The structure of the 5′-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation

Analysis of the human protein-tyrosine phosphatase (PTP) PTPRJ mRNA detected three in-frame AUGs at the 5′-end (starting at nt +14, +191 and +356) with no intervening stop codons. This tandem AUG arrangement is conserved between humans and the mouse and is unique among the genes of the classical PTPs. Until now it was assumed that the principal open reading frame (ORF) starts at AUG356. Our experiments showed that: (i) translation of the mRNA synthesized under the PTPRJ promoter starts predominantly at AUG191, leading to the generation of a 55 amino acid sequence preceding the signal peptide; (ii) the longer form is being likewise correctly processed into mature PTPRJ; (iii) the translation of the region between AUG191 and AUG356 inhibits the overall expression, a feature which depends on the sequence of the encoded peptide. Specifically, a sequence of 13 amino acids containing multiple arginine residues (RRTGWRRRRRRRR) confers the inhibition. In the absence of uORF these previously unrecognized characteristics of the 5′-end of the mRNA present a novel mechanism to suppress, and potentially to regulate translation

Experimental studies of proton-neutron mixed symmetry states in the mass A ∼ 130 region

Considerable progress has been achieved recently in the experimental investigation of quadrupole-collective isovector excitations in the valence shell, the so called mixed-symmetry states (MSSs), in the mass A ≈ 130 region. This is due to a new experimental technique for study MSSs which is based on the observation of low-multiplicity γ-ray events from inverse kinematics Coulomb excitation with the large 4π spectrometer, such as Gammasphere. The obtained experimental information for the MSSs of stable N 80 isotones indicates that for low-collective vibrational nuclei the underlying single-particle structure can be the most important factor for preserving or fragmenting the MSSs through the mechanism of shell stabilization. The evolution of the MSSs from 134Xe to 138Ce is also used to determine the local strength of the proton-neutron interaction derived for first time from states with symmetric and antisymmetric nature

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection

The translation attenuating arginine-rich sequence in the extended signal peptide of the protein-tyrosine phosphatase PTPRJ/DEP1 is conserved in mammals.

The signal peptides, present at the N-terminus of many proteins, guide the proteins into cell membranes. In some proteins, the signal peptide is with an extended N-terminal region. Previously, it was demonstrated that the N-terminally extended signal peptide of the human PTPRJ contains a cluster of arginine residues, which attenuates translation. The analysis of the mammalian orthologous sequences revealed that this sequence is highly conserved. The PTPRJ transcripts in placentals, marsupials, and monotremes encode a stretch of 10-14 arginine residues, positioned 11-12 codons downstream of the initiating AUG. The remarkable conservation of the repeated arginine residues in the PTPRJ signal peptides points to their key role. Further, the presence of an arginine cluster in the extended signal peptides of other proteins (E3 ubiquitin-protein ligase, NOTCH3) is noted and indicates a more general importance of this cis-acting mechanism of translational suppression

Expression and localization of PCSK9 in rat hepatic cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), recently cloned in several laboratories, including ours, causes a third form of autosomal dominant hypercholesterolemia. Its mechanism of action remains unclear. We studied the expression and subcellular localization of PCSK9 in fetal and adult rat tissues associated with cholesterol homeostasis using quantitative reverse transcriptase--PCR, Western blot analysis, subcellular fractionation, and confocal immunofluorescent microscopy. PCSK9 mRNA is most abundant in yolk sac and fetal liver, but the highest expression of the protein was found in differentiated hepatoma FAO-1 cell line, which also shows the highest expression of LDLR. In FAO-1 cells PCSK9 expression is downregulated by cholesterol and 25-hydroxycholesterol and upregulated in the absence of sterols following the same pattern of expression as HMG-CoA reductase, synthase, and LDLR. Subcellular fractionation, combined with Western blotting, showed that PCSK9 is localized in the ER and intermediate vesicular compartment of the cell but not in Golgi cisternae. The mature enzyme is secreted from the liver and hepatoma cells. Double labeling with antibodies to PCSK9 and LDLR or clathrin revealed some colocalization of PCSK9 with clathrin-coated vesicles and LDLR. In conclusion, our results show that PCSK9 is processed in the ER, and the mature convertase is secreted in the plasma

A single amino acid exchange inverts susceptibility of related receptor tyrosine kinases for the ATP-site inhibitor STI-571

The tyrosine kinase inhibitor STI-571 potently blocks BCR-Abl, platelet-derived growth factor (PDGF) a- and b-receptors, and c-Kit kinase activity. However Flt3, a receptor tyrosine kinase closely related to PDGF receptors and c-Kit, is not inhibited by STI-571. Sequence alignments of different kinases and indications from the crystal structure of the STI-571 Abl kinase complex revealed amino acid residues that are probably crucial for this activity profile. It was predicted that Flt3 Phe-691 in the b5 strand may sterically prevent interaction with STI-571. The point mutants Flt3 F691T and PDGFb-receptor T681F were constructed, and kinase assays showed that the Flt3 mutant but not the PDGFb-receptor mutant is inhibited by STI-571. Docking of STI-571 into computer models of the PDGFb-receptor and Flt3 kinase domains and comparison with the crystal structure of the STI-571 Abl kinase complex indicated very similar binding sites among the three nonphosphorylated kinases, suggesting corresponding courses of their Asp-Phe-Gly motifs and activation loops. Accordingly, we obsd. reduced sensitivity of preactivated compared with nonactivated PDGFR-b for the inhibition by STI-571. Courses of the activation loop that collide with STI-571 binding explain its inactivity toward other kinases such as the insulin receptor. The binding site models of PDGFR-b and Flt3 were applied to predict structural approaches for more selective PDGFb-receptor inhibitors

Experimental studies of proton-neutron mixed symmetry states in the mass A ≈ 130 region

Considerable progress has been achieved recently in the experimental investigation of quadrupole-collective isovector excitations in the valence shell, the so called mixed-symmetry states (MSSs), in the mass A ≈ 130 region. This is due to a new experimental technique for study MSSs which is based on the observation of low-multiplicity γ-ray events from inverse kinematics Coulomb excitation with the large 4π spectrometer, such as Gammasphere. The obtained experimental information for the MSSs of stable N = 80 isotones indicates that for low-collective vibrational nuclei the underlying single-particle structure can be the most important factor for preserving or fragmenting the MSSs through the mechanism of shell stabilization. The evolution of the MSSs from ¹³⁴Xe to ¹³⁸Ce is also used to determine the local strength of the proton-neutron interaction derived for first time from states with symmetric and antisymmetric nature