Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Selective destabilization of polypeptides synthesized from NMD-targeted transcripts

The translation of mRNAs that contain a premature termination codon (PTC) generates truncated proteins that may have toxic dominant negative effects. Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that degrades PTC-containing mRNAs to limit the production of truncated proteins. NMD activation requires a ribosome terminating translation at a PTC, but what happens to the polypeptides synthesized during the translation cycle needed to activate NMD is incompletely understood. Here, by establishing reporter systems that encode the same polypeptide sequence before a normal termination codon or PTC, we show that termination of protein synthesis at a PTC is sufficient to selectively destabilize polypeptides in mammalian cells. Proteasome inhibition specifically rescues the levels of nascent polypeptides produced from PTC-containing mRNAs within an hour, but also disrupts mRNA homeostasis within a few hours. PTC-terminated polypeptide destabilization is also alleviated by depleting the central NMD factor UPF1 or SMG1, the kinase that phosphorylates UPF1 to activate NMD, but not by inhibiting SMG1 kinase activity. Our results suggest that polypeptide degradation is linked to PTC recognition in mammalian cells and clarify a framework to investigate these mechanisms.     

Organization of early region 1B of human adenovirus type 2: identification of four differentially spliced mRNAs.

The mRNAs from early region 1B of adenovirus type 2 have been studied by Northern blot, S1 nuclease, and cDNA analysis. Two novel mRNAs, designated 14S and 14.5S, have been observed in addition to the previously identified 9S, 13S, and 22S mRNAs. They are 1.26 and 1.31 kilobases long and differ from the 13S and 22S mRNAs in being composed of three exons instead of two. Their two terminal exons are the same as those present in the 13S mRNA, whereas the middle exon is unique to each of the two novel mRNA species. The structures of the 14S and 14.5S mRNAs allow the prediction of their coding capacities: both mRNA species, like the 22S and 13S mRNAs, contain an uninterrupted translational reading frame encoding a 21,000-molecular-weight (21K) polypeptide. The 14S mRNA can, in addition, encode a 16.5K polypeptide which shares N-terminal and C-terminal sequences with the 55K polypeptide, known to be encoded by the 22S mRNA. The 14.5S mRNA species encodes a hypothetical 9.2K polypeptide which has the same N terminus as the 55K polypeptide but a unique C terminus. The two mRNAs differ in their kinetics of appearance; the 14.5S mRNA is preferentially expressed late after infection in contrast to the 14S mRNA, which is present in approximately equal amounts early and late after infection. Taken together with previously published information the results suggest that early region 1B of adenovirus type 2 encodes five proteins in addition to virion polypeptide IX. These have predicted molecular weights of 55,000, 21,000, 16,500, 9,200, and 8,100.     

Tetracycline resistance determined by pBR322 is mediated by one polypeptide

Only one polypeptide specified by plasmid pBR322 is necessary to determine tetracycline resistance. Small deletions in pBR322 constructed in vitro which result in the lack of ability to confer tetracycline resistance in vivo also result in the absence or alteration of this polypeptide in vivo. Other deletions define the extent of material necessary to encode this polypeptide. A correction to the DNA sequence of the tetracycline resistance cistron has been determined which confirms these observations.     

Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma

Using the rabbit reticulocyte cell-free translation system we have estimated during ontogenesis the proportions of in vitro translatable alpha and gamma brain enolase mRNAs, which are two minor mRNA species. No polypeptide precursor to these enzyme subunits appears to be synthesized during translation in vitro. During brain development, the changes in translatable alpha and gamma mRNA content seem to parallel those of the corresponding antigens. The proportion of each of the enolase mRNAs is highest in adult mouse brain. Mechanisms controlling alpha and gamma antigen expression are discussed. In order to prepare the specific cDNA probes, purification of alpha and gamma mRNAs was undertaken.     

Molecular characterization of oat seed globulins

We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a highly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin).     

Targeting of mRNAs to domains of the endoplasmic reticulum

The targeting of proteins to specific regions of the cell by signal elements within the polypeptide sequence has received much attention, but proteins can also be directed to their appropriate cellular locations by localization of their mRNAs. This mechanism is seen clearly in polar cells like germ and embryonic cells, neurons and epithelia. Recent evidence indicates that mRNAs may also be localized to morphologically and functionally distinct endoplasmic reticulum membranes, thereby facilitating sorting of the proteins they encode to subdomains of the reticulum or to polarized plasma membranes.     

Respiratory syncytial virus mRNA coding assignments.

The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.     

