Kunio Inoue

A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG

Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue‐specific RNA‐binding protein, Fox‐1, which regulates alternative splicing in vertebrates. Fox‐1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox‐1 is expressed in heart and skeletal muscles. As candidates for muscle‐specific targets of Fox‐1, we considered two genes, the human mitochondrial ATP synthase γ‐subunit gene (F1γ) and the rat α‐actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox‐1 induced muscle‐specific exon skipping of the F1γ gene via binding to GCAUG sequences upstream of the regulated exon. Fox‐1 also regulated mutually exclusive splicing of the α‐actinin gene, antagonizing the repressive effect of polypyrimidine tract‐binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox‐1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox‐1 plays key roles in both positive and negative regulation of tissue‐specific splicing via GCAUG.

Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence.

Maternal mRNA localization of zebrafish DAZ-like gene.

Members of the DAZ gene family encode RNA-binding proteins and have been shown to play a pivotal role in gametogenesis. In Xenopus, a DAZ-like gene encodes an RNA component of the germ plasm. We have identified a zebrafish DAZ homologue, zDazl. zDazl mRNA was expressed in gonads of both sexes. In ovary, it was localized in the cortex of oocytes. At the onset of embryogenesis, maternal zDazl mRNA was detected at the vegetal pole. It migrated toward blastomeres through cytoplasmic streams as early embryogenesis proceeded. This is the first report showing maternal mRNA localization at the vegetal pole in fish and the existence of mRNA streams in the yolk cytoplasm.

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression. Methodology/Principal Findings Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430. Conclusions/Significance Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.

Zebrafish DAZ-like protein controls translation via the sequence 'GUUC'.

Background: In many species, DAZ homologous genes encode RNA‐binding proteins containing two conserved motifs, namely the RNA‐recognition motif (RRM) and the DAZ motif. Genetic analysis and gene disruption studies have demonstrated that DAZ family proteins play important roles in gametogenesis. However, little is known about the biochemical functions of DAZ family proteins.

Regulation of alternative splicing of α‐actinin transcript by Bruno‐like proteins

Background: The Bruno‐like or CELF proteins, such as mammalian CUGBP1 and Etr‐3, Xenopus EDEN‐BP, and Drosophila Bruno (Bru), are regulators of gene expression at the post‐transcriptional level, and contain three RNA‐recognition motifs (RRMs). It has been shown that mammalian CUGBP1 and Etr‐3 regulate alternative splicing of cardiac troponin T pre‐mRNA via binding to CUG‐triplet repeats.

Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core, Catalyzed by the GlcNAc-1-phosphate Transferase WecA, by the Novel Caprazamycin Derivative CPZEN-45

Background: Because CPZEN-45 is a promising antituberculous drug candidate, the identification of the target is required. Results: CPZEN-45 inhibits the decaprenyl-phosphate–GlcNAc-1-phosphate transferase of Mycobacterium tuberculosis and the corresponding enzyme of Bacillus subtilis responsible for initiation of cell wall synthesis. Conclusion: CPZEN-45 inhibits a novel target in cell wall assembly. Significance: This study is critical for launching CPZEN-45 and for exploitation toward new antituberculous drugs. Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate–GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.

Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5′ cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

Vegetal localization of the maternal mRNA encoding an EDEN-BP/Bruno-like protein in zebrafish.

Asymmetric distribution of maternal mRNAs has not been well documented in zebrafish. Recently, we have shown that dazl mRNA is localized at the vegetal pole. Here we report a novel zebrafish gene, bruno-like (brul), which provides another example of vegetal mRNA localization. brul encodes an Elav-type RNA-binding protein that belongs to the Bruno-like family that includes mammalian CUG-BP, Xenopus EDEN-BP, and Drosophila Bruno. At 24 hpf, brul mRNA was abundant in lens fiber cells. At the onset of embryogenesis, maternal brul mRNA was detected at the vegetal pole, and it then migrated rapidly toward the blastoderm through yolk cytoplasmic streams. During oogenesis, brul mRNA became localized at the vegetal cortex at stage II, later than dazl mRNA. We found that anchoring of brul mRNA was dependent on microfilaments.

Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1γ gene

Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U)GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U)GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1γ gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.