Pan-archaeal analysis of C/D box sRNA biogenesis and methylation targets

Post-transcriptional modifications of RNA molecules occur in all three domains of life and influence RNA stability and functionality. The most numerous modifications are 2'-O-methylations at the ribose moiety and pseudouridylations. In archaea, modified bases are abundant in ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). The introduction of both modifications is guided by small RNAs that are incorporated into ribonucleoprotein complexes (RNPs). 2'-O-methylations are guided by C/D box sRNAs in archaea. C/D box sRNAs are characterized by the conserved sequence elements boxC/C' (consensus sequence: RUGAUGA) and boxD/D' (consensus sequence: CUGA). Upon C/D box sRNA folding, both sequence elements base-pair (boxC with boxD and boxC' with boxD'), which results in the formation of two kink-turn motifs that are stabilized by binding of the protein L7Ae. The sequences between the two kink-turn elements show complementarity to the sequences of the target RNA and thereby serve as guide sequences that determine the sites of 2'-O-methylation. The modifications are introduced site-specifically at the nucleotide of the target RNA that is complementary to the fifth nucleotide upstream of the boxD/D' motif by the methyltransferase fibrillarin. Based on the guide sequences, C/D box sRNA targets of seven archaea were predicted and mapped onto the consensus structure of the 16S and 23S rRNA. Conserved methylation hotspots were observed in ancient core regions of the rRNAs that are important for ribosome integrity and functionality and that are not protected by ribosomal proteins. Therefore, the modifications might contribute to the folding, structural stabilization and function of the rRNAs. The biogenesis of archaeal C/D box sRNAs is largely unknown as independent promoters cannot be identified for the majority of the C/D box sRNA genes. The analysis of C/D box sRNA genes in six archaeal model organisms revealed diverse genetic contexts, providing opportunities for transcription without the necessity of an independent promoter. C/D box sRNA genes localize e.g. in the 5' or 3'-UTR of flanking protein-coding regions and polycistronic C/D box sRNA transcripts exist. Plasmid-based C/D box sRNA in vivo analyses were performed in Sulfolobus acidocaldarius in which C/D box sRNA genes variants with their native or random upstream and downstream sequences were used to identify C/D box sRNA stabilization and maturation requirements. The analyses revealed that the maturation of C/D box sRNAs occurs independently of the upstream and downstream sequences. The integrity of the k-turn is important for C/D box sRNA stability. Archaeal C/D box sRNAs exhibit a transcriptional plasticity and their maturation is suggested to include the action of unspecific exoribonucleases. Complete degradation might be prevented by co-transcriptional L7Ae binding or complete C/D box sRNP assembly. Circular forms of the C/D box sRNAs were identified in several hyperthermophilic archaea and the circularization reaction should protect RNAs from degradation. C/D box sRNA gene upstream and downstream sequences were shown not to be required for circularization but the responsible RNA ligase remains to be identified. Thus, this thesis provides insights into the transcription and maturation of archaeal C/D box sRNAs and highlights conserved 2'-O-methylation pattern in archaeal rRNAs

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Pan-archaeal analysis of C/D box sRNA biogenesis and methylation targets

Post-transcriptional modifications of RNA molecules occur in all three domains of life and influence RNA stability and functionality. The most numerous modifications are 2'-O-methylations at the ribose moiety and pseudouridylations. In archaea, modified bases are abundant in ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). The introduction of both modifications is guided by small RNAs that are incorporated into ribonucleoprotein complexes (RNPs). 2'-O-methylations are guided by C/D box sRNAs in archaea. C/D box sRNAs are characterized by the conserved sequence elements boxC/C' (consensus sequence: RUGAUGA) and boxD/D' (consensus sequence: CUGA). Upon C/D box sRNA folding, both sequence elements base-pair (boxC with boxD and boxC' with boxD'), which results in the formation of two kink-turn motifs that are stabilized by binding of the protein L7Ae. The sequences between the two kink-turn elements show complementarity to the sequences of the target RNA and thereby serve as guide sequences that determine the sites of 2'-O-methylation. The modifications are introduced site-specifically at the nucleotide of the target RNA that is complementary to the fifth nucleotide upstream of the boxD/D' motif by the methyltransferase fibrillarin. Based on the guide sequences, C/D box sRNA targets of seven archaea were predicted and mapped onto the consensus structure of the 16S and 23S rRNA. Conserved methylation hotspots were observed in ancient core regions of the rRNAs that are important for ribosome integrity and functionality and that are not protected by ribosomal proteins. Therefore, the modifications might contribute to the folding, structural stabilization and function of the rRNAs. The biogenesis of archaeal C/D box sRNAs is largely unknown as independent promoters cannot be identified for the majority of the C/D box sRNA genes. The analysis of C/D box sRNA genes in six archaeal model organisms revealed diverse genetic contexts, providing opportunities for transcription without the necessity of an independent promoter. C/D box sRNA genes localize e.g. in the 5' or 3'-UTR of flanking protein-coding regions and polycistronic C/D box sRNA transcripts exist. Plasmid-based C/D box sRNA in vivo analyses were performed in Sulfolobus acidocaldarius in which C/D box sRNA genes variants with their native or random upstream and downstream sequences were used to identify C/D box sRNA stabilization and maturation requirements. The analyses revealed that the maturation of C/D box sRNAs occurs independently of the upstream and downstream sequences. The integrity of the k-turn is important for C/D box sRNA stability. Archaeal C/D box sRNAs exhibit a transcriptional plasticity and their maturation is suggested to include the action of unspecific exoribonucleases. Complete degradation might be prevented by co-transcriptional L7Ae binding or complete C/D box sRNP assembly. Circular forms of the C/D box sRNAs were identified in several hyperthermophilic archaea and the circularization reaction should protect RNAs from degradation. C/D box sRNA gene upstream and downstream sequences were shown not to be required for circularization but the responsible RNA ligase remains to be identified. Thus, this thesis provides insights into the transcription and maturation of archaeal C/D box sRNAs and highlights conserved 2'-O-methylation pattern in archaeal rRNAs