Effect of high salt treatment on influenza B viral protein synthesis in MDCK cells

Based on the information that high salt inhibits the initiation of cellular mRNA translation which depends on the function of the 5'-terminal structure of mRNA, we compared the effect of high salt on translation of host cellular mRNAs and influenza viral mRNAs, both of which are of 5'-terminal structure. Brief exposure of influenza B virus-infected MDCK cells to high salt medium resulted in a dose-dependent inhibition of viral polypeptide synthesis as well as of cellular polypeptide synthesis, but it had less effect on synthesis of viral polypeptides, particularly nonstructural protein (NS). Under these conditions the Na+ content of the infected cells was significantly increased. A similar salt effect on in vitro translation of viral and cellular mRNAs extracted from infected cells was also observed. There was no significant difference in sensitivity to hypertonic block of in vivo translation of influenza viral mRNAs and vesicular stomatitis virus mRNAs, the latter of which possess a virus-directed structure at the 5'-terminus.     

The alpha 1 chain of type XIII collagen consists of three collagenous and four noncollagenous domains, and its primary transcript undergoes complex alternative splicing.

We have recently reported a characterization of cDNA clones that encode an apparently novel human collagen that undergoes alternative splicing. These cDNAs covered one-third of the corresponding 2.5-2.8-kilobase mRNAs. We have now determined the complete primary structure of the protein encoded by several overlapping cDNAs isolated from a human endothelial cell library. Since the deduced translation product of the cDNAs is different in structure from all other collagen types, we have given the collagen chain encoded by the cDNAs the designation alpha 1 (XIII). The deduced polypeptide consists of three collagenous domains and four noncollagenous domains, two of them separating the collagenous domains and two located at the N-terminal and C-terminal ends of the polypeptide. Cysteine residues are found in three of the noncollagenous domains and also in the extreme N-terminal collagenous domain. Surprisingly, comparison of the nucleotide sequences encoded by the overlapping cDNA clones demonstrates that there are several alpha 1 (XIII) collagen mRNAs in HT-1080 human fibrosarcoma cells and human endothelial cells which differ in coding potential. Nuclease S1 mapping experiments suggest that these different mRNAs arise through alternative splicing of the precursor RNA at five locations within the coding region. This property makes type XIII collagen unique among all the collagen types studied so far. Its polypeptide length, therefore, may vary between 614 and 526 amino acids, depending on what internal splicing has taken place.     

Expression and developmental regulation of two unique mRNAs specific to brain membrane-bound polyribosomes.

Translation in vitro of membrane-bound polyribosomal mRNAs from rat brain has shown several to be developmentally regulated [Hall & Lim (1981) Biochem. J. 196, 327-336]. Here we describe the isolation and characterization of cDNAs corresponding to two such brain mRNAs. One cDNA (M444) hybrid-selected a 0.95 kb mRNA directing the synthesis in vitro of a 21 kDa pI-6.3 polypeptide, which was processed in vitro by microsomal membranes. A second cDNA (M1622) hybridized to a 2.2 kb mRNA directing the synthesis of a 55 kDa pI-5.8 polypeptide. Both mRNAs were specific to membrane-bound polyribosomes. Restriction maps of the corresponding genomic DNA sequences are consistent with both being single copy. The two mRNAs were present in astrocytic and neuronal cultures, but not in liver or spleen or in neuroblastoma or glioma cells. The two mRNAs were differently regulated during brain development. In the developing forebrain there was a gradual and sustained increase in M444 mRNA during the first 3 weeks post partum, whereas M1622 mRNA appeared earlier and showed no further increase after day 10. In the cerebellum the developmental increase in M444 mRNA was biphasic. After a small initial increase there was a decrease in this mRNA at day 10, coincident with high amounts of M1622 mRNA. This was followed by a second, larger, increase in M444 mRNA, when amounts of M1622 mRNA were constant. The contrasting changes in these two mRNAs in the developing cerebellum are of particular interest, since they occur during an intensive period of cell proliferation, migration and altering neural connectivity. As these mRNAs are specific to differentiated neural tissue, they represent useful molecular markers for studying brain differentiation.     

Vaccinia virus induces cellular mRNA degradation.

The infection of mouse L cells with vaccinia virus induced a rapid inhibition of cellular polypeptide synthesis and a diversion of protein synthesis to the exclusive production of viral polypeptides. This shutoff of cell-specific protein synthesis was achieved by a novel mechanism by which the virus induced the rapid degradation of cellular mRNAs. Concurrent with the degradation of cellular mRNA, the virus proceeds in the orderly temporal expression of its own genetic information. The effect of vaccinia virus infection upon two abundant L-cell mRNAs was assessed by using the highly conserved cDNA sequences that encode chicken beta-actin and rat alpha-tubulin. Hybridization analyses demonstrated that throughout infection there is a rapid and progressive degradation of both of these mRNAs. In fact, after 3 h of infection they are reduced to less than 50% of their concentration in uninfected L cells, and between 8 to 10 h they are almost entirely degraded. This observation explains in part the mechanism by which vaccinia virus inhibits host cell protein synthesis.     

