분열효모인 S. pombe에서 mRNA export와 관련된 rsm1 유전자의 기능에 대한 연구

Abstract

fission yeast 인 Schizosaccharomyces pombe에서 mRNA의 nuclear export에 관여하는 factors를 밝히기 위해 spmex67 null mutant allele와 synthetic lethality를 보이는 3종류의 돌연변이주를 screening하였다. 이들 돌연변이주는 기존에 S. pombe에서 밝혀진 mRNA export factors인 Gle1, Rae1, Nup184, Elf3, Crp79, Npp106가 아닌 다른 유전자에서 mutation이 일어난 것임을 확인하였다. 이들 SLmex1, SLmex3, SLmex9에서 각각의 cognate 유전자들을 cloning 하였다. 이들 중 SLmex1 으로부터 cloning한 rsm1 (the mRNA export defect and Synthetic lethality with Mex67)유전자는 chromosome III 상에 존재하고 그 기능이 알려져 있지 않은 hypothetical protein을 encoding하고 있다. Rsm1 유전자의 기능을 연구하기 위해 야생형 haploid 균주에 이 유전자가 Ura4+/Knar로 치환된 knock-out 돌연변이주를 만들었다. 그 결과rsm1은 성장에 필수 유전자는 아니지만 mRNA export에 결함을 보이는 새로운 mRNA export factor임을 알 수 있었다. Rsm1유전자가 mRNA export와 관련된 중요 유전자들과 유전학적으로 서로 연관되어 있는 지를 알아 보기 위해 double mutants를 제조, synthetic lethality를 조사 했다. Synthetic lethality를 보이는 유전자는 찾을 수 없었지만, double mutants의 growth defect정도와 in situ hybridization을 통한 mRNA export의 결함을 조사한 결과 Δrsm1 돌연변이가 mRNA export에 관여하는 것으로 알려진 다른 유전자들의 돌연변이와 함께 있는 경우, mRNA export 결함을 악화시키던지(Δmex67, Δnpp106), 약화시키는(Δthp1) 것으로 보아, rsm1은 여러 mRNA export factors 들과 상호작용 하면서 mRNA export에 관련이 있음을 암시한다.|In eukaryote, nucleocytoplasmic transport of macromolecules generally requires soluble transport receptors that specifically recognize and transport their cargoes through nuclear pore complexes (NPC) embedded in the nuclear envelope (Mattaj and Englmeier, 1998; Strasser and Hurt, 1999). These receptors mediate transport through nuclear pore complexes (NPCs), and the directionality of transport is controlled by the small GTPase Ran. However, the export of mRNA from the nucleus to the cytoplasm is different from the other nucleocytoplasmic transport routes and is significantly complicated. mRNA export is a conserved process. mRNA is exported by the heterodimeric NXF-NXT class of receptors. The homolog of large subunit (NXF) is Mex67 and the homolog of small subunit (NXT) is Mtr2 in yeast, and whose conserved metazoan counterparts are TAP and p15, respectively. Although mex67 is not essential in Schizosaccharomyces pombe, spMex67p binds to mRNA and functions to export mRNA. In order to identity mutations in genes that are functionally linked to mex67 in mRNA export, three synthetic lethal mutants with the spmex67 null allele present in S. pombe were isolated. A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex1. The rsm1 gene contains no introns and encodes a 296 amino-acid long protein with the RING finger domain, a C3HC4 in the N-terminal half. The Δrsm1 null mutant is viable, but it showed a poly(A)+ RNA accumulation in the nucleus. Although the combination of Δrsm1 and Δspmex67 mutations not showd synthetic lethality, don’t appear expectation, they were shown to cause more significantly defect in the process of mRNA export. The other deletion mutants of mRNA export factors (Δp15, Δmlo3, Δnpp106, Δnup184, Δthp1) and ts mutant (rae1-167) not showed synthetic lethality between absence rsm1. However, thp1 deletion mutant suppressed the mRNA export defect of rsm1 mutation. Also, npp106 and mex67 null mutations affected more strongly growth defect in Δrsm1. These data suggested that rsm1 is involved in the mRNA export and interacted genetically with mRNA export factors (mex67, thp1, npp106, nup184) in S. pombe.