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1 Korean Journal of Microbiology (2017) Vol. 53, No. 4, pp pissn DOI eissn Copyright c 2017, The Microbiological Society of Korea Note 분열효모에서 THO 복합체의구성요소인 Tex1/THOC3 가생장및 mrna 방출에미치는영향 배수정 고은진 윤진호 * 성신여자대학교생명과학 화학부 Effect of Tex1/THOC3, a component of THO complex, on growth and mrna export in fission yeast Soo Jeong Bae, Eun-Jin Koh, and Jin Ho Yoon* School of Biological Science and Chemistry, Sungshin Women's University, Seoul 01133, Republic of Korea (Received November 22, 2017; Revised December 11, 2017; Accepted December 12, 2017) In eukaryote, THO/TREX complex plays a critical role in transcriptional elongation, pre-mrna processing, and nuclear mrna export. This complex is evolutionally well- conserved, but there are some differences in composition and function according to organisms. Here we showed that sptex1, a component of THO/TREX complex, is not essential for growth and mrna export in a fission yeast, Schizosaccharomyces pombe, which is more similar to higher eukaryote than budding yeast. Deletion and overexpression of the sptex1 gene do not lead to any detectable growth phenotype and accumulation of poly(a)+ RNA in the nucleus. And the sptex1-gfp protein is localized mainly in the nucleus. Yeast two-hybrid and Coimmunoprecipitation analysis showed that the sptex1 protein interacted with sphpr1 (THOC1) and sptho2 (THOC2), main subunits of THO complex. We conclude that the S. pombe Tex1 is a component of THO/TREX complex, but does not plays important roles in growth and bulk mrna export from the nucleus. Keywords: Schizosaccharomyces pombe, mrna export, Tex1/ THOC3, THO/TREX complex *For correspondence. jhoyoon@sungshin.ac.kr; Tel.: ; Fax: 진핵생물에서단백질을암호화하고있는유전자는 RNA 중합효소 II에의해 pre-mrna로전사되고 mrna 가공 (5 캡핑, 스플라이싱, 3 절단및폴리아데닐화 ) 과정을거친후핵공복합체 (nuclear pore complex) 를통해핵에서세포질로방출된다. mrna는핵안에서전사되는순간부터수많은단백질들이결합하여 mrnp (messenger ribonucleoprotein) 복합체를형성하며, 결합단백질들은 mrna의생성, 성숙, 방출등에관여한다 (Stewart, 2010). 유전자발현의각단계가진행되면서 mrna 분자의변형과함께 mrna에결합하는특이적단백질들의조합도달라지므로, mrnp는고정된것이아니라역동적으로변하는조성및구조를가진다 (Rodriguez-Navarro and Hurt, 2011; Katahira, 2015). 이렇게복잡한일련의유전자발현과정들은조절되고조직화되어서로영향을주고받는데, TREX (Transcription and Export) 는 mrna에결합하는다중단백질복합체로유전자발현과정에핵심적인역할을담당한다 (Heath, 2016). 출아효모 (Saccharomyces cerevisiae), 초파리 (Drosophila), 애기장대 (Arabidopsis), 개구리 (Xenopus), 사람등대부분의진핵생물에보존되어있는 TREX는 THO 복합체와추가적인인자들로나눌수있다. THO 복합체는출아효모인 S. cerevisiae 에서전사신장과정에중요한역할을담당하는것으로처음알려졌는데, RNA 중합효소 II의인산화된 C-말단영역 (CTD) 과직접결합함으로써전사가활성화된유전자부위에모여든

분열효모에서의 mrna export 293 Fig. 2. Localization of sptex1 protein fused to GFP. sptex1 was tagged with GFP at its carboxyl-terminus (sptex1-gfp).

Green fluorescent image (GFP) and coincident differential interference contrast image (DIC), and the merged image of both are shown. Fig. 1. S.

The entire sptex1 (SPCC18B5.10c) coding region was substituted with the marker genes, ura4 + or kan r, using one-step gene disruption method.