Inhibition of translation in L-cell lysates by free polyadenylic acid: differences in sensitivity among different mRNAs and possible involvement of an initiation factor

Free polyadenylic acid specifically inhibits in vitro translation of naturally polyadenylated mRNAs in L-cell lysates. The polynucleotide affects the initiation of protein synthesis but has no apparent effect on elongation of polypeptide chains. Reovirus mRNA, naturally devoid of a poly(A) tail, is much less sensitive to this inhibition than are naturally polyadenylated mRNAs. Reovirus mRNA that was polyadenylated in vitro is not more sensitive than normal reovirus mRNA. The degree of inhibition of translation varies for the different reovirus mRNA species. The addition of proteins contained in a high salt wash of ribosomes can mitigate the inhibition of translation of naturally polyadenylated mRNAs by free polyadenylic acid. Altogether these results suggest that the inhibition by polyadenylic acid may be mediated by its interaction with a cellular (initiation) factor. The various sensitivities exhibited by different mRNAs may indicate differences in requirement for this factor.     

Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules.

We have characterized the distinct polypeptides, primary translation products and mRNAs encoding glutamine synthetase (GS) in the various organs of pea. Western blot analysis of soluble protein has identified five distinct GS polypeptides which are expressed at different relative levels in leaves, roots and nodules of pea. Of the two GS polypeptides in leaves (44 and 38 kd), the 44-kd GS polypeptide is predominant and is localized to the chloroplast stroma. In roots, the predominant GS polypeptide is 38 kd. Upon Rhizobium infection of roots, three 37-kd GS polypeptides increase in abundance in the nodules relative to uninfected roots. cDNA clones encoding three different GS mRNAs have been characterized. Hybrid-select translation has identified three different GS primary translation products (49, 38 and 37 kd). Two cDNA clones (pGS134 and pGS341) are homologous to GS mRNAs most abundant in nodules which encode the 38- and 37-kd GS primary translation products. A third cDNA (pGS197) corresponds to a larger GS mRNA species specific to leaf poly(A) RNA, which encodes a 49-kd putative precursor to the mature chloroplast GS polypeptide. cDNA sequence analysis and Southern blot analysis of pea nuclear DNA identifies at least three genes encoding GS in pea which are related but distinct in structure and in vivo pattern of expression.     

Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo

We have shown that the individual members of the plant gene family for glutamine synthetase (GS) are differentially expressed in vivo, and each encode distinct GS polypeptides which are targeted to different subcellular compartments (chloroplast or cytosol). At the polypeptide level, chloroplast GS (GS2) and cytosolic GS (GS1 and GSn) are distinct and show an organ-specific distribution. We have characterized full length cDNA clones encoding chloroplast or cytosolic GS of pea. In vitro translation products encoded by three different GS cDNA clones, correspond to the mature GS2, GS1, and GSn polypeptides present in vivo. pGS185 encodes a precursor to the chloroplast GS2 polypeptide as shown by in vitro chloroplast uptake experiments. The pGS185 translation product is imported into the chloroplast stroma and processed to a polypeptide which corresponds in size and charge to that of mature chloroplast stromal GS2 (44 kDa). The 49 amino terminal amino acids encoded by pGS185 are designated as a chloroplast transit peptide by functionality in vitro, and amino acid homology to other transit peptides. The cytosolic forms of GS (GS1 and GSn) are encoded by highly homologous but distinct mRNAs. pGS299 encodes the cytosolic GS1 polypeptide (38 kDa), while pGS341 (Tingey, S. V., Walker, E. L., and Coruzzi, G. M. (1987) EMBO. J. 6, 1-9) encodes a cytosolic GSn polypeptide (37 kDa). The homologous nuclear genes for chloroplast and cytosolic GS show different patterns of expression in vivo. GS2 expression in leaves is modulated by light, at the level of steady state mRNA and protein, while the expression of cytosolic GS is unaffected by light. The light-induced expression of GS2 is due at least in part to a phytochrome mediated response. Nucleotide sequence analysis indicates that chloroplast and cytosolic GS have evolved from a common ancestor and suggest a molecular mechanism for chloroplast evolution.     

Identification of lactate dehydrogenase-M polypeptide translated in vitro from human and mouse tumor cell poly(A)-containing messenger RNA

Rabbit anti-human lactate dehydrogenase-5(M4) antisera were raised which cross-reacted with mouse lactate dehydrogenase M polypeptide. The antisera were used for identification of human and mouse LDH-M polypeptides synthesized using an in vitro system directed by the mRNAs. The in vitro translation products directed by both mRNAs were similar in size and immunologically identical to the authentic LDH-M polypeptides. The sizes of the mRNAs encoding for both human and mouse LDH-M polypeptides were similar, about 15S (1445 nucleotides) and were shorter than the corresponding rat mRNA which is about 18S (1765 nucleotides).