RESUMEN
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.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , ARN Mensajero , Saccharomyces cerevisiae , ARN , Catálisis , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa , NucleotidiltransferasasRESUMEN
The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3'-5' exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.