2 분열효모에서의 mrna export 293 Fig. 2. Localization of sptex1 protein fused to GFP. sptex1 was tagged with GFP at its carboxyl-terminus (sptex1-gfp). The sptex1-gfp::kan r DNA fragment was integrated at the sptex1 gene locus, and the localization of the fusion protein was determined by confocal microscope. Green fluorescent image (GFP) and coincident differential interference contrast image (DIC), and the merged image of both are shown. Fig. 1. S. pombe Tex1/THOC3 (sptex1) is not essential for growth and mrna export. (A) A schematic diagram shows the wild-type sptex1 allele and the knockout allele ( sptex1) in S. pombe. The entire sptex1 (SPCC18B5.10c) coding region was substituted with the marker genes, ura4 + or kan r, using one-step gene disruption method. The coding region is represented by an open box and six introns are indicated by vertical thick line in the open box. The arrow above the sptex1 allele shows the direction of transcription. (B) sptex1 deletion did not lead to any detectable growth defects. Wild type sptex1 + (WT) cells and sptex1 null mutant cells were monitored by spot assay. Cells were serially diluted and spotted on YES plates, and incubated for 5 days at 22 C, 3 days at 30 C, and 37 C, respectively. (C) The sptex1 null mutant cells showed no defect of mrna export. Cells were grown to the mid-log phase in appropriately supplemented EMM medium at 30 C and fixed. FISH for poly(a) + RNA were performed using Oligo-(dT) 50 labelled with an -digoxygenin at the 3 end as the hybridization probe, and using FITC-anti-digoxygenin Fab antibody for detecting the hybridization probe. DAPI staining are used to identify the nucleus in the right panels. 다 (Chavez et al., 2000; Meinel et al., 2013). 그후 THO는 mrna 방출인자인 Yra1 (RNA-결합단백질 ) 및 Sub2 (DEAD box RNA helicase) 와함께 TREX 복합체를형성하여 mrna 에결합하고, mrna 방출운반체 (export receptor) 인 Mex67- Mtr2를 mrnp에불려들여성숙한 mrna의방출에관여함이밝혀졌다 (Strasser et al., 2002; Zenklusen et al., 2002). TREX 복합체는이외에도 mrna의가공과정에관여하는여러단백질과도결합하는것이알려짐으로써 (Roug le et al., 2008; Chanarat et al., 2011), TREX가유전자발현의높은정확도를유지하도록 mrna 생성, 가공, 방출등의각단계들을통합조절하는인터페이스로작용하는것으로추정된다. 흥미롭게도진화적으로보존된 TREX 복합체는연구된생명체에서모두유전자발현에중요한역할을담당하지만, 출아효모와고등생명체의 TREX는조성과기능에서차이를보인다. THO 구성요소중에서핵심소단위의이종상동체 (ortholog) 인 Hpr1( 출아효모 )/THOC1( 고등생명체 ) 와 Tho2/THOC2, 그리고 Tex1/THOC3는대부분의진핵생물에서잘보존되어있지만, Mft1와 Thp2는출아효모에만특이적이고고등생물에는대신 THOC5, THOC6, THOC7가특이적으로존재한다 (Strasser et al., 2002; Rehwinkel et al., 2004). THO 이외에 TREX를구성하는인자도 Yra1/ALY (REF, THOC4), Sub2/ UAP56 (DDX39B), Tho1/SARNP 등은공통적이지만, 고등생물에서발견되는 CHTOP, POLDIP3와 ZC3H11A 등은출아효모에서는발견되지않는다 (Heath, 2016). 또한 RNA 중합효소 II와직접적인결합을통해 mrna로결합하는출아효모와는다르게, 고등생물의 THO/TREX는 RNA 중합효소 II와는상호작용이없고대신스플라이싱과연관된 Exon junction complex와캡-결합단백질 (CBC20과 CBC80) 과의상호작용을통해 pre-mrna에결합한다 (Masuda et al., 2005; Cheng et al., 2006). 분열효모인 Schizosaccharomyces pombe의유전체데이터베이스인 PomBase ( 에서 THO 복합체의구성인자를찾아보면, 진화적으로보존된 Hpr1/THOC 1, Tho2/THOC2, Tex1/THOC3 등은역시존재하지만출아효모특이적인 Mft1과 Thp2의이종상동체는존재하지않고고등생물특이적인THOC5와 THOC7이종상동체가존재하였다 (Koh and Yoon, 2015). 본연구에서는 THO 복합체의구성인자중아직연구하지않은 Tex1/THOC3의분열효모이종상동체 (sptex1으로명명 ) 를암호화하는 SPCC18B5.10c 유전자가생장및 mrna 방출에미치는영향을알아보았다. 또한 sptex1 단백질가세포어디에위치하는지, 그리고실제로 THO/TREX Korean Journal of Microbiology, Vol. 53, No. 4

294 Bae et al. Table 1. Yeast two-hybrid analysis AD- His3 expression AD- BD- BD- His3 expression a X Tex1 - Tex1 X - Fig. 3. Association of sptex1 with sphpr1 in S. pombe extracts.

