Preeclampsia is a major contributor to maternal and fetal morbidity and mortality. The disorder can be classified into early- and late-onset subtypes, both of which evolve in two stages. The first stage comprises the development of pre-clinical, utero-placental malperfusion. Early and late utero-placental malperfusion have different causes and time courses. Early-onset preeclampsia (20 % of cases) is driven by dysfunctional placentation in the first half of pregnancy. In late-onset preeclampsia (80 % of cases), malperfusion is a consequence of placental compression within the confines of a limited uterine cavity. In both subtypes, the malperfused placenta releases stress signals into the maternal circulation. These stress signals trigger onset of the clinical syndrome (the second stage). Small RNA molecules, which are implicated in cellular stress responses in general, may be involved at different stages. Micro RNAs contribute to abnormal trophoblast invasion, immune dysregulation, angiogenic imbalance, and syncytiotrophoblast-derived extracellular vesicle signalling in preeclampsia. Transfer RNA fragments are placental signals known to be specifically involved in cell stress responses. Disorder-specific differences in small nucleolar RNAs and piwi-interacting RNAs have also been reported. Here, we summarise key small RNA advances in preeclampsia pathogenesis. We propose that existing small RNA classifications are unhelpful and that non-biased assessment of RNA expression, incorporation of non-annotated molecules and consideration of chemical modifications to RNAs may be important in elucidating preeclampsia pathogenesis.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Mitochondrial transfer RNA mutation is one of the most important causes of hereditary hearing loss in humans. Mitochondrial transfer RNASer (UCN) gene is another hot spot for mutations associated with non-syndromic hearing loss, besides the 12S ribosomal RNA gene. In this study, we assessed the clinical phenotype and the molecular characteristics of two Chinese families with non-syndromic hearing loss. Mutational analysis revealed that 7445A > G and 7510T > C mutations in the mitochondrial transfer RNASer (UCN) gene were the molecular etiology of Family 1 and Family 2, respectively. However, the clinical and genetic characteristics of the two families carrying the above mutations in the transfer RNASer (UCN) gene exhibited a variable expression of hearing loss and an incomplete penetrance. Sequencing analysis of the complete mitochondrial genome showed the presence of transfer RNATrp 5568A > G and NADH-ubiquinone oxidoreductase chain 4 11696G > A mutations in Family 1. The mitochondrial haplotype analysis showed that the two families belonged to Asian D4 and M80\'D haplotypes, respectively, and no pathogenic variations were found in the nuclear genes. To our knowledge, our study is the first to report 7445A > G and 7510T > C mutations in the mitochondrial transfer RNASer (UCN) gene, in multi-generation non-syndromic hearing loss pedigrees from China. Our study suggests that 5568A > G and 11696G > A mutations may enhance the penetrance of hearing loss in Chinese Family 1, while mitochondrial haplotypes and known nuclear genes may not be modifiers for the phenotypic expression of 7445A > G and 7510T > C mutations in these Chinese families.

UNASSIGNED: The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterized. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the layer of placenta in direct contact with maternal blood. Such stress alters the content and increases the release of syncytiotrophoblast extracellular vesicles (STB-EVs) into the maternal circulation. We have previously shown 5\'-tRNA fragments (5\'-tRFs) constitute the majority of small RNA in STB-EVs in healthy pregnancy. 5\'-tRFs are produced in response to stress. We hypothesized STB-EV 5\'-tRF release might change in preeclampsia.
UNASSIGNED: We perfused placentas from 8 women with early-onset preeclampsia and 6 controls, comparing small RNA expression in STB-EVs. We used membrane-affinity columns to isolate maternal plasma vesicles and investigate placental 5\'-tRFs in vivo. We quantified 5\'-tRFs from circulating STB-EVs using a placental alkaline phosphatase immunoassay. 5\'-tRFs and scrambled RNA controls were added to monocyte, macrophage and endothelial cells in culture to investigate transcriptional responses.
UNASSIGNED: 5\'-tRFs constitute the majority of small RNA in STB-EVs from both preeclampsia and normal pregnancies. More than 900 small RNA fragments are differentially expressed in preeclampsia STB-EVs. Preeclampsia-dysregulated 5\'-tRFs are detectable in maternal plasma, where we identified a placentally derived load. 5\'-tRF-Glu-CTC, the most abundant preeclampsia-upregulated 5\'-tRF in perfusion STB-EVs, is also increased in preeclampsia STB-EVs from maternal plasma. 5\'-tRF-Glu-CTC induced inflammation in macrophages but not monocytes. The conditioned media from 5\'-tRF-Glu-CTC-activated macrophages reduced eNOS (endothelial NO synthase) expression in endothelial cells.
UNASSIGNED: Increased release of syncytiotrophoblast-derived vesicle-bound 5\'-tRF-Glu-CTC contributes to preeclampsia pathophysiology.

The endoribonuclease RNase P is responsible for tRNA 5\' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

OBJECTIVE: Ribonuclease P RNA component H1 (RPPH1) is a long non-coding RNA (lncRNA) associated with cancer progression. Higher RPPH1 expression in breast and cervical cancer samples than that in normal tissues were observed through the lncRNASNP2 database; therefore, silencing RPPH1 expression might be a potential strategy for cancer treatments, even though RPPH1 is also an RNA subunit of ribonuclease P involved in processing transfer RNA (tRNA) precursors and the effect of RPPH1 knockdown is not yet fully understood.
METHODS: Differentially expressed genes (DEGs) were identified through RNA sequencing in each shRNA-transfected RPPH1 knockdown MDA-MB-231, RPPH1 knockdown HeLa cell, and respective control cells, then the gene ontology enrichment analysis was performed by IPA and MetaCore database according to these DEGs, with further in vitro experiments validating the effect of RPPH1 silencing in MDA-MB-231 and HeLa cells.
RESULTS: Hundreds of down-regulated DEGs were identified in RPPH1 knockdown MDA-MB-231 and HeLa cells while bioinformatics analysis revealed that these genes were involved in pathways related to immune response and cancerogenesis. Compared to mock- and vector-transfected cells, the production of mature tRNAs, cell proliferation and migration capacity were inhibited in RPPH1-silenced HeLa and MDA-MB-231 cells. Additionally, RPPH1 knockdown promoted G1 cell cycle arrest mainly through the down-regulation of cyclin D1, although glycolytic pathways were only affected in RPPH1 knockdown HeLa cells but not MDA-MB-231 cells.
CONCLUSIONS: This study demonstrated that knockdown RPPH1 affected tRNA production, cell proliferation and metabolism. Our findings might provide insight into the role of RPPH1 in tumor development.

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA (mRNA) codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codons\' decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we identified that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.

Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA m1A22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (m1A), 3-methylcytosine (m3C), 3-methyluridine (m3U), 1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G).

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

Megalurothrips usitatus (Bagnall, 1913) (Thysanoptera: Thripidae) is a widely distributed pest in Asia that primarily affects the production of snap beans and cowpea. The complete mitochondrial genome of Megalurothrips usitatus has been sequenced and annotated in this study, which is 17,209 bp long and contains 13 protein-coding genes (PCGs), two rRNAs, and 22 tRNA genes. Most of the protein-coding genes (PCGs) start with ATG except ND4 using TTG. Meanwhile, eight PCGs stop with TAA, four PCGs have an incomplete stop codon, and the gene Cytb ends with TAG. Phylogenetic analysis showed that M. usitatus is closely related to Frankliniella intonsa and F. occidentalis, providing a basis for the study of the mitochondrial evolution of Thripinae.