Mitsuo Sekine

Snurportin1, an m3G‐cap‐specific nuclear import receptor with a novel domain structure

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5′‐2,2,7‐terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G‐cap but not m7G‐cap structures. Snurportin1 enhances the m3G‐capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin‐permeabilized HeLa cells, demonstrating that it functions as an snRNP‐specific nuclear import receptor. Interestingly, solely the m3G‐cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N‐terminal importin β binding (IBB) domain, essential for function, and a C‐terminal m3G‐cap‐binding region with no structural similarity to the arm repeat domain of importin α.

Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap.

We have investigated the nuclear transport of U1 and U5 snRNPs by microinjection studies in oocytes from Xenopus laevis using snRNP particles prepared by reconstitution in vitro. Competition studies with snRNPs showed that the Sm core domain of U1 snRNPs contains a nuclear location signal that acts independently of the m3G cap. The transport of U1 snRNP can be blocked by saturation with competitor U1 snRNPs or by U5 snRNPs, which indicates that the signals on the respective Sm core domains interact with the same transport receptors. Further, by using a minimal U1 snRNP particle reconstituted in vitro and containing only the Sm core RNP domain and lacking stem‐loops I to III of U1 RNA, we show that this is targeted actively to the nucleus, in spite of the absence of the m3G cap. This indicates that under certain conditions the NLS in the Sm core domain not only is an essential, but may also be a sufficient condition for nuclear targeting. We propose that the RNA structure of a given snRNP particle determines at least in part whether the particles m3G cap is required for nuclear transport or can be dispensed with.

Efficient synthesis of γ-methyl-capped guanosine 5′-triphosphate as a 5′-terminal unique structure of U6 RNA via a new triphosphate bond formation involving activation of methyl phosphorimidazolidate using ZnCl2 as a catalyst in DMF under anhydrous conditions

P1-Methyl P3-(5′-guanosyl) triphosphate (CH3pppG), which exists as a unique structure at the 5′-terminal region of U6 RNA, was synthesized in an excellent yield by methyl phosphorimidazolidate (2c) with the bis(tetrabutylammonium) salt of GDP in the presence of ZnCl2 as an effective catalyst in dry DMF.

Complete Genome Sequence of the Soil Actinomycete Kocuria rhizophila

The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae, a divergent bacterial group for which only a limited amount of genomic information is currently available. K. rhizophila is also important in industrial applications; e.g., it is commonly used as a standard quality control strain for antimicrobial susceptibility testing. Sequencing and annotation of the genome of K. rhizophila DC2201 (NBRC 103217) revealed a single circular chromosome (2,697,540 bp; G+C content of 71.16%) containing 2,357 predicted protein-coding genes. Most of the predicted proteins (87.7%) were orthologous to actinobacterial proteins, and the genome showed fairly good conservation of synteny with taxonomically related actinobacterial genomes. On the other hand, the genome seems to encode much smaller numbers of proteins necessary for secondary metabolism (one each of nonribosomal peptide synthetase and type III polyketide synthase), transcriptional regulation, and lateral gene transfer, reflecting the small genome size. The presence of probable metabolic pathways for the transformation of phenolic compounds generated from the decomposition of plant materials, and the presence of a large number of genes associated with membrane transport, particularly amino acid transporters and drug efflux pumps, may contribute to the organisms utilization of root exudates, as well as the tolerance to various organic compounds.

2-(Azidomethyl)benzoyl as a new protecting group in nucleosides ☆

Abstract A new protecting group, 2-(azidomethyl)benzoyl (AZMB), which can be easily removed by treatment with MePPh 2 in dioxane–H 2 O or reduction with HCOONH 4 –Pd/C in dioxane–MeOH, was developed for protection of the hydroxy and amino functions of deoxyribonucleosides.

New guanosine derivatives: facile O6-phosphorylation, thiophosphinylation sulfonylation and silylation of guanosine derivatives by 4-dimethylaminopyridine catalized reaction

Abstract Appropriately protected guanosine derivatives were successfully converted to the corresponding O 6 -substituted guanosine derivatives by treatment with dialkyl- or diaryl-phosphoryl halides, dialkyl- or diaryl-phosphinothioyl halides, arenesulfonyl chlorides, and trialkylsilyl chlorides.

One-step synthesis of 5′-azido-nucleosides

Regioselective azidation of unprotected or appropriately protected nucleosides was conducted by means of the reagent triphenylphosphine–carbon tetrabromide–lithium azide. By use of this reagent, 5′-azido-5′-deoxynucleosides were prepared conveniently in one step from nucleosides in high yields. Secondary hydroxy-groups of appropriately 5′-protected nucleosides were also converted by the reagent to azido-functions with complete inversion.

Synthesis and properties of 2-azidodeoxyadenosine and its incorporation into oligodeoxynucleotides

Abstract 2-Azidodeoxyadenosine ( 7 ) was conveniently synthesized from deoxyguanosine ( 2 ) by use of a combined reagent of TMSN 3 –BuONO. The structure of the tautomer of the azido derivative was determined by 1 H NMR. Reaction of 7 with i Pr 2 NP(OEt) 2 gave an intermediate 10 of the Staudinger reaction. Incorporation of 7 into a DNA 13mer resulted in a significant decrease of the T m value of the DNA duplex upon hybridization with the complementary strand. The thermal stability was discussed based on the hydrogen bond energy and electrostatic repulsion.

Diphenylcarbamoyl and propionyl groups: a new combination of protecting groups for the guanine residue

Abstract Protection of the guanine residue with the O 5 -diphenylcarbamoyl and N 2 -propionyl groups is described. These protecting groups could be readily introduced and removed simultaneously. They were used to demonstrate the synthesis of the deoxyguanylate dimer in high yield.

Phthaloyl group : a new amino protecting group of deoxyadenosine in oligonucleotide synthesis

Abstract A phthaloyl group has been introduced into the N 6 -amino group of deoxyadenosine via silylation followed by acylation. The phthaloyl group resulted in remarkable retarding effects on depurination, while it could be removed under milder conditions than the benzoyl group. Thus, a tetradeoxyadenylate has been successfully synthesized in high yield.