A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast <em>Schizosaccharomyces pombe</em>

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

Treating Osteoporotic Spinal Fractures in Elderly Patients <em>via</em> a Modified Clinical Bone Cement Injection Technique

The minimally invasive injection of bone cement (MIIBC) is an effective way to treat senile osteoporotic spinal fractures (OSF) in clinical practice. ...

Wake Forest University

The University of Texas Medical Branch

Linfen People’s Hospital of Shanxi Medical University

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3'-5' exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5'-3' exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome. 

A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5'-3' Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe.

Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes.

The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis.

An important aspect of RNAi-mediated heterochromatin assembly is the production of short interfering RNAs (siRNAs) in cis at the heterochromatic target. These populations of small RNAs (20-30 nt) can be detected via northern blot analysis with radiolabeled RNA probes corresponding to the siRNAs. Most heterochromatin-associated siRNAs originate from both strands of the pericentromeric dg and dh repeats. Thus, production of RNA probes by in vitro transcription utilizes a DNA template with sequences corresponding to either dg or dh repeats. Alternatively, radiolabeled DNA probes, which are somewhat easier to prepare, may be used. A control probe against a small RNA, such as a tRNA, may also be prepared for loading. More recently genome-scale analysis using next-generation sequencing platforms has enabled the detection of rare siRNAs at sites other than the major heterochromatin domains. This protocol describes procedures for the extraction of small RNAs from Schizosaccharomyces pombe and their detection by northern blot analysis.

Analysis of Schizosaccharomyces pombe Heterochromatin-Associated Short Interfering RNAs.

A healthy individual may carry a detrimental genetic trait that is masked by another genetic mutation. Such suppressive genetic interactions, in which a mutant allele either partially or completely restores the fitness defect of a particular mutant, tend to occur between genes that have a confined functional connection. Here we investigate a self-recovery phenotype in , mediated by suppressive genetic interactions that can be amplified during cell culture. Cells without Elf1, an AAA+ family ATPase, have severe growth defects initially, but quickly recover growth rates near to those of wild-type strains by acquiring suppressor mutations.  cells accumulate RNAs within the nucleus and display effects of genome instability such as sensitivity to DNA damage, increased incidence of lagging chromosomes, and mini-chromosome loss. Notably, the rate of phenotypic recovery was further enhanced in  cells when RNase H activities were abolished and significantly reduced upon overexpression of RNase H1, suggesting that loss of Elf1-related genome instability can be resolved by RNase H activities, likely through eliminating the potentially mutagenic DNA-RNA hybrids caused by RNA nuclear accumulation. Using whole genome sequencing, we mapped a few consistent suppressors of  including mutated Cue2, Rpl2702, and SPBPJ4664.02, suggesting previously unknown functional connections between Elf1 and these proteins. Our findings describe a mechanism by which cells bearing mutations that cause fitness defects and genome instability may accelerate the fitness recovery of their population through quickly acquiring suppressors. We propose that this mechanism may be universally applicable to all microorganisms in large-population cultures.

Ke Zhang

Department of Biology,
Center for Molecular Communication and Signaling

Wake Forest University

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Treating Osteoporotic Spinal Fractures in Elderly Patients via a Modified Clinical Bone Cement Injection Technique

Ke Zhang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology,Center for Molecular Communication and Signaling

Wake Forest University

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Treating Osteoporotic Spinal Fractures in Elderly Patients via a Modified Clinical Bone Cement Injection Technique

Department of Biology,
Center for Molecular Communication and Signaling