (tRNAs). These tRNAs are stabilized by m7G46, and this increases the translation of mRNAs containing the cognate codons. METTL1 is often over-expressed in cancer tissue, and its gene locus is frequently amplified in specific tumor types. Recent studies have shown that reducing METTL1 expression leads to tumor growth inhibition highlighting this enzyme as a potential novel target for anti-cancer drugs. We optimized small molecule inhibitors of METTL1 from a HTS hit using structure-guided medicinal chemistry. Representative compounds of two distinct chemical series exhibit potent in vitro METTL1 inhibition at low nanomolar concentrations while displaying exquisite selectivity over other RNA and protein methyltransferases. The effects of inhibiting METTL1 were measured by cellular mechanistic assays and modification-induced misincorporation tRNA sequencing (mim-tRNAseq). As expected, pharmacological inhibition led to decreased m7G46 methylation, lower levels of a subgroup of tRNAs and reduced cancer cell proliferation.The efficacy of METTL1 inhibition in a panel of cancer cell lines representing a wide range of solid and hematologic tumor types was determined. Cell proliferation was impaired in a subset of cell lines originating from various cancer types, including lymphoma, sarcoma, esophageal squamous cell carcinoma, glioblastoma, colorectal and lung adenocarcinoma. Importantly, viability was not affected in non-transformed cell lines, indicating a highly selective mechanism of action. The phenotypic consequences of METTL1 inhibition in selected cell lines were further investigated by flow cytometry and western blotting. Cell lines sensitive to METTL1 inhibition displayed reduced cell cycle progression and lowered expression of cell cycle regulators. RNA sequencing and proteome analyses identified molecular changes at both transcript and protein level. METTL1 inhibition led to induction of ATF4 expression and activation of the integrated stress response, as well as global changes to the translation machinery. Studies on cell-line derived xenograft or syngeneic in vivo mouse models were conducted to assess tumor growth inhibition after oral or intraperitoneal administration of the potent METTL1 small molecule inhibitor STM9005. Inhibition of METTL1 by this compound reduced tumor growth in both immune-deficient and immune-competent mouse models, effects accompanied by dose-dependent changes in pharmacodynamic biomarkers. Here, we describe the discovery of first-in-class inhibitors of METTL1 tRNA methyltransferase. To our knowledge, our data provide the first evidence that pharmacological inhibition of a tRNA methyltransferase reduces tumor growth in vivo. Citation Format: Alexandra Sapetschnig, Beth Thomas, Eliza Yankova, Harry Fischl, Aleksandra Azevedo, Sarah Bucknell, Richard Fosbeary, Sapphire Sawyer, Sian Evans, Carmen Livi, Byron Andrews, Jack Rogan, Natalie Webster, Matthew Fyfe, Konstantinos Tzelepis, Oliver Rausch. First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB171.

New insights into the mechanism of electrotransfer of small nucleic acids

RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA and DNA. Although the mechanism of electrotransfer of small nucleic acids has been studied previously, too little is known about the potential effects of significantly larger pDNA on this process. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism, we have shown that the electrotransfer of oligonucleotides and siRNA is driven by both electrophoretic forces and diffusion after EP, followed by subsequent entry into the nucleus within 5 min after treatment. The study also revealed that the efficiency of siRNA electrotransfer decreases in response to an increase in pDNA concentration. Overall, the study provides new insights into the mechanism of electrotransfer of small nucleic acids which may have broader implications for the future application of RNAi-based strategies.

The complete chloroplast genome of Keteleeria evelyniana Mast var. pendula Hsüeh (Pinaceae), a species with extremely small populations in China

Keteleeria evelyniana Mast var. pendula Hsüeh, a typical plant species of extremely small population, is faced to be endangered. The complete chloroplast (cp) genome of K. evelyniana var. pendula has been assembled and annotated for the first time in this study. The complete genome in length was found to be 117,139 bp. The genome annotation revealed a total of 118 genes, including 34 transfer RNA (tRNA) genes, 4 ribosomal RNA (rRNA) genes, and 80 protein-coding genes. The maximum-likelihood phylogenetic tree supported that K. evelyniana var. pendula, K. fortune, K. evelyniana, and K. davidiana are clustered in one branch. This complete chloroplast genome helped us to understand the evolution of K. evelyniana var. pendula. These results laid the foundation for future studies on the conservation of this species.

