Classification and Structural Comparison of Full-Length cDNAs for Pathogenesis-Related Proteins

Fourteen cDNA clones of pathogenesis-related (PR) proteins, PR1a and PR1b of tobacco were obtained and classified into six groups based on restriction enzyme maps. To assign the groups to different classes of PR1 proteins, all the clones were partially sequenced and compared with amino acid sequences of PR1a and PR1b. Two groups of these corresponded to PR1a and four to PR1b. The results indicate that there are at least two kinds of PR1a mRNAs and four kinds of PR1b mRNAs. In fact, one cDNA insert hybridized to at least six to seven DNA fragments in restriction enzyme fragments of Samsun NN genomic DNA, indicating that the PR1 protein genes exist as a multigene family in the tobacco genome. Two sequences of essentially full-length cDNAs for PR1a and PR1b were determined and compared. The coding sequences of two cDNAs share 93% homology and the deduced amino acid sequences of PR1a and PR1b precursors, which are synthesized as larger precursors containing signal peptides, are 91% homologous. The homology of mature PR1a and PR1b regions is higher than that of larger precursors, 94% in the nucleotide sequence and 93% in the amino acid sequence, whereas that of the signal peptide regions is 80 and 90%, respectively. The hydropathy patterns and the secondary structures predicted by Chou-Fasman rules are similar to tomato PR protein in the half-side of the C terminus, which suggests that the half-C terminus side is important for the function of PR1 proteins.     

Highly selective actions of HuR in antagonizing AU-rich element-mediated mRNA destabilization

Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.     

Biosynthesis of the light-harvesting chlorophyll a/b protein. Control of messenger RNA activity by light

1. Antibodies raised against the 26000-Mr polypeptides of the light-harvesting chlorophyll a/b proteins of pea leaves specifically immunoprecipitated two 32000-Mr polypeptides synthesized when pea leaf poly(A)-containing RNA was translated in vitro. On the basis of immunochemical relatedness and by comparison of their partial tryptic digestion products, the 32000-Mr products formed in vitro are identified as precursors to the authentic polypeptides of the light-harvesting chlorophyll a/b complex. 2. The specificity of the immunoprecipitation permitted the development of an assay for the cellular levels of translationally active light-harvesting protein mRNA in plants exposed to different light regimes. Low levels of the mRNAs were detectable in dark-grown plants. Exposure to continuous illumination caused these levels to increase by at least ten-fold and led to the appearance of large quantities of the light-harvesting chlorophyll a/b complex. In plants exposed to intermittent illumination (2 min of white light every 2 h for 2 days), the light-harvesting complex did not accumulate, although levels of mRNA specifying the polypeptides of the complex were high (50% of those in continuously illuminated plants). 3. Messenger RNAs encoding the light-harvesting proteins were detected in polysomes of intermittently illuminated leaves. These polysomes were active in a wheat-germ 100 000 X g supernatant "run-off" system, to form light-harvesting protein precursors, under conditions when only nascent polypeptide chains initiated in vivo were elongated and terminated. These results demonstrate that the inability of intermittently illuminated leaves to accumulate the light-harvesting proteins is not due to a selective inhibition of the translation of the corresponding mRNAs. 4. Intermittently illuminated leaves were labelled with [35S]methionine in darkness, and incorporation of radioisotope into the light-harvesting proteins and their precursors was assayed immunologically. No pool of untransported or unprocessed 32000-Mr precursor polypeptides could be detected in the soluble fraction (cytoplasm and stroma). However, low levels of the mature 26000-Mr polypeptides were detected in the membrane fraction. It is concluded that the newly synthesized light-harvesting chlorophyll a/b protein fail to accumulate in intermittently illuminated leaves because they undergo rapid turnover. The site of light-harvesting protein breakdown is probably the thylakoid membrane, and the cause of breakdown is probably the absence of chlorophyll a and chlorophyll b molecules that are required for eventual stabilization of the proteins within the photosynthetic membrane.     

Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize

Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.     

Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs

The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 [35S]methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.     

Virus-induced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) induces a number of host-encoded, so-called pathogenesis-related (PR-) proteins, which are found in the intercellular space of the leaf and are associated with induced resistance. By immunoprecipitation of their in vitro translation products we were able to detect the mRNAs corresponding to a number of PR-proteins in TMV-infected tobacco, but not in healthy plants. Analysis by the Northern blot technique using cloned cDNA of PR1-mRNAs as probe showed that the mRNAs for the closely related proteins PR1a, 1b and 1c occur at a low level in healthy tobacco; upon TMV infection this level is increased > 100-fold. The PR1-specific probe did no hybridize to mRNAs corresponding to other PR-proteins. Sequencing of the 5'-terminal region of PR1-mRNAs showed that PR1-proteins are derived from precursors by removal of an N-terminal signal peptide of 30 amino acids.     

Cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells studied by in vitro translation

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2- phase cells by centrifugal elutriation with less than 10% cross-contamination. Isolation of poly(A+) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). In vitro translation of poly(A+) RNA from asynchronous and phase synchronous cells. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.     

Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia

Diapausing pupae of the Cecropia moth (Hyalophora cecropia) respond to an injection of live bacteria by the selective synthesis of certain types of RNA and immune proteins (designated P1-P9). The in vitro translation products of RNA from both injured and infected pupae showed specific patterns with a defined number of extra bands. Some proteins characteristic of the normal RNA were reduced in the immune RNA translation products. Antibody reaction was used to show the selective synthesis of immune proteins P4 and P5 with mRNA from pupae subjected to injury or infection. The protein synthesized in vitro, which cross-reacted with P5 antibodies, is most likely a precursor of the attacins described in the preceding paper. A cDNA clone bank was prepared and two clones were isolated and shown to contain 750 bp corresponding to P4 and 250 bp of attacin information. These clones were used to estimate the sizes of the mRNAs by Northern blotting and to estimate, by RNA/DNA hybridization, the levels of P4 and P5 mRNA. In vivo incorporation of [35S]methionine into attacins and P4 during different conditions was compared with the levels of the corresponding mRNA.     

Yar1 protects the ribosomal protein Rps3 from aggregation

2000 ribosomes have to be synthesized in yeast every minute. Therefore the fast production of ribosomal proteins, their efficient delivery to the nucleus and correct incorporation into ribosomal subunits are prerequisites for optimal growth rates. Here, we report that the ankyrin repeat protein Yar1 directly interacts with the small ribosomal subunit protein Rps3 and accompanies newly synthesized Rps3 from the cytoplasm into the nucleus where Rps3 is assembled into pre-ribosomal subunits. A yar1 deletion strain displays a similar phenotype as an rps3 mutant strain, showing an accumulation of 20S pre-rRNA and a 40S export defect. The combination of an rps3 mutation with a yar1 deletion leads to an enhancement of these phenotypes, while increased expression of RPS3 suppresses the defects of a yar1 deletion strain. We further show that Yar1 protects Rps3 from aggregation in vitro and increases its solubility in vivo. Our data suggest that Yar1 is a specific chaperone for Rps3, which serves to keep Rps3 soluble until its incorporation into the pre-ribosome.     

HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.     

Cleavage of eIF4G by HIV-1 protease: effects on translation

We have recently reported that HIV-1 protease (PR) cleaves the initiation factor of translation eIF4GI [Ventoso et al., Proc. Natl. Acad. Sci. USA 98 (2001) 12966-12971]. Here, we analyze the proteolytic activity of HIV-1 PR on eIF4GI and eIF4GII and its implications for the translation of mRNAs. HIV-1 PR efficiently cleaves eIF4GI, but not eIF4GII, in cell-free systems as well as in transfected mammalian cells. This specific proteolytic activity of the retroviral protease on eIF4GI was more selective than that observed with poliovirus 2A(pro). Despite the presence of an intact endogenous eIF4GII, cleavage of eIF4GI by HIV-1 PR was sufficient to impair drastically the translation of capped and uncapped mRNAs. In contrast, poliovirus IRES-driven translation was unaffected or even enhanced by HIV-1 PR after cleavage of eIF4GI. Further support for these in vitro results has been provided by the expression of HIV-1 PR in COS cells from a Gag-PR precursor. Our present findings suggest that eIF4GI intactness is necessary to maintain cap-dependent translation, not only in cell-free systems but also in mammalian cells.     

Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens.

The effects of temperature on protein synthesis by Escherichia coli, a mesophile, and Pseudomonas fluorescens, a psychotroph, were investigated by using whole-cell and cell extract preparations. After shifts to 5 degrees C, protein was synthesized at a slowly decreasing rate for 1 h by both organisms, after which P. fluorescens synthesized protein at a new rate corresponding to its 5 degrees growth rate, in contrast to E. coli which did not synthesize protein at a measurable rate. In vitro protein-synthesizing systems using MS-2 RNA, endogenous mRNA, and purified polysomes were utilized to investigate initiation of translation at 5 degrees C. In these systems, P. fluorescens cell extracts synthesized protein at linear rates for up to 2 h at 5 degrees C, whereas E. coli cell extracts synthesized protein for only 25 min at 5 degrees C. The rates of polypeptide elongation, as tested by the incorporation of phenylalanine into polyphenylalanine by cell extract protein-synthesizing systems from both organisms, were identical over the range of 25 to 0 degrees C. The polysome profiles of E. coli whole cells shifted from 37 to 5 degrees C showed accumulation of 70S ribosomal particles and ribosomal subunits at the expense of polysomes. Similar experiements done with P. fluorescens resulted in polysome reformation at 5 degrees C. In vitro experiments demonstrated that the 70S ribosomal particles, which accumulated in E. coli at 5 degrees C, were capable of synthesizing protein in vitro in the absence of added mRNA. These in vivo and in vitro results suggest that incubation of E. coli at subminimal temperatures results in a block in initiation of translation causing polysomal runoff and the accumulation of 70S particles, some of which are 70S monosomes.     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA₃ plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

Stress mRNA metabolism in canavanine-treated chicken embryo cells.

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.     

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.