Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export

The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5' end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

Two enzymes concerned in peptide hormone alpha-amidation are synthesized from a single mRNA.

By expressing truncated rat pituitary 'peptidylglycine alpha-amidating enzyme' cDNAs in COS-7 cells, we found that the two reactions concerned in peptide carboxyl-terminal amidation, namely the peptidylglycine alpha-hydroxylation reaction and the peptidyl-hydroxyglycine amidation reaction, were catalyzed by 37- and 53-K proteins, which were derived from the 5'- and 3'-coding sequences, respectively. The full-length cDNA directed the expression of both the 37- and 53-K enzymes, and in the combined presence of the two enzymes the full conversion of a glycine-extended peptide into the amidated product was achieved. These results indicated that two enzymes concerned in peptide hormone alpha-amidation are generated from a common precursor protein encoded by a single mRNA.     

Molecular orientation of tropoelastin is determined by surface hydrophobicity

Tropoelastin is the precursor of the extracellular protein elastin and is utilized in tissue engineering and implant technology by adapting the interface presented by surface-bound tropoelastin. The preferred orientation of the surface bound protein is relevant to biointerface interactions, as the C-terminus of tropoelastin is known to be a binding target for cells. Using recombinant human tropoelastin we monitored the binding of tropoelastin on hydrophilic silica and on silica made hydrophobic by depositing a self-assembled monolayer of octadecyl trichlorosilane. The layered organization of deposited tropoelastin was probed using neutron and X-ray reflectometry under aqueous and dried conditions. In a wet environment, tropoelastin retained a solution-like structure when adsorbed on silica but adopted a brush-like structure when on hydrophobized silica. The orientation of the surface-bound tropoelastin was investigated using cell binding assays and it was found that the C-terminus of tropoelastin faced the bulk solvent when bound to the hydrophobic surface, but a mixture of orientations was adopted when tropoelastin was bound to the hydrophilic surface. Drying the tropoelastin-coated surfaces irreversibly altered these protein structures for both hydrophilic and hydrophobic surfaces.     

Partial characterization of a tropoelastin precursor isolated from chick aorta.

Evidence is presented that indicates tropoelastin is derived from a soluble elastin with a molecular weight of 95000. Tropoelastin and its proposed precursor were isolated from the aortas of copper-deficient chicks. Although it is doubtful that the proposed precursor is an initial product of elastin translation, i.e., a proelastin, it is proposed to be at least a truncated form of proelastin that is converted to tropoelastin. The key to its isolation was the presence of alpha 1-antitrypsin at each step in the purification procedure. The first 11 amino acid residues at the NH2 terminal of the proposed tropoelastin precursor (GGVPGVAVPGGV) are the same as those for tropoelastin. Its amino acid composition is similar to that of tropoelastin, except for higher amounts of acidic amino acid residues. Further, the proposed precursor contains a limited number of aldehydic functions, presumably in the form of peptidyl allysine. This was taken as an indication that the proposed precursor serves as a substract for lysyl oxidase. Under the conditions used for the isolation, the precursor appeared to be in higher concentrations than tropoelastin in aorta extracts from copper-deficient chicks.     

Human fibronectin is synthesized as a pre-propolypeptide

Fibronectins (FNs) are extracellular glycoproteins consisting of dimers or multimers of similar but not identical subunits. The subunit differences result from variations in internal primary sequence due to alternative splicing in at least 2 regions of the pre-mRNA. The complete amino acid sequence of mature human cellular FN has been reported recently from cDNA cloning and sequencing. The same approach has now enabled us to deduce, for the first time, that FN has a 26 amino acid signal peptide and that it undergoes proteolytic processing at its N-terminus to eliminate a 5 amino acid pro-sequence (Ser-Lys-Ser-Lys-Arg). The signal sequence matches the consensus format, while this pro-sequence is a distinctive, very hydrophilic and basic peptide.     

Additional evidence for a proform to tropoelastin from chick aorta.

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10--20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.     

Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers.

A number of reports point to the presence of proteoglycans and/or glycosaminoglycans within elastic fibers in normal and in pathological conditions. We present data that heparan sulphate (HS)-containing proteoglycans are associated with normal elastic fibers in human dermis and that isolated HS chains interact in vitro with recombinant tropoelastin and with peptides encoded by distinct exons of the human tropoelastin gene (EDPs). By immunocytochemistry, HS chains were identified as associated with the amorphous elastin component in the human dermis and remained associated with the residual elastin in the partially degenerated fibers of old subjects. HS appeared particularly concentrated in the mineralization front of elastic fibers in the dermis of patients affected by pseudoxanthoma elasticum (PXE). In in vitro experiments, HS induced substantial changes in the coacervation temperature and in the aggregation properties of recombinant tropoelastin and of synthetic peptides (EDPs) corresponding to sequences encoded by exons 18, 20, 24 and 30 of the human tropoelastin gene. In particular, HS modified the coacervation temperature and favoured the aggregation into ordered structures of tropoelastin molecules and of EDPs 18, 20 and 24, but not of EDP30. These data strongly indicate that HS-elastin interactions may play a role in tissue elastin fibrogenesis as well as modulating elastin stability with time and in diseases.     

Wheat-germ agglutinin is synthesized as a glycosylated precursor

The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [³⁵S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with M_r 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of M_r 23 000 and the mature monomer of M_r 18000. When [³H]mannose was used in labeling studies, only the polypeptide of M_r 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of M_r 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745–6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - M_r relative molecular mass - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Concanavalin A is synthesized as a glycoprotein precursor

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (M_r) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with M_r 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-M_r polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 M_r. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [³H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, -mannosidase or -N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (M_r=30000). The 4000 decrease in M_r is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.Abbreviations Con A concanavalin A - Glc glucose - GlcNAc N-acetylglucosamine - IgG immunoglobulin G - Man mannose - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis