Introduction

　RNA splicing is one of the major steps in the control of gene expression in eukaryotes.Genes are often interrupted by intervening sequences(IVSs or introns)that must be removed by a two-step transesterification mechanism,and the functional sequences(exons)are joined together before they are translated into proteins.A defect in splicing of certain introns results in production of aberrant proteins.In the cells,some molecular mechanisms have been identified to be responsible for preventing pre-mRNAs from being exported to the cytoplasm before they are correctly processed.For example, the branchpoint splicing factor,BBP/SF1,and the newly identified RES complex have been shown to be required for pre-mRNA retention in the nucleus.Most recently,Mlp1,a protein localized at the nuclear periphery,plays an important role in preventing pre-mRNAs from being exported to the cytoplasm.To date,there are several lines of evidence for the link between these error-induced mRNAs and human diseases.For example,retinitis pigmentosa(RP),a retinal degeneration that causes blindness,is due to mutations in the autosomal dominant RP(adRP)genes.However,the molecular mechanisms underlying the pathogenesis of RP are not well understood.Many RP genes are expressed predominantly in retina,and four nonretina-specific adRP genes have been shown to encode pre-mRNA splicing factors involved in the spliceosome assembly.It has been reported that some mutations in several RP genes are responsible for RNA processing defects and retinal degeneration.Thus elucidation of the mechanism of mRNA processing will help find new therapeutic strategies for genetic disease treatments and for a better understanding of genetic information and gene expression.

　Classical genetics,which starts from the identification of particular mutations,is one of the best approaches for understanding the cellular function of a protein.In recent years,chemical genetics involving the use of small molecules that inhibit the function of proteins has been widely used for unveiling the biological systems forming complicated networks.

　FR901464(FR)is a novel antitumor substance,which promotes the transcription of the SV40 early gene.FR has also been shown to induce arrest in G1 and G2/M phases in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as for activation of the SV40 promoter-dependent cellular transcription in the reporter-introduced MCF7(M-8)cells.In this laboratory,FR induced accumulation of a C-terminal truncated form of one of the CDK inhibitors,p27.Extension studies have shown that the short form of p27 was intron-containing mRNA transcripts.However,the mechanism by which FR induces translation of the unspliced mRNAs is yet to be elucidated.In this study,fission yeast Schizosaccharomyces pombe was used as a genetic model system to identify the molecular target of FR.

　Biotinylated-FR binding proteins

　For determination of the mode of action of FR that inhibits pre-mRNA processing,a biotinylated FR(bio-FR)derivative was synthesized as an affinity probe for identification of cellular FR-binding proteins.Prp10,a spliceosome-associated factor in the SF3b complex,was isolated to be the FR binding protein by LC-MS/MS.Studies have shown that the SF3b complex in the mammalian cells contains SAP49/SF3b50, SAP130/SF3b130,SAP145/SF3b145,SAP155/SF3b155,and two other small proteins,SF3b14b and SF3b10.The homologues corresponding to these subunits are identified in S.pombe as Sap49,Prp12,Sap145,Prp10,Ini1,and SPBC211.05,respectively.These proteins associate with one another in purified U2 snRNP in the nuclear extracts and play an important role in excision of introns from pre-mRNA.

　The ability of FR to bind to other SF3b components was analyzed by pull-down assay using green fluorescent protein(GFP)-tagged Prp12 and Sap145.As a result,bio-FR also bound to GFP-tagged Prp12.Furthermore,a stronger GFP signal was detected when the binding assay was performed in high salt condition(300 mM NaCl).A previous study reported that the yeast SF3b complex was released from the larger U2snRNP complex when the KCl concentration was increased from 150 mM to 500 mM.In this study,an increase in the salt concentration may release the SF3b complex from U2snRNP,thereby allowing FR to bind to the target proteins.However,the binding of bio-FR to GFP-tagged Sap145 was yet to be detected.In competition-binding assay,the binding was completely blocked when the active FR was added in the assay 1 h before the addition of bio-FR,indicating that the active FR competed with bio-FR for binding to target proteins.Similar experiments showed that inactive FR did not compete with bio-FR,suggesting that bio-FR specifically binds to Prp1O and Prp12.In addition, the association was unstable in the presence of SDS,indicating that the interaction may be mediated by non-covalent binding to FR.

　FR901464 induced pre-mRNA accumulation and translation in fission yeast.

　Effects of the FR binding to SF3b on in vivo splicing in S.pombe were examined by using reverse transcriptase-PCR.Because the wild-type cells shows resistance to FR,pmdl,a gene encoding an ATP-binding cassette(ABC) transporter,was disrupted.The mutants were viable and exhibited hypersensitivity to FR.RT-PCR analysis on the TFIID gene containing three introns showed accumulation of the TFIID pre-mRNA in the presence of FR.The occurrence of unspliced mRNA was accompanied by a strong decrease in the mature mRNA level.

　If the accumulated pre-mRNAs are allowed to be translated,the proteins containing polypeptides derived from the intron sequences should be generated.For detection of proteins containing intron-derived sequences,a reporter was designed(Fig. 2),in which only unspliced mRNA could be translated into the functional enzyme,β-galactosidase.This reporter was introduced into the pmdlΔ mutant strain.The β-galactosidase assay indicated that unspliced mRNAs were exported and translated into the enzyme in FR-treated cells.

　Abnormal nuclear morphology induced by FR901464.

　FR also induced abnormal nuclear morphology in fission yeast.The result of DAPI-staining 3 h after the FR challenge showed that the nucleus was deformed in a way that the nucleolar region was expanded.For further investigation of the mechanism underlying the abnormal nuclear structure,the localization of several nucleolar proteins tagged with YFP was observed.Some of the YFP-tagged proteins appeared to have increased in the intensity of the fluorescence after treatment with FR,thereby causing the abnormal nucleolar structure.In contrast,cellular distribution of proteins that are normally localized in the nuclear envelope was not changed.

　Pre-mRNA processing mutants

　Prp10 and prp12 of the fission yeast encode the essential proteins highly homologous with human splicing factors.Temperature-sensitive(ts) mutations of prp10 and prp12 cause defects in pre-mRNA splicing at the nonpermissive temperature.In this study,FR was found to bind to Prp10 and Prp12 in the bio-FR binding assay.Therefore,it seems likely that the mutations allow translation of the pre-mRNAs.In support of this hypothesis,the two prp mutants were transformed with the reporter and β-galactosidase activity was assayed after a 3-h incubation at the restrictive temperature.As expected, the β-galactosidase activity at a relatively high level was detected in the mutants,indicating that unspliced mRNAs accumulated in the cells were translated into the proteins.Several other prp ts mutants transformed with the reporter also exhibited β-galactosidase activity after shifting to the nonpermissive temperature.

　Conclusions

1. Splicing factor SF3b was identified as the FR target in this study.

2. Temperature-sensitive mutations of prp10 and prp12 caused accumulation and translation of pre-mRNAs.

3. Aberrant nuclear structure was observed in FR-treated cells, accompanied by the expanding of nucleolar region that might cause the abnormality in the nuclear structure.

Fig.1 FR901464

Fig.2 A reporter system was used to detect pre-mRNA translation.

FR901464は微生物の培養液から単離された低分子化合物であり、SV40プロモータを介した転写の活性化、強い抗腫瘍活性、細胞周期停止作用など、様々な興味深い作用が動物細胞を用いた実験より明らかになっている。また、FR901464処理細胞中ではpre-mRNAが蓄積し、そのpre-mRNAから異常なタンパク質が翻訳される。しかし、その標的分子や作用メカニズムは明らかになっていない。本論文は、比較的ゲノムサイズが小さく、遺伝学的操作が可能であり、また、45％の遺伝子にイントロンが含まれ、様々な細胞内メカニズムが出芽酵母に比べ高等真核生物に近い分裂酵母を用い、FR901464の標的分子の探索、ならびにその作用メカニズムの解明を述べたものである。

(1)　分裂酵母に対するSSAの影響

　実際の実験においては、FR901464のメチルアセタール体であるspliceostatin A(SSA)を用いた。SSAはFR901464と同等の活性を持ち、FR901464より安定である。分裂酵母の細胞増殖に対するSSAの効果を調べたところ、2.5μg/mlのSSAで増殖が阻害された。動物細胞同様にSSA処理細胞中では、pre-mRNAの蓄積が観察された。また、イントロンを含んだpre-mRNAからの翻訳を検出するシステムを構築し、システムを導入した細胞をSSA処理したところ、pre-mRNAからの翻訳が観察された。以上のことから、分裂酵母においても動物細胞と同様のメカニズムが存在することが示唆された。そこで、分裂酵母細胞抽出液からSSA標的タンパク質を同定することとした。

(2)　SSA標的タンパク質の同定

　ビオチン化SSAを、分裂酵母細胞抽出液に加え、ストレプトアビジンビーズを用いSSA結合タンパク質を精製し、質量分析により同定したところ、スプライシング因子であるPrp10pがSSA結合タンパク質として同定された。次に確かにPrp10pがSSA結合タンパク質であるかを確かめるために、GFPタグを付加したPrp10pを発現させ、SSAならびにストレプトアビジンビーズを用いた沈降実験を行なったところ、GFP-Prp10pが沈降した。Prp10pは7つのタンパク質からなるSF3b複合体の構成タンパク質であり、SF3b複合体はスプライシング反応に必要なスプライソソームの構成因子である。そこで、SF3b複合体の他の構成因子であるPrp12pともSSAが結合するかを確かめたところ、Prp10pと同様の結果が得られた。この結果から、SSAはSF3b複合体と結合することが強く示唆された。

(3)　prp変異株におけるpre-mRNAの蓄積ならびにその翻訳

　現在までに、pre-mRNAのスプライシングに関わるprp遺伝子が数多く単離されており、前述のprp10、prp12もその中に含まれる。そこで、prp10、prp12を含むいくつかのprp変異株においてpre-mRNAが蓄積するかを確かめたところ、非許容温度においてSSA処理細胞と同様にpre-mRNAが蓄積していた。さらに、これらの変異株中ではpre-mRNAの翻訳も観察された。他のprp変異株中、prp1、prp2においてはprp10、prp12と同様の結果が得られたものの、prp4、prp8に関しては非許容温度でのpre-mRNAの蓄積、ならびにその翻訳は認められなかった。これらの結果から、pre-mRNAが核内に蓄積すればその一部が細胞質に漏れだし翻訳されるという可能性は捨てきれないものの、Prp10p、Prp12pはPrp1pやPrp2pなどとともに、pre-mRNAスプライシングのみならず、pre-mRNAの核内繋留にも機能していることが示唆された。

　以上の結果から、FR901464ならびにspliceostatin Aは、スプライソソームの構成因子であるPrp10pやPrp12pに結合することにより、その機能を阻害し、その結果pre-mRNAの蓄積ならびに翻訳を引き起こしていることが強く示唆された。

　本研究から得られた知見は、翻訳されるべきmRNAのみを翻訳するという、生物にとって必要不可欠な品質管理機構を理解する上で重要な情報を与えるものである。よって、審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。