3 294 Bae et al. Table 1. Yeast two-hybrid analysis AD- His3 expression AD- BD- BD- His3 expression a X Tex1 - Tex1 X - Fig. 3. Association of sptex1 with sphpr1 in S. pombe extracts. The sphrp1-tap and sphrp1 (control) cells were transformed with HA-spTex1 plasmid. Whole cell extracts from the strains expressing sphrp1-tap fusion (lane 1) or sphrp1 (lane 4) are denoted. The sphpr1-tap associated proteins were captured on IgG-Sepharose beads and separated on SDS-PAGE, transferred onto PVDF membrane. Western blot were performed using antibodies against HA or protein A. Input whole cell extracts (WCE), supernatants (Sup), and eluents from IgG bead (IgG Elute) are shown as indicated. 복합체의구성인자인지조사하였다. S. pombe의 3번염색체에존재하는 SPCC18B5.10c 유전자는인트론이 6개있으며 (Fig. 1A), 예상분자량 kda, 등전점이 ph5.21인단백질을암호화하고있다. 309개아미노산으로이루어진이단백질은대부분영역이여러단백질과의결합에관여하는것으로알려진 WD40 도메인을형성하고있다. NCBI (National Center for Biotechnology Information) 에서제공하는다중서열정렬 (multiple sequence alignment) 도구인 COBALT를사용하여분석하면, sptex1 는출아효모의 Tex1보다고등생물의 THOC3 단백질과더유사하다. 먼저 SPCC18B5.10c 유전자가생장및 mrna 방출에미치는영향을알아보기위해, sptex1 결실돌연변이균주를제작하였다. 반수체야생형균주인 AY217 (h - leu1-32 ura4-d18) 에선별유전자로 ura4 + 또는 kan r 유전자를사용하여 sptex1::ura4 + 또는 sptex1::kan r 결실돌연변이를제작하여선별하였다 (Fig. 1A). 이렇게별개의반수체 sptex1 결실돌연변이를모두얻음으로써, sptex1 유전자가생장에필수적이지않다는것을확인하였다. 다양한온도에서 sptex1 결실돌연변이의생장을 spot assay로확인한결과, 측정한모든온도 (22, 34, 37 C) 에서대조군인야생형 (WT) 과비슷하게정상적인생장속도를보였다 (Fig. 1B). 다음으로 fluorescence in situ hybridization (FISH) 방법을사용하여 (Yoon et al., 2000), sptex1단백질이 mrna의핵에서세포질로의방출에관여하는지알아보았다. 야생형 (WT) 균주의 poly(a) + RNA의분포는세포전체에거의균일하게퍼져있는데, sptex1 결실돌연변이균주에서도비슷한양상이관찰되었다 (Fig. 1C). 즉, sptex1 결실돌연변이균주도 bulk mrna의방출에는이상이없다는것을의미 Hpr1 Tex1 + Tex1 Hpr1 + Tho2 Tex1 + Tex1 Tho2 + THOC5 Tex1 - Tex1 THOC5 - THOC7 Tex1 - Tex1 THOC7 + Uap56 Tex1 - Tex1 Uap56 - Mlo3 Tex1 - Tex1 Mlo3 - Mex67 Tex1 - Tex1 Mex67 - Rae1 Tex1 - Tex1 Rae1 + Sac3 Tex1 - Tex1 Sac3 - Pci2 Tex1 - Tex1 Pci2 - Dss1 Tex1 - Tex1 Dss1 + Cdc31 Tex1 - Tex1 Cdc31 - Sus1 Tex1 - Tex1 Sus1 + a X indicates the empty vector. 한다. 