Systematic Screening And Mechanisms Of Suppressing Essential Gene Of Schizosaccharomyces

In unicellular organisms, essential genes are usually defined as genes required for cell survival in normal growth conditions. It has been shown that essential genes are usually involved in core biological processes and are also highly conserved among species[1]. The essentiality of certain genes differs in different organisms, suggesting that the essentiality of genes could be influenced by genetic context[3]. Furthermore, the lethality caused by the deletion of an essential gene can sometimes be rescued by a single mutation, which is called a bypass suppressor. We here tried to systematically investigate this phenomenon by determining the bypassability of 165 essential genes on the left arm of Chr â…¡.Using the overexpression plasmid library created by the Yoshida laboratory [4], we first established a pipeline for screening the overexpression-mediated bypass-of-essential-gene (O-BOE) suppressors in S. pombe, and then successfully performed screens on 128 essential genes with this pipeline and verified the O-BOE screen results.We found that about 12.5% of the essential genes in S. pombe could be bypassed by an overexpression plasmid, while for other essential genes we could not obtain an O-BOE suppressor. The difference in bypassability suggests that essential genes are not equally important. We further found that the overexpression bypassable essential genes tend to evolve faster and be less conserved compared with non-bypassable essential genes. In physical interaction networks, they also tend to have lower degrees and smaller clustering coefficient, implying that they are not as important as non-bypassable essential genes in network. All these characteristics make bypassable essential genes more similar to non-essential genes.By analyzing the patterns of bypass genetic interactions, we found that genes encoding proteins in the same protein complex or acting in the same pathway tend to share O-BOE suppressors, suggesting that large scale bypass suppressor maaping might help reveal essential modules in the cell. Furthermore, O-BOE suppressors could help uncovering the essential functions of essential genes. For example, we found that resl could be bypassed by a plasmid overexpressing Cdc18, suggesting that Cdc18 is the essential target of Resl in G1/S cell cycle transition; and we also found phospholipase overexpression could rescue the lethality of deleting an ERMES complex gene, implying the essential function of the ERMES complex is related to lipid regulation. Besides, we also found that mitochondrial ribosomal subunit genes mrp21 and mrps28 could be bypassed by the overexpression of the Dicer protein Dcr1 whose function is related to small RNA metabolism, indicating that O-BOE genetic interactions could help revealing functional relationships between different bioprocesses. All these results demonstrate that systematical O-BOE suppressor analysis not only help wire connections between different biological processes, but also provide valuable clues for studying the functions of genes.SUMO-targeted ubiquitin ligases (STUbLs) are a family of proteins conserved from yeast to human. The discovery of these proteins indicates that SUMO-modified proteins could be delivered to proteasome for degradation. Some cellular substrates of STUbLs have been discovered in previous studies, but which biological processes in the cell are mainly regulated by STUbLs and what the biological meaning of crosstalk between ubiquitin and SUMO is still remain elusive.In S. pombe, the only reported STUbL is the Slx8-Rfp1/Rfp2 protein complex. Both slx8 deletion and rfp1 rfp2 double knockout make cell inviable in normal growth conditions, which means STUbL is essential for cell gowth in S. pombe. Because essential genes are usually in control of basic and core biological processes in the cell, unraveling the essential function of Slx8 may not only help us understand the most important function of Slx8 in S. pombe, but may also help shed light on the function of the whole STUbL family.A piggyBac transposon and an overexpression library mediated bypass suppressor screen found that the lethality of slx8 deletion could be rescued by mutation or overexpression of several genes. We tried to study the essential function of Slx8 based on the transposon-mediated bypass-of-essential-gene (T-BOE) suppressors. Mutation of enzymes in SUMO modification cycle (Pli1 and Fub2) could bypass slx8 by inhibiting the global SUMOylation in the cell, suggesting that some SUMO-modified proteins might be toxic in slx8 deleted cell. Nucleoporin mutations such as nup132 deletion and nup189_C_ins could bypass slx8. These mutations and another T-BOE suppressor, mutation of the SUMO protease gene ulp2, could delocalize the SUMO protease Ulp1 from nuclear envelope. These results intimate that the essential function of Slx8 might also be closely related to nuclear pore complex and Ulp1. We further demonstrated that mutations affecting the Y shape nuclear pore complex (Nup132 and Nup138) and the SUMO protease Ulp2 might bypass slx8 by altering the localization of Ulp1 at nuclear envelope.Mutations of rrp2, whose function is unclear in S. pombe, were also identified as T-BOE suppressors of slx8 in our screen. The SUMO binding motifs at the N terminal regions of Rrp2 are important for its toxicity in slx8â–³ cells, implying that Rrp2 might bind SUMO modified substrates and produce toxicity. Our study also found that Rrp2 may interact with itself through its N terminal region. ChIP-seq of Rrp2 revealed binding of Rrp2 at long gene-free regions in the genome, suggesting a relationship between the non-coding portion of the genome and Slx8 essentiality. Overall, our study suggests that the essential function of Slx8 in S. pombe may be regulating a connection between chromosome-bound Rrp2 and nuclear-envel op-localized Ulp1.