Chemical Genetics Laboratory

Outline

“Chemical Genetics” is one of the new molecular genetic fields in which mutations in classical genetics are replaced by specific chemicals called bioprobes, and by which their unique cellular phenotypes are analyzed at the molecular level. This approach is providing more and more new insights into the molecular mechanisms underlying cellular response, cell cycle control and molecular recognition. Our laboratory aims to establish comprehensive and systematic chemical genetics as a basis for chemical genomics by integrating genomics, proteomics, informatics and bioimaging. By this approach, we have identified novel modes of action of various bioactive natural products that target unprecedented molecules such as the spliceosome. Furthermore, using these bioprobes we elucidate cellular functions of molecules identified as the targets and their roles in the cell cycle, replication, signal transduction, aging and primary and secondary metabolisms. In particular, we focus on regulation of protein post-translational modifications and their interplays.

Recent Research Topic

Discovery of a splicing inhibitor to open a new avenue to understanding higher eukaryotic gene expression

Fig. 1 Molecular mechanism by which spliceostatin A inhibits splicing and nuclear retention of pre-mRNA

Fig. 2 Change in the morphology of nuclear speckles. (A)control, (B)drug-treated

In eukaryotes, protein-coding sequences (exons) are interrupted by intervening sequences (introns), which must be eliminated by splicing in the nucleus from pre-mRNA before nuclear export and translation. Splicing occurs by two sequential transesterification reactions. The precise excision of introns from pre-mRNA is carried out by a multi-protein complex known as the spliceosome1,4. The spliceosome comprises five small nuclear RNAs (snRNAs), U1, U2, U4/U6 and U5, which form the catalytic core of the splicing reaction, and over 150 proteins. Because introns often contain termination codons in frame with the upstream protein coding sequences, leakage of unspliced pre-mRNA into the cytoplasm can result in the production of aberrant, truncated and potentially deleterious proteins. Therefore, there are several systems to prevent pre-mRNA from being exported to the cytoplasm and translated.

The natural product FR901464 was isolated from the fermentation broth of a bacterium as a potent anticancer compound that enhances the transcriptional activity of the SV40 promoter and causes cell cycle arrest at the G1 and G2/M phases. Despite its high potency in inducing viral gene promoters, FR901464 paradoxically reduces the mRNA levels of several endogenous genes. This unprecedented pharmacological profile of FR901464 has drawn considerable interest, prompting us to further investigate its mechanism of action. Our studies showed that FR901464 and its methylated derivative spliceostatin A inhibit pre-mRNA splicing by binding non-covalently to the SF3b sub-complex in the U2 snRNP. Furthermore, our results uncover a surprising ability of spliceostatin A to leak pre-mRNA to the cytoplasm, thereby allowing translation of unspliced mRNAs. Thus, spliceostatin A is the first specific inhibitor of splicing and nuclear retention of pre-mRNA.

The snRNPs and some non-snRNP splicing factors are localized in the nuclear speckles, which are intensely stained and irregularly shaped regions of the nucleus. An early study showed that microinjection of oligonucleotides targeted to snRNA or antibodies that inhibit splicing in vitro resulted in reorganization of the nuclear speckles. The irregularly shaped speckles became larger, rounded, and decreased in number, suggesting that in vivo splicing inhibition causes reorganization of splicing factors in the nucleus. Spliceostatin A induced the altered morphology of the nuclear speckles. Undoubtedly, the dissection of splicing itself and its relation to nuclear speckle organization would greatly benefit from spliceostatin A.