Yoshiro Shimura

Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein

The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) messenger RNA precursor by binding to the tra polypyrimidine tract during the sex-determination process. The crystal structure has now been determined at 2.6 Å resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The two RNA-binding domains have their β-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers the first insight, to our knowledge, into how a protein binds specifically to a cognate RNA without any intramolecular base-pairing.

Role of a positive regulator of root hair development, CAPRICE ,in Arabidopsis root epidermal cell differentiation

In Arabidopsis, root hairs are formed only from a set of epidermal cells named trichoblasts or hair-forming cells. Previous studies showed CAPRICE (CPC) promotes differentiation of hair-forming cells by controlling a negative regulator, GLABRA2 (GL2), which is preferentially expressed in hairless cells. Here, we show that CPC is also predominantly expressed in the hairless cells, but not in the neighboring hair-forming cells, and that CPC protein moves to the hair-forming cells and represses the GL2 expression. We also show that the N terminus of bHLH protein interacts with CPC and is responsible for the GL2 expression. We propose a model in which CPC plays a key role in the fate-determination of hair-forming cells.

Polypurine sequences within a downstream exon function as a splicing enhancer.

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.

Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila

Sex-specific alternative processing of doublesex (dsx) precursor messenger RNA (pre-mRNA) regulates somatic sexual differentiation in Drosophila melanogaster. Cotransfection analyses in which the dsx gene and the female-specific transformer (tra) and transformer-2 (tra-2) complementary DNAs were expressed in Drosophila Kc cells revealed that female-specific splicing of the dsx transcript was positively regulated by the products of the tra and tra-2 genes. Furthermore, analyses of mutant constructs of dsx showed that a portion of the female-specific exon sequence was required for regulation of dsx pre-messenger RNA splicing.

FILAMENTOUS FLOWER CONTROLS THE FORMATION AND DEVELOPMENT OF ARABIDOPSIS INFLORESCENCES AND FLORAL MERISTEMS

Phenotypic analysis of single and multiple mutants as well as in situ localization analysis of the expression patterns of floral genes have revealed that the FILAMENTOUS FLOWER (FIL) gene plays important roles in establishing the inflorescence in Arabidopsis. As previously reported, the fil mutant generates clusters of both filamentous structures and flowers with floral organs of altered number and shape. The structural resemblance of the filamentous structures to peduncles and the expression pattern of the APETALA1 (AP1) gene have shown that these filamentous structures are underdeveloped flowers that fail to form receptacles and floral organs, indicating that one of the roles of the FIL gene is to support the development of the floral meristem. That FIL also is involved in fate determination in the floral meristem is demonstrated by the homeotic conversion of flowers to inflorescences in fil ap1 double mutants and in fil ap1 cauliflower triple mutants. In double mutants with flowering-time loci (i.e., ft or fwa), leafy (lfy), and unusual floral organs (ufo), filamentous structures are formed, but very few or no flowers with floral organs develop. The enhanced phenotype in the fil ap1 and the fil lfy double mutants suggests that the FIL protein may work together with AP1 and LFY proteins. The FIL gene also may be involved in the cell fate determination of floral organ primordia, possibly by controlling the spatial expression patterns of the class A and C floral organ identity genes.

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and se-quenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22.

In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned. Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes. However, any such splicing factor containing the helicase motifs in vertebrates has not been identified. To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in a S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family. We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. We speculate that HRH1 might be targeted to the spliceosome through this interaction.

The primary structure of iodopsin, a chicken red-sensitive cone pigment

A purified iodopsin was digested by CNBr or several proteolytic enzymes into fragments, the amino acid sequences of which were determined. A partial sequence of the C‐terminal fragment was utilized for synthesizing an oligonucleotide probe which identified the iodopsin cDNA (1339 bases). The deduced amino acid sequence (362 residues) had 80%, 42% or 43% homology to that of human red‐sensitive cone pigment, cattle or chicken rhodospin, respectively. Although the hydropathy profile implies that iodopsin, like rhodopsin, has 7 transmembrane α‐helical segments, iodopsin may have a hydrophilic pocket near the seventh segment on the basis of the unexpected cleavages in the middle of the segment VII by chymotrypsin under nondenaturing conditions.

Mutant tyrosine tRNA of altered amino acid specificity

Each set of tRNA molecules accepting a particular amino acid are recognized and acylated by an amino acyl tRNA synthetase. This enzyme must recognize some feature in this set of tRNA molecules which distinguishes them from all other tRNAs. In this communication we describe the isolation and initial characterization of a set of E. coli tyr tRNA mutants that have altered amino acid specificity. These mutants should eventually lead to an understanding of which features of the tRNA are recognized by the tyrosine tRNA synthetase. Sequence analysis of one of the mutants (SU+III~-“~) has revealed that a single base change (A82 + G) near the amino acid acceptor end of the molecule is sufficient to alter its specificity.

Precursor molecules of Escherichia coli transfer RNAs accumulated in a temperature-sensitive mutant☆

Abstract Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su+ 3 tRNA1Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.