Abstract Background As in eukaryotes, precursor transfer RNAs in Archaea often contain introns that are removed in tRNA maturation. Two unrelated archaeal species display unique pre-tRNA processing complexity in the form of split tRNA genes, in which two to three segments of tRNAs are transcribed from different loci, then trans-spliced to form a mature tRNA. Another rare type of pre-tRNA, found only in eukaryotic algae, is permuted, where the 3' half is encoded upstream of the 5' half, and must be processed to be functional. Results Using an improved version of the gene-finding program tRNAscan-SE, comparative analyses and experimental verifications, we have now identified four novel trans-spliced tRNA genes, each in a different species of the Desulfurococcales branch of the Archaea: tRNAAsp(GUC) in Aeropyrum pernix and Thermosphaera aggregans, and tRNALys(CUU) in Staphylothermus hellenicus and Staphylothermus marinus. Each of these includes features surprisingly similar to previously studied split tRNAs, yet comparative genomic context analysis and phylogenetic distribution suggest several independent, relatively recent splitting events. Additionally, we identified the first examples of permuted tRNA genes in Archaea: tRNAiMet(CAU) and tRNATyr(GUA) in Thermofilum pendens, which appear to be permuted in the same arrangement seen previously in red alga. Conclusions Our findings illustrate that split tRNAs are sporadically spread across a major branch of the Archaea, and that permuted tRNAs are a new shared characteristic between archaeal and eukaryotic species. The split tRNA discoveries also provide new clues to their evolutionary history, supporting hypotheses for recent acquisition via viral or other mobile elements.

N ¹-methyladenosine (m¹A), N ³-methylcytidine (m³C), and N ¹-methylguanosine (m¹G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m¹A, m³C, or m¹G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Small RNAs have the potential to store a secondary layer of labile biological information in the form of modified nucleotides. Emerging evidence has shown that small RNAs including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and tRNA-derived small RNAs (tsRNAs) harbor a diversity of RNA modifications. These findings highlight the importance of RNA modifications in the modulation of basic properties such as RNA stability and other complex physiological processes involved in stress responses, metabolism, immunity, and epigenetic inheritance of environmentally acquired traits, among others. High-resolution, high-throughput methods for detecting, mapping and screening these small RNA modifications now provide opportunities to uncover their diagnostic potential as sensitive disease markers.

Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative analyses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, reveals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illuminated some of the genome dynamics potentially shared with other species in this genus.

COVID-19 increased the prevalence of clinically significant anxiety in the United States. To investigate contributing factors we analyzed anxiety, reported online via monthly Generalized Anxiety Disorders-7 (GAD-7) surveys between April 2020 and May 2022, in association with self-reported worry about the health effects of COVID-19, economic difficulty, personal COVID-19 experience, and subjective social status. 333,292 anxiety surveys from 50,172 participants (82% non-Hispanic white; 73% female; median age 55, IQR 42-66) showed high levels of anxiety, especially early in the pandemic. Anxiety scores showed strong independent associations with worry about the health effects of COVID-19 for oneself or family members (GAD-7 score +3.28 for highest vs. lowest category; 95% confidence interval: 3.24, 3.33; p<0.0001 for trend) and with difficulty paying for basic living expenses (+2.06; 1.97, 2.15, p<0.0001) in multivariable regression models after adjusting for demographic characteristics, COVID-19 case rates and death rates, and personal COVID-19 experience. High levels of COVID-19 health worry and economic stress were each more common among participants reporting lower subjective social status, and median anxiety scores for those experiencing both were in the range considered indicative of moderate to severe clinical anxiety disorders. In summary, health worry and economic difficulty both contributed to high rates of anxiety during the first two years of the COVID-19 pandemic in the US, especially in disadvantaged socioeconomic groups. Programs to address both health concerns and economic insecurity in vulnerable populations could help mitigate pandemic impacts on anxiety and mental health.