A regulatory RNA is involved in RNA duplex formation and biofilm regulation in sulfolobus acidocaldarius

Non-coding RNAs (ncRNA) are involved in essential biological processes in all three domains of life. The regulatory potential of ncRNAs in Archaea is, however, not fully explored. In this study, RNA-seq analyses identified a set of 29 ncRNA transcripts in the hyperthermophilic archaeon Sulfolobus acidocaldarius that were differentially expressed in response to biofilm formation. The most abundant ncRNA of this set was found to be resistant to RNase R treatment (RNase R resistant RNA, RrrR(+)) due to duplex formation with a reverse complementary RNA (RrrR(-)). The deletion of the RrrR(+) gene resulted in significantly impaired biofilm formation, while its overproduction increased biofilm yield. RrrR(+) was found to act as an antisense RNA against the mRNA of a hypothetical membrane protein. The RrrR(+) transcript was shown to be stabilized by the presence of the RrrR(-) strand in S. acidocaldarius cell extracts. The accumulation of these RrrR duplexes correlates with an apparent absence of dsRNA degrading RNase III domains in archaeal proteins.Deutsche Forschungsgemeinschaft DFG RA 2169/3-1 Comision Nacional de Ciencia y Tecnologia de Chile FONDECYT 1130229 Max Planck Societ

Additonal file 4:

Methylation predictions in 16S rRNA alignment. The 16S rRNA sequences from the seven archaeal species used in this study were aligned using Infernal with manual adjustments; a consensus sequence (CON) is given at the bottom. The 16S rRNA sequence from Escherichia coli was included in the alignment for reference with positions numbered separately. Highlights and abbreviations are as listed in the legend to Additional file 3. (DOCX 57 kb

Additional file 3: of C/D box sRNA-guided 2′-O-methylation patterns of archaeal rRNA molecules

Methylation predictions in 23S rRNA alignment. The 23S rRNA sequences from the seven archaeal species used in this study were aligned using Infernal with manual adjustments; a consensus sequence (CON) is given at the bottom. The 23S rRNA sequence from Escherichia coli was included in the alignment for reference with positions numbered separately. Highlighted bases in the alignment are as follows: (green) D guide predictions; (blue) D′ guide predictions; (magenta) sites that are predicted by more than one sRNA guide; (red) predictions that contain a mismatch base pair at the site of modification in the region of guide-target complementarity. The sRNAs responsible for each prediction are listed above the highlighted bases (shaded in grey) in the alignment; species abbreviations for sRNA designations are (S) Sac; (P) Pca; (K) Mka; (M) Mma, (N) Neq; (I) Iho; (T)Tte. (DOCX 80 kb

Additional file 2: of C/D box sRNA-guided 2′-O-methylation patterns of archaeal rRNA molecules

Conservation of C and D boxes. The conservation of C/C′ and D/D′ box sequences is detailed using sequence logos. The overall height of the stacks of nucleic acid symbols indicates sequence conservation and the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position [41]. (EPS 4363 kb

Education and Public outreach Programmes for the Faulkes Telescope Project

We describe the current programmes and future plans of the Faulkes Telescope Project (in the UK and Europe) and Las Cumbres Global Telescope. Within the UK FTP is running a number of educational programmes, including Down-to-Earth, Astronomy to a Captive Audience, and the National Redshift Project that complement the original telescope-based education activities. By engaging learners in "real science", and potentially working alongside "real scientists", we hope to motivate and inspire the study of science, technology, engineering and mathematics (STEM) subjects