Pervasive mRNA uridylation in fission yeast is catalysed by both Cid1 and Cid16 terminal uridyltransferases

Messenger RNA uridylation is pervasive and conserved among eukaryotes, but the consequences of this modification for mRNA fate are still under debate. Utilising a simple model organism to study uridylation may facilitate efforts to understand the cellular function of this process. Here we demonstrate that uridylation can be detected using simple bioinformatics approach. We utilise it to unravel widespread transcript uridylation in fission yeast and demonstrate the contribution of both Cid1 and Cid16, the only two annotated terminal uridyltransferases (TUT-ases) in this yeast. To detect uridylation in transcriptome data, we used a RNA-sequencing (RNA-seq) library preparation protocol involving initial linker ligation to fragmented RNA-an approach borrowed from small RNA sequencing that was commonly used in older RNA-seq protocols. We next explored the data to detect uridylation marks. Our analysis show that uridylation in yeast is pervasive, similarly to the one in multicellular organisms. Importantly, our results confirm the role of the cytoplasmic uridyltransferase Cid1 as the primary uridylation catalyst. However, we also observed an auxiliary role of the second uridyltransferase, Cid16. Thus both fission yeast uridyltransferases are involved in mRNA uridylation. Intriguingly, we found no physiological phenotype of the single and double deletion mutants of cid1 and cid16 and only minimal impact of uridylation on steady-state mRNA levels. Our work establishes fission yeast as a potent model to study uridylation in a simple eukaryote, and we demonstrate that it is possible to detect uridylation marks in RNA-seq data without the need for specific methodologies

Izolacija, biokemijske značajke i identifikacija termotolerantnih i celulolitičkih bakterija Paenibacillus lactis i Bacillus licheniformis

Research background. Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that the polymer molecule is degraded to smaller particles and used as a carbon source by microorganisms. Because of the frequently applied methods of pretreatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. Experimental approach. We were looking for cellulolytic microorganisms able to grow at 50 °C and we described their morphological features and biochemical characteristics based on carboxymethyl cellulase (CMCase) activity and the API® ZYM system. The growth curves during incubation at 50 °C were examined using the BioLector® microbioreactor. Results and conclusions. Forty bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganisms. The CMCase activity was confirmed in 27 strains. Hydrolysis capacities were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was observed, among others, for BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. Novelty and scientific contribution. We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P. lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of circular bioeconomy.Pozadina istraživanja. Celuloza je sastojak otpadnih tvari koji se može preraditi u korisne spojeve. To je moguće ako se molekule polimera razgrade na manje čestice koje mikroorganizmi mogu iskoristiti kao izvor ugljika. Celulaze proizvedene s pomoću termofilnih mikroorganizama su naročito zanimljive zbog njihove učestale primjene u prethodnoj obradi lignoceluloznog materijala. Eksperimentalni pristup. Istražili smo celulolitičke mikroorganizme koji rastu na 50 °C te opisali njihove morfološke i biokemijske značajke na osnovi aktivnosti karboksimetil celulaze i ostalih enzima ispitanih sustavom API® ZYM. Krivulje rasta tijekom inkubacije pri 50 °C ispitane su uzgojem bakterija u mikrobioreaktoru BioLector®. Rezultati i zaključci. Četrdeset sojeva bakterija izolirano je iz fermentirane slame, geotermičkog izvora u kršu, termalnog vrela i geotermalnog jezera s temperaturom od 50 °C. Većina bakterija bile su Gram-pozitivne i štapićastog oblika, a najviša temperatura pri kojoj su rasle je iznosila najmanje 50 °C. Također smo potvrdili veliku raznolikost biokemijskih značajki među ispitanim mikroorganizmima. Aktivnost karboksimetil celulaze potvrđena je u 27 sojeva. Sojevi bakterija sa značajnim hidrolitičkim kapacitetom bili su: BBLN1 s 2,74; BSO6 s 1,62; BSO10 s 1,30; BSO13 s 1.38 i BSO14 s kapacitetom od 8,02. Sojevi BBLN1, BSO10, BSO13 i BSO14 rasli su brzo i stabilno. Ispunjavali su odabrane preduvjete pa su identificirani 16S rDNA sekvenciranjem. Sojevi BBLN1, BSO10 i BSO13 klasificirani su kao sojevi bakterije Bacillus licheniformis, dok je soj BSO14 potvrđen kao soj Paenibacillus lactis. Novina i znanstveni doprinos. Opisali smo celulolitičku aktivnost i biokemijska svojstva velikog broja bakterija izoliranih iz lokacija gdje vladaju velike vrućine. Prvi smo izvijestili o celulolitičkoj aktivnosti termotolerantne bakterije P. lactis. Opisani bi sojevi mogli biti izvor novih termički stabilnih celulaza, što je izuzetno poželjno u različitim granama cirkularne bioekonomije

Pervasive mRNA uridylation in fission yeast is catalysed by both Cid1 and Cid16 terminal uridyltransferases.

Messenger RNA uridylation is pervasive and conserved among eukaryotes, but the consequences of this modification for mRNA fate are still under debate. Utilising a simple model organism to study uridylation may facilitate efforts to understand the cellular function of this process. Here we demonstrate that uridylation can be detected using simple bioinformatics approach. We utilise it to unravel widespread transcript uridylation in fission yeast and demonstrate the contribution of both Cid1 and Cid16, the only two annotated terminal uridyltransferases (TUT-ases) in this yeast. To detect uridylation in transcriptome data, we used a RNA-sequencing (RNA-seq) library preparation protocol involving initial linker ligation to fragmented RNA-an approach borrowed from small RNA sequencing that was commonly used in older RNA-seq protocols. We next explored the data to detect uridylation marks. Our analysis show that uridylation in yeast is pervasive, similarly to the one in multicellular organisms. Importantly, our results confirm the role of the cytoplasmic uridyltransferase Cid1 as the primary uridylation catalyst. However, we also observed an auxiliary role of the second uridyltransferase, Cid16. Thus both fission yeast uridyltransferases are involved in mRNA uridylation. Intriguingly, we found no physiological phenotype of the single and double deletion mutants of cid1 and cid16 and only minimal impact of uridylation on steady-state mRNA levels. Our work establishes fission yeast as a potent model to study uridylation in a simple eukaryote, and we demonstrate that it is possible to detect uridylation marks in RNA-seq data without the need for specific methodologies