이전연구에서 S. pombe THO 복합체의구성요소중진 화적으로잘보존된 hpr1와 tho2 유전자는생존과 bulk mrna 의방출에필수적이고 (Lee and Yoon, 2012; Koh and Yoon, 2015), thoc5와 thoc7는필수적이지않지만결실되면생장과 mrna의방출에영향을미친다는것을관찰하였다 (Koh and Yoon, 2014). 이러한결과들은 S. pombe THO 복합체의다른 구성요소들과는다르게 Tex1/THOC3 상동체는생장과 bulk mrna의방출에별다른영향을미치지않는것을보여준다. 또한분열효모의 THO 복합체는모든구성요소들이항상하 나의복합체로작용하지않거나복합체내에서역할이다르다 는것도암시한다. sptex1 유전자의결실이생장과 mrna 방 출에영향을주지않았으므로, 반대로 sptex1를과발현 (overexpression) 시키면표현형에어떠한영향을미치는지알아보 았다. 강력한야생형 nmt1 프로모터를가진 prep3x 벡터를 이용하여, sptex1 단백질을정상적인상황보다훨씬많이과 발현하는균주를제작하였다. 하지만 sptex1 유전자가과발현 되더라도생장및 mrna 방출은야생형균주와별다른차이 가없이정상적이었다 ( 자료미제시 ). sptex1 단백질의세포내위치를알아보기위하여 GFP 유 전자를 sptex1의 ORF의 3 말단에붙인 sptex1-gfp::kan r DNA 절편을제작한후반수체인야생형균주인 AY217에형질전환 하여 sptex1 유전자위치에삽입된균주를선별하였다. 공초점 현미경으로 sptex1-gfp가발현되는균주를관찰한결과, sptex1-gfp 단백질은대부분핵안에서존재하였다 (Fig. 2). 미생물학회지제 53 권제 4 호

4 분열효모에서의 mrna export 295 전사, mrna 가공및방출에관여하는 THO/TREX 복합체의역할은핵안에한정된다는것을고려하면, sptex1가 THO/ TREX 복합체의구성요소라는가정을지지하는결과이다. Tex1/THOC3의출아효모상동체가정상적인생장과 mrna 방출에직접적으로관여하지않는것으로보이므로, 실제로다른 THO/TREX 및 TREX-2 구성요소와상호결합하는지를알아보기위해 yeast two-hybrid (Y2H) 분석을하였다. 이를위해이유전자들의 cdna를각각 lexa 작동자의 DNA 결합영역 (BD) 을가진 ptlexa4 벡터, 그리고 GAL4 활성화영역 (AD) 을가진 pgad424 벡터 (Clontech Laboratories, Inc.) 에클로닝하였다. 이렇게제작된벡터들은 DNA 염기서열분석을통해이상이없음을확인하였다. 제작한벡터들을 L40 균주 (MATa his3δ200 trp1-901 leu ade2 LYS::4lexAop- HIS3 URA3::8lexAop-LacZ GAL4) 에형질전환하였다. 먼저 GAL4 활성화영역과융합된 sptex1 (AD-Tex1) 또는 lexa 작동자의 DNA 결합영역 (BD) 과융합된 sptex1 (BD-Tex1) 이홀로존재할때, 리포터인 His3 유전자를발현시키지못하는것을확인하였다. AD-Tex1과 BD-X (DNA 결합영역을가진빈 ptlexa4 벡터 ) 를함께가진형질전환체, 그리고 AD-X ( 활성화영역을가진빈 pgad424 벡터 ) 와 BD-Tex1 를함께가진경우기대한바와같이모두 His3 유전자가발현되지않았다 (Table 1). Y2H 분석결과는 Tex1이 THO/TREX 구성인자중 Hpr1/THOC1과 Tho2/THOC2에각각상호작용하는것으로나타났으며, 다른 THO/TREX 및 TREX-2 구성인자들과는상호작용하지않는다는것을확인하였다. AD-Tex1 과 BD-THOC7, BD-Rae1, BD-Dss1, BD-Sus1의조합에서각각 His3 유전자가발현되었지만, THOC7, Rae1, Dss1, Sus1은 DNA 결합영역에융합되었을때, AD-Tex1 없이도홀로 His3 유전자를발현시킬수있었으므로 (Bae and Yoon, 2017), Tex1과의상호작용의결과로해석할수없었다. Tex1과 Hpr1/THOC1와의상호결합을더확인하기위해, 공동침전 (co-immunopreciptation, Co-IP) 실험을수행하였다. 이를위해 sphpr1 cdna의 3 말단에 TAP tag을붙인 sphpr1-tap::kan r DNA 절편이 sphpr1 유전자위치에삽입된 Hpr1-TAP 균주를사용하였다 (Koh and Yoon, 2015). 한편 sptex1 단백질의 N 말단에는 HA tag을붙이기위해 pslf273 벡터에이유전자의 cdna를클로닝하였다 (Forsburg and Sherman, 1997). 