Characterization of the complete chloroplast genome sequence of Camellia tetracocca (Theaceae)

Camellia tetracocca H.T. Chang 1981 is an important wild relative of cultivated tea plants. Its leaves are widely used by local people to make tea, showing great economic and breeding values. We here report the complete chloroplast genome of C. tetracocca using Illumina sequencing technology. The complete chloroplast genome of C. tetracocca is 157,026 bp in length, and structurally contains a pair of inverted repeat regions (IRa and IRb, 26,052 bp) separated by a large single-copy region (LSC, 86,669 bp) and a small single-copy region (SSC, 18,253 bp). It is composed of 131 predicted genes, including 86 protein-coding genes, 37 transfer RNA genes, and eight ribosomal RNA genes. The overall GC content is 37.31%. Phylogenetic analysis among four Camellia species and 11 other close species reveals a close relationship between C. tetracocca and C. gymnogyna.

Characterization of the complete mitochondrial genome of the Libelloides sibiricus (Neuroptera, Ascalaphidae)

Libelloides sibiricus (Eversmann, 1850) is widely distributed in China, Korea and eastern Russia. To date, few studies have been conducted on this species, with the exception of morphological taxonomy studies. In this study, we sequenced the complete mitochondrial genome (mitogenome) of Libelloides sibiricus, which is 15,811 bp in length, with an overall A + T content of 74.8%, encoding 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a control region. The gene arrangement and components of L. sibiricus are identical to those of most other Neuropteran species. TAA is utilized as the termination codon for most PCGs and TAG for nd1, however, nd6 and atp6 used the incomplete termination codon TA- and cox1, cox2, nd5, cytb had termination codons consisting of only T–. In addition, we selected all known 59 species of Neuroptera from NCBI, and used Sialis hamata, Sialis melania, Sialis longidens and Sialis jiyuni (Megaloptera: Sialidae) as the outgroup. Phylogenetic analysis suggested that the mitogenome of L. sibiricus was the most closely related to L. macaronius and all the owlflies formed the monophyletic group within the superfamily Myrmeleontoidea.

The complete mitochondrial genome of freshwater gammarid Gammarus nipponensis (Crustacea: Amphipoda: Gammaridae)

This study presents the complete mitochondrial genome sequence of Gammarus nipponensis, a freshwater crustacean found in the western regions of Honshu, Shikoku and Kyushu in Japan. The entire genome is 16,429 bp in length, encoding a standard set of 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes, as well as the putative control regions. The mitochondrial genome of G. nipponensis is characterized by a high concentration of A and T nucleotides (67.1%). Notably, the mitogenome contains long TATTTTA repeats in the control region 2 at 686 bp long. This newly available genome information will be useful for studying the evolutionary relationships within the genus Gammarus and for understanding diversification among G. nipponensis populations.

The complete mitogenome of the pond wolf spider Pardosa pseudoannulata, with phylogenetic implications for the Lycosidae

The pond wolf spider Pardosa pseudoannulata Bösenberg & Strand, 1906 (Araneae: Lycosidae) is an important predator of agricultural pests in southern, eastern and southeastern Asia. Here, we report the complete mitogenome of this spider reconstructed from Illumina sequencing data. The circular mitogenome length is 14,533 bp with the nucleotide composition A (33.3%), C (8.2%), G (15.2%), and T (43.3%). The P. pseudoannulata mitogenome comprises 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. Phylogenetic analyses of Lycosidae mitogenomes supported the monophyly of the subfamily Pardosinae and the two genera Pardosa and Alopecosa, and indicated the polyphyly of the subfamily Lycosinae and the paraphyly of its type genus Lycosa. In this study, P. pseudoannulata is the closest relative to P. pusiola. These results provide useful genetic information for future studies on the diversity, phylogeny, and evolution for wolf spiders.

