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An antisense RNA-mediated mechanism eliminates a meiosis-specific copper -regulated transcript in mitotic cells

Significance Statement

Eukaryotic cell division undergoes either mitosis or meiosis.  In the case of meiosis, this process allows diploid precursor cells to produce haploid gametes, which are required for sexual reproduction.  Because several specialized proteins are involved in meiosis-specific mechanisms such as meiotic homologous recombination, meiotic nuclear divisions, and forespore morphogenesis, their presence is detrimental to cells proliferating in mitosis.  Cells have therefore developed different mechanisms to prevent or eliminate meiosis-specific transcripts during mitosis.  One mechanism of mRNA elimination involves genes that are organized into convergent pairs.  In this case two genes are in proximity of one another with their transcription orientated one toward the other.  We have recently discovered that Schizosaccharomyces pombe iss1+ and cum1+ genes are arranged with their transcription orientated convergently.  The iss1+ gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1+ sense transcript.  When the production of the long iss1+ antisense transcript was disrupted by insertion of a polyadenylation signal immediately downstream of iss1+, results showed high levels of cum1+ sense mRNA during mitosis, which were copper starvation- and Cuf1-dependent.  This finding suggested that inhibition of expression of cum1+ sense mRNA occurs due to the presence of the long iss1+ antisense RNA.  In fission yeast, sense/antisense RNA duplexes accumulate in G1 phase of the cell cycle, especially in regions where convergent genes are present.  In G1, transcription of several convergent genes fails to terminate after their proximal cleavage and polyadenylation sites, thereby resulting in a transcriptional read-through that produces long antisense/sense transcripts.  Accumulation of long antisense/sense RNA duplexes activates the RNAi pathway, which leads to gene silencing and heterochromatin formation over convergent gene regions.  To assess involvement of the RNAi machinery in silencing cum1+ transcription, we probed cum1+ mRNA levels in four mutants that affect the RNAi pathway.  These mutants were dcr1D (dicer), ago1D (argonaute), rdp1D (RNA-dependent RNA polymerase) and clr4D (histone methyltransferase).  Using G1-synchronized wild-type and dcr1D, ago1D, rpd1D, and clr4D mutant cells, we determined that components of the RNAi pathway are involved in antisense-mediated repression of cum1+ transcription, especially during the G1 phase of the cell cycle.  In this study, we have uncovered a mechanism by which a copper- and meiosis-specific transcript is selectively inhibited in growing cells, preventing its untimely expression during mitosis.  To our knowledge, this is the first report to show an antisense transcription control of a copper-regulated transcript under physiological low copper conditions. 

About The Author

Vincent Normant obtained his bachelor’s degree (2012) in Biology and Immunology from the Université de Nantes in France.  He continued his training as a Master I and Master II student in the program of Master Biologie Santé at the Université de Nantes in France.  He then joined the laboratory of Dr. S. Labbé at the Department of Biochemistry at the Université de Sherbrooke (Canada) as a doctoral student in 2013.  His research focuses on metal-dependent mechanisms of meiotic gene regulation in the fission yeast Schizosaccharomyces pombe.  

About The Author

Dr. Simon Labbé is a Professor of Biochemistry in the Faculty of Medicine and Health Sciences at the Université de Sherbrooke in Québec, Canada.  He received his bachelor’s degree in Microbiology from the Université Laval (Québec) in 1987.  He undertook graduate studies at Laval Université and received his Master (1989) and Ph.D. (1995) in Microbiology and Molecular-cellular biology, respectively.  Dr. Labbé continued his training as a post-doctoral fellow with Dr. Dennis J. Thiele at the Department of Biological Chemistry at the University of Michigan in Ann Arbor, MI, USA.  He was supported by Fellowship awards, including the prestigious Centennial Fellowship from the Medical Research Council of Canada.  Over the past sixteen years he has been at the Université de Sherbrooke where he has established his own laboratory.  As an independent investigator, he has received the New Investigator Award from the Canadian Institutes of Health Research, the Junior II and Senior Investigator Awards from the Fonds de la Recherche en Santé du Québec.  His research group has developed a productive yeast model (Schizosaccharomyces pombe) for investigating copper and iron metabolism at the molecular level.  Because S. pombe provides a genetically tractable model that permits the drawing of parallels with other eukaryotic living systems, especially filamentous yeasts, Dr. Labbé’s research has contributed to the identification of molecular mechanisms that regulate copper and iron transport, as well as uncovering different strategies that yeasts have acquired to take up copper and iron from their environment and/or hosts.

Figure Legend: Illustration of a proposed model for elimination of cum1+ meiotic transcripts in mitotic cells.

Global Medical Discovery, antisense RNA-mediated mechanism eliminates a meiosis-specific copper regulated transcript in mitotic cells

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

J Biol Chem. 2015;290(37):22622-37.

Normant V1, Beaudoin J1, Labbé S2.

Show Affiliations
  1. Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, J1E 4K8, Canada.
  2. Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1E 4K8, Canada [email protected]

Abstract

Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1(+) gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1(+) sense transcript, inhibiting cum1(+) expression in vegetative cells. Inhibition of cum1(+) transcription was not at the level of its initiation because fusion of the cum1(+) promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1(+)-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1(+) led to the exclusive production of a shorter iss1(+) mRNA isoform, thereby allowing accumulation of cum1(+) sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1(+) antisense  mRNA could pair with cum1(+) sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1(+) sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells.

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