이벡터를 Hpr1-TAP 균주그리고 sphpr1-tap 단백질이발현되지않은대조군균주 (Hpr1) 에각각형질전환하였다. 형질전환된두균주에서세포추출물을추출하여 sphpr1-tap 단백질을 IgG-Sepharose beads로포획하였다. 대조군에는 sphpr1-tap 단백질이발현되지않으므로, HA로표지된 sptex1 단백질이정상적으로발 현되더라도 (Fig. 3, Lane 4), IgG-Sepharose beads로포획되지않았다 (Fig. 3, Lane 6). 반면, sphpr1-tap 단백질이 IgG beads 에포획된경우, HA-spTex1 은같이포획되었다 (Fig. 3, Lanes 1~3). 이결과는분열효모의 sptex1 단백질이 sphpr1 단백질과강하게결합한다는것을의미한다. Y2H와 Co-IP 결과들을종합하면 sptex1 도분열효모에서 THO/TREX 복합체의구성요소임을강력히시사한다. 적요 진핵생물에서 THO/TREX 복합체는전사신장, pre-mrna 가공및 mrna의핵에서방출에중요한역할을담당한다. 이복합체는진화적으로잘보존되어있지만, 생명체에따라구성성분과기능에차이가존재한다. 이논문에서는출아효모보다는고등생물과더유사한분열효모 Schizosaccharomyces pombe에서 THO/TREX 복합체의한구성요소인 sptex1가생장과 mrna의방출에필수적이지않다는것을보였다. sptex1 유전자의결실과과발현어느것도생장의결함과 poly(a) + RNA가핵안에축적되는현상을거의보이지않았다. 또한 sptex1-gfp 단백질은주로핵안에위치하였다. Yeast two-hybrid와 Co-immunoprecipitation 분석에서 S. pombe Tex1 은 THO/TREX 복합체의주요구성인자인 sphpr1 (THOC1), sptho2 (THOC2) 과상호작용을하였다. 이와같은결과들은 S. pombe의 Tex1도 THO/TREX 복합체의구성인자이지만, 생장과 mrna 방출에는중요한역할을하지않음을의미한다. 감사의말 이논문은 2015년도성신여자대학교학술연구조성비지원에의하여연구되었음. References Bae, S.J. and Yoon, J.H Effects of Sus1, a component of TREX-2 complex, on growth and mrna export in fission yeast. Korean J. Microbiol. 53, Chanarat, S., Seizl, M., and Strässer, K The Prp19 complex is a novel transcription elongation factor required for TREX occupancy at transcribed genes. Genes Dev. 25, Chavez, S., Beilharz, T., Rondon, A.G., Erdjument-Bromage, H., Tempst, P., Svejstrup, J.Q., Lithgow, T., and Aguilera, A Korean Journal of Microbiology, Vol. 53, No. 4

5 296 Bae et al. A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae. EMBO J. 19, Cheng, H., Dufu, K., Lee, C.S., Hsu, J.L., Dias, A., and Reed, R Human mrna export machinery recruited to the 5' end of mrna. Cell 127, Heath, C.G., Viphakone, N., and Wilson, S.A The role of TREX in gene expression and disease. Biochem. J. 473, Katahira, J Nuclear export of messenger RNA. Gene 6, Koh, E. and Yoon, J.H Effects of spthoc7 deletion on growth and mrna export in fission yeast. Korean J. Microbiol. 50, Koh, E. and Yoon, J.H Effects of Tho2, a component of THO complex, on growth and mrna export in fission yeast. Korean J. Microbiol. 