The complete mitochondrial genome of the ice krill Euphausia crystallorophias Holt & Tattersall, 1906 (Euphausiacea, Euphausiidae), from the Ross Sea, Antarctica

The mitogenome of Euphausia crystallorophias collected from the Ross Sea Region Marine Protected Area (RSR MPA) is described for the first time. The assembled mitogenome was 17,291 bp in length and consisted of two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), and noncoding regions, all of which were identical to those of other euphausiid species. The most common start codon for the 13 PCGs was ATG, and the most common termination codon was TAA. The overall G + C content was 33.2% in the heavy strand. Euphausia crystallorophias was sister to E. superba in the phylogenetic analysis. The mitogenome of E. crystallorophias provided significant DNA molecular data for further identification and phylogenetic analysis within the euphausiids.

The complete chloroplast genome sequence and phylogenetic analysis of Strobilanthes dalzielii (W.W.Sm.) Benoist 1935 (Acanthaceae)

Strobilanthes dalzielii of Acanthaceae is an herb species with potentially extensive applications for its pharmaceutical and ornamental values. Due to taxonomic complications and limited genetic information, the structural characteristics, and phylogenetic relationships of the S. dalzielii chloroplast genome were assembled and characterized here for the first time. The complete chloroplast genome of S. dalzielii was 144,580 bp in length. The genome is quadripartite in structure and consists of a large single-copy region (92,137 bp) and a small single-copy region (17,669 bp), which are separated by a pair of inverted repeats (each 17,387 bp). A total of 125 genes were annotated, including 80 protein-coding, 37 transfer RNA, and eight ribosomal RNA genes. The overall GC content was 36.4%. Phylogenetic analysis based on the complete chloroplast genome sequence of 21 taxa within the tribe Ruellieae of Acanthaceae using the maximum likelihood and Bayesian inference methods revealed that Strobilanthes diverged after Ruellia; S. dalzielii is closely related to S. tonkinensis. The genomic data obtained from this study will serve as valuable information to the species delimitation and genetic classification of Strobilanthes.

The complete mitochondrial genome of Hypsipetes amaurotis (Passeriformes: Pycnonotidae)

The brown-eared bulbul (Hypsipetes amaurotis) is a medium-sized songbird native to East Asia and characterized by its prominent reddish-brown ear-coverts. Previous studies on it have primarily been from the taxonomic and morphological aspects, with limited research in the realm of molecular biology. In this study, we sequenced and annotated the complete mitochondrial genome of H. amaurotis, which was the first reported complete mitogenome of the genus Hypsipetes. The mitogenome of H. amaurotis is 17,871 bp in length and was predicted to encode 37 typical mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs). Specifically, this mitogenome contains two D-loop control regions that are of similar length and sequencing pattern. A total of 8 Pycnonotidae and six outgroup taxa were used to determine the phylogenetic placement with two methods: Maximum Likelihood Approximation (IQ-TREE) and Bayesian inference (MrBayes). Our findings reveal that H. amaurotis is phylogenetically closely related to Ixos mcclellandii. The outcomes are generally consistent with the phylogenetic trees constructed in previous studies. The data gathered from this research provides valuable insights for future genomic investigations into the evolution, ecology, and conservation of this species.

The complete mitochondrial genome of Rhipicephalus haemaphysaloides and its phylogenetic analysis

We conducted an analysis of the complete mitochondrial genome of Rhipicephalus haemaphysaloides, a tick species known for transmitting various bacteria and viruses. The mitochondrial genome of R. haemaphysaloides has a length of 14,739 bp and consists of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 2 control regions. By utilizing the maximum likelihood method, we established the phylogenetic relationship among R. haemaphysaloides and other species within the Rhipicephalus genus of the Ixodidae family. This analysis revealed that R. haemaphysaloides and other Rhipicephalus species belong to the same clade, further affirming the taxonomic placement of R. haemaphysaloides within the Rhipicephalus genus. Furthermore, we compared the mitochondrial genomes of R. haemaphysaloides isolates from Changning, Yunnan Province, China, with isolates from Yangxin, Ganzhou, and Yingtan, Hubei Province, China. In summary, our investigation offers genetic proof endorsing the taxonomic categorization and phylogenetic placement of Ixodidae by assessing the entire mitochondrial genome of R. haemaphysaloides.

