RESUMEN
Non-coding RNAs (ncRNAs) ubiquitously exist in normal and cancer cells. Despite their prevalent distribution, the functions of most long ncRNAs remain uncharacterized. The fission yeast Schizosaccharomyces pombe expresses >1800 ncRNAs annotated to date, but most unconventional ncRNAs (excluding tRNA, rRNA, snRNA and snoRNA) remain uncharacterized. To discover the functional ncRNAs, here we performed a combinatory screening of computational and biological tests. First, all S. pombe ncRNAs were screened in silico for those showing conservation in sequence as well as in secondary structure with ncRNAs in closely related species. Almost a half of the 151 selected conserved ncRNA genes were uncharacterized. Twelve ncRNA genes that did not overlap with protein-coding sequences were next chosen for biological screening that examines defects in growth or sexual differentiation, as well as sensitivities to drugs and stresses. Finally, we highlighted an ncRNA transcribed from SPNCRNA.1669, which inhibited untimely initiation of sexual differentiation. A domain that was predicted as conserved secondary structure by the computational operations was essential for the ncRNA to function. Thus, this study demonstrates that in silico selection focusing on conservation of the secondary structure over species is a powerful method to pinpoint novel functional ncRNAs.
Asunto(s)
Schizosaccharomyces , Schizosaccharomyces/genética , Diferenciación Sexual , ARN no Traducido/genética , ARN no Traducido/química , ARN Nucleolar Pequeño/genética , Sistemas de Lectura AbiertaRESUMEN
Dormancy breaking is a common physiological phenomenon that is shared by eukaryotes. Germination of spores in fungi is one of the most representative cases of dormancy breaking. Understanding the mechanisms of spore germination is therefore fundamental to basic studies on the control of cell proliferation and differentiation, as well as agricultural applications and medical investigation of fungal pathogenesis. In fission yeast, spores are generated as a consequence of sexual differentiation under nutrient starvation, remaining dormant until further nourishment, but little is known about how dormant spores germinate in response to environmental change. In a breakthrough, methods for single-cell-based gene expression profiling have recently been introduced. Several mRNA expression profiles were assembled from single spore cells during dormancy or germination. Single-cell RNA-seq profiles were aligned sequentially according to their similarities. The alignment of transcriptomes visualised how gene expression varies over time upon dormancy breaking. In this review, we revisit knowledge from previous studies on germination, select candidate genes that may be involved in germination, and query their expression from the temporal transcriptomic dataset so that studies on S. pombe germination can be extended further.
Asunto(s)
Germinación/genética , Esporas Fúngicas/genética , Transcriptoma/genética , Regulación Fúngica de la Expresión Génica/genética , RNA-Seq , Análisis de la Célula Individual , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/ultraestructura , Imagen de Lapso de TiempoRESUMEN
How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial 'wake-up' trigger at dormancy breaking.
Asunto(s)
Histonas/genética , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transcriptoma , División Celular/genética , División Celular/fisiología , Biología Computacional , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Germinación/fisiología , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismoRESUMEN
The fission yeast Schizosaccharomyces pombe is a powerful model organism for cell biology and molecular biology, as genetic manipulation is easily achieved. Introduction of exogenous genes cloned in episomal plasmids into yeast cells can be done through well-established transformation methods. For expression of genes in S. pombe cells, the multi-copy plasmid pREP1 and its derivatives, including pREP41 and pREP81, have been widely used as vectors. Although recent advancement of technology brought a number of useful genetic elements such as new promoters, selection marker genes and fluorescent protein tags, introduction of those elements into conventional pREP1 requires a large commitment of both time and effort because cloning procedures need to be repeated until the final products are constructed. Here, we introduce materials and methods to construct many pREP1-type plasmids easily and systematically using the Golden Gate shuffling method, which enables one-step ligation of many DNA fragments into a plasmid. These materials and methods support creation of expression plasmids employing a variety of novel genetic elements, which will further facilitate genetic studies using S. pombe.