51, Lee, H. and Yoon, J.H Effects of the repression of sphpr1 expression on growth and mrna export in fission yeast. Korean J. Microbiol. 48, Masuda, S., Das, R., Cheng, H., Hurt, E., Dorman, N., and Reed, R Recruitment of the human TREX complex to mrna during splicing. Genes Dev. 19, Meinel, D.M., Burkert-Kautzsch, C., Kieser, A., O'Duibhir, E., Siebert, M., Mayer, A., Cramer, P., Söding, J., Holstege, F.C., and Sträßer, K Recruitment of TREX to the transcription machinery by its direct binding to the phospho-ctd of RNA polymerase II. PLoS Genet. 9, e Rehwinkel, J., Herold, A., Gari, K., Köcher, T., Rode, M., Ciccarelli, F.L., Wilm, M., and Izaurralde, E Genome-wide analysis of mrnas regulated by the THO complex in Drosophila melanogaster. Nat. Struct. Mol. Biol. 11, Rodriguez-Navarro, S. and Hurt, E Linking gene regulation to mrna production and export. Curr. Opin. Cell Biol. 23, Roug le, M., Dieppois, G., Kisseleva-Romanova, E., Gudipati, R.K., Lemoine, S., Blugeon, C., Boulay, J., Jensen, T.H., Stutz, F., Devaux, F., et al THO/Sub2p functions to coordinate 3'-end processing with gene-nuclear pore association. Cell 135, Stewart, M Nuclear export of mrna. Trends Biochem. Sci. 35, Strasser, K., Masuda, S., Mason, P., Pfannstiel, J., Oppizzi, M., Rodriguez-Navarro, S., Rondon, A.G., Aguilera, A., Struhl, K., Reed, R., et al TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, Yoon, J.H., Love, D., Guhathakurta, A., Hanover, J.A., and Dhar, R Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mrna export. Mol. Cell. Biol. 20, Zenklusen, D., Vinciguerra, P., Wyss, J.C., and Stutz, F Stable mrnp formation and export require cotranscriptional recruitment of the mrna export factors Yra1p and Sub2p by Hpr1p. Mol. Cell. Biol. 22, 미생물학회지제 53 권제 4 호

Korean Journal of Microbiology (2015) Vol. 51, No. 4, pp pissn DOI eissn Copyrigh

Korean Journal of Microbiology (2015) Vol. 51, No. 4, pp. 435-439 pissn 0440-2413 DOI http://dx.doi.org/10.7845/kjm.2015.5058 eissn 2383-9902 Copyright c 2015, The Microbiological Society of Korea 단보 THOC5