Characterization of the complete chloroplast genome sequence of &lt;i&gt;Hypericum petiolulatum&lt;/i&gt; (Hypericaceae)

&lt;i&gt;Hypericum&lt;/i&gt; L. (Hypericaceae) is one of the best-selling herbal medicines in the world comprising ca. 500 species of herbs, shrubs, and small trees. &lt;i&gt;Hypericum petiolulatum&lt;/i&gt; Hook. f. &amp; Thomson ex Dyer is widely distributed in China, Vietnam, Myanmar, Nepal, India, Malaysia, and Bhutan and is used as a traditional herb to treat hemoptysis and inflammation. In this study, we sequenced and assembled the complete chloroplast (cp) genome of &lt;i&gt;H. petiolulatum&lt;/i&gt;. The complete plastome of &lt;i&gt;H. petiolulatum&lt;/i&gt; was 136,105 bp in length, with a large single copy region (LSC) of 93,709 bp, a small single copy region (SSC) of 11088 bp and two identical inverted repeats (IRs) of 15,654 bp. The overall GC content of the plastome was 37.0%, while GC contents of the LSC, SSC, and each IR were 35.5%, 31.0%, and 43.8%, respectively. In addition, 116 genes consisting of 76 protein-coding genes, six ribosomal RNA genes, and 34 transfer RNA genes were identified. A phylogenetic analysis of 14 taxa inferred based on cp genome sequences revealed a close relationship between &lt;i&gt;H. petiolulatum&lt;/i&gt; and &lt;i&gt;H. perforatum&lt;/i&gt;. The complete cp genome sequence of &lt;i&gt;H. petiolulatum&lt;/i&gt; reported in this paper will facilitate population and phylogenomics studies of this medicinal plant group.

The first complete chloroplast genome of &lt;i&gt;Polygonatum hirtellum&lt;/i&gt;: Genome features and phylogenetic relationships

&lt;i&gt;Polygonatum hirtellum&lt;/i&gt; is a perennial herb within the family Asparagaceae. In the present study, the chloroplast (cp) genome of &lt;i&gt;P. hirtellum&lt;/i&gt; is reported for the first time, and its phylogenetic relationships are also investigated. The findings revealed that the cp genome was 155,697 bp and exhibited a typical quadripartite structure, consisting of a large single copy (84,468 bp), a small single copy (18,491 bp), and a pair of invert repeat sequences (26,369 bp). The GC content of the cp genomes amounted to 37.7%; 130 genes were identified, consisting of 84 protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. A phylogenetic analysis based on the cp genome and coding sequence showed that &lt;i&gt;P. hirtellum&lt;/i&gt; was a member of section &lt;i&gt;Verticillata&lt;/i&gt; and could be distinguished from other species of the genus &lt;i&gt;Polygonatum&lt;/i&gt; used in the analysis.

Analysis of the Overlength Main Noncoding Region in Metacarcinus magister (Decapoda: Brachyura) and a Phylogenetic Study of the Cancroidea Species.

Complete mitochondrial genomes (mitogenomes) can provide important information regarding the molecular evolution and phylogenetic relationships of marine invertebrates, especially in Brachyura. Only one Cancroidea species of mitogenomes has been sequenced before; in this research, the mitogenomic characteristics of Metacarcinus magister (Cancridae: Cancroidea) are newly studied. The length of the M. magister mitogenome was 48,820 bp, and it contained the typical 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. We performed a series of analyses on the characteristics of the mNCR of M. magister. The phylogenetics, life circumstances, and selective pressures were all analyzed to explain the formation of this length, which revealed the length of the M. magister mitogenome to be approximately three times greater than the normal length of Brachyuran mitogenomes. Phylogenetic analyses based on a dataset of 215 Decapodan mitogenomes indicated that all Eriphioidea crabs were clustered together as a group. Moreover, the rearrangement mechanism of the Cancroidea species was predicted to provide stronger evidence for the phylogenetic analysis. In general, the results obtained in this study will contribute to a better understanding of the cause of the unusual length of the M. magister mitogenome and provide new insights into the phylogeny of Brachyura.

Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm2C), respectively, in the anticodon of tRNAIle to decipher AUA codon. L and agm2C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAsIle recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N6-threonylcarbamoyladenosine (ct6A), another tRNA modification, and revealed a molecular basis how ct6A contributes to efficient decoding.