RESUMO
The recent development of epitranscriptomics revealed a new fundamental layer of gene expression, but the mapping of most RNA modifications remains technically challenging. Here, we describe our protocol for Rho-Seq, which enables the mapping of dihydrouridine RNA modification at single-nucleotide resolution. Rho-Seq relies on specific rhodamine-labeling of a subset of modified nucleotides that hinders reverse transcription. Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022).
Assuntos
Nucleotídeos , Tiouridina , RNA/genética , Rodaminas , Análise de Sequência de RNA/métodosRESUMO
The universal dihydrouridine (D) epitranscriptomic mark results from a reduction of uridine by the Dus family of NADPH-dependent reductases and is typically found within the eponym D-loop of tRNAs. Despite its apparent simplicity, D is structurally unique, with the potential to deeply affect the RNA backbone and many, if not all, RNA-connected processes. The first landscape of its occupancy within the tRNAome was reported 20 years ago. Its potential biological significance was highlighted by observations ranging from a strong bias in its ecological distribution to the predictive nature of Dus enzymes overexpression for worse cancer patient outcomes. The exquisite specificity of the Dus enzymes revealed by a structure-function analyses and accumulating clues that the D distribution may expand beyond tRNAs recently led to the development of new high-resolution mapping methods, including Rho-seq that established the presence of D within mRNAs and led to the demonstration of its critical physiological relevance.
Assuntos
Oxirredutases , RNA de Transferência , Humanos , Oxirredutases/genética , RNA/química , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Uridina/químicaRESUMO
The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.
Assuntos
Segregação de Cromossomos , Escherichia coli/genética , Meiose , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Uridina/metabolismo , Cromossomos Bacterianos , Cromossomos Fúngicos , Cromossomos Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Células HCT116 , Humanos , Oxirredução , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análise de Sequência de RNA , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
The cell fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which encodes the central inducer of mating and gametogenesis, known as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. During vegetative growth, rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates cis repression at the ste11 promoter through SET3C-dependent histone deacetylation. The absence of rse1 bypasses the starvation-induced signaling and induces gametogenesis in the presence of nutrients. Our data reveal that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differentiation.
