Binding of a 50-kD Protein to a U-Rich Sequence in an mRNA Encoding a Proline-Rich Protein That Is Destabilized by Fungal Elicitor

The mRNA encoding the bean proline-rich protein PvPRP1 has been shown previously to be destabilized in elicitor-treated cells. In this study, we identified a 50-kD protein in cellular extracts that binds specifically to the PvPRP1 mRNA by UV cross-linking assays. Using 32P-labeled RNAs transcribed in vitro from a series of 5[prime] deleted PvPRP1 cDNA clones, we demonstrated that the PvPRP1 mRNA binding protein (PRP-BP) binds to a 27-nucleotide U-rich (~60%) domain in the 3[prime] untranslated region. Poly(U) and, to a lesser extent, poly(A-U) competed for the PRP-BP binding activity. PRP-BP activity is redox regulated in vitro, as shown by the effects of sulfhydryl-modifying reagents on the RNA binding activity. Treatment of cellular extracts with the reducing agents DTT and [beta]-mercaptoethanol increased binding activity, whereas treatment with the oxidizing agent diamide and the alkylating agent N-ethylmaleimide inhibited binding. In extracts from elicitor-treated cells, PRP-BP activity increased approximately fivefold prior to rapid PvPRP1 mRNA degradation. The increase in PRP-BP activity was apparently due to post-translational regulation because control and elicitor-treated cell extracts supplemented with DTT showed high comparable levels of RNA binding activity. The kinetics of PRP-BP activation after elicitor treatment and its capacity for redox regulation in vitro suggested that PRP-BP may function in the elicitor-induced destabilization of PvPRP1 mRNA.     

Metabolic Properties of Early and Late Vaccinia Virus Messenger Ribonucleic Acid

In vaccinia-infected cells, 60% of the viral messenger ribonucleic acid (mRNA) was associated with polyribosomes, and the remainder sedimented in a broad peak in the 30 to 74S region. The quantity of mRNA in polyribosomes was sharply reduced late in the infectious cycle [9 hr postinfection (PI)] to less than 30% of the 2-hr value. However, protein synthesis proceeded at a nearly constant rate from 2 to 13 hr PI. This ability of small quantities of late mRNA to support as much protein synthesis as do the much larger quantities of early mRNA was not due to an increase in stability, since late mRNA decays with a half-life of 13 min, whereas early mRNA has a half-life of 120 min. A similar decrease in viral mRNA synthesis without an accompanying decrease in viral protein synthesis was observed when deoxyribonucleic acid synthesis is inhibited. In contrast to the rapid decay of the late mRNA which was present in polyribosomes, the mRNA which sedimented in the 30 to 74S region remained unchanged even after a 2-hr period of exposure to actinomycin. The rate at which infected cells lose the capacity to synthesize specific viral proteins after exposure to actinomycin D was consistent with the half-life values of early and late mRNA that were observed.     

Negative and positive regulation of a novel proline-rich protein mRNA by fungal elicitor and wounding.

The structure and expression of a cDNA clone (PvPRP1) isolated from a cDNA library prepared from bean (Phaseolus vulgaris) cells treated with fungal elicitor have been characterized. Sequence analysis of the 1.1 kb insert revealed a complete open reading frame which encodes a 32 kDa protein. The protein resembles other proline-rich proteins in plants but possesses several unique features: (i) the N-terminal half of the protein is proline rich and contains three identical repeats of Pro-Val-His-Pro-Pro-Val-Lys-Pro-Pro-Val and six related repeats; (ii) the proline-rich region contains two tracts of six histidine residues; and (iii) the C-terminal half is low in proline and lacks repeats. Genomic blotting experiments suggest the presence of a single PvPRP1 gene as well as more distantly related genes within the bean genome. A dramatic decrease in PvPRP1 mRNA levels occurs within 2 h of elicitor treatment of cell cultures. The PvPRP1 mRNA is present at a moderate level in hypocotyls. Upon wounding, the mRNA level initially decreases over 5 h and then accumulates over 25 h to levels which are higher than the basal level in unwounded hypocotyls. Based on the similarity to other proline-rich proteins with repeated motifs, including the presence of a putative signal peptide, it is likely that the PvPRP1 protein is targetted to the cell wall. The expression of the PvPRP1 gene appears to be integrated with the remodeling of the plant cell wall during the defense response.     

Mechanisms of regulating tubulin synthesis in cultured mammalian cells.

Colchicine and nocadazole both depolymerize microtubules in cultured fibroblasts and lead to a rapid inhibition of tubulin synthesis. The level of translatable tubulin mRNA is greatly reduced in drug-treated cells as demonstrated by translation in a reticulocyte-derived in vitro protein synthesizing system. A model of tubulin synthesis regulation is proposed in which the elevated level of unpolymerized tubulin in drug-treated cells inhibits the formation of new tubulin mRNA and the preexisting message decays rapidly. In agreement with this model, tubulin message is found to be short-lived and has an approximately 2 hr half-life in cells treated with actinomycin D. Another prediction of the proposed model is that destabilization of microtubules without a concomitant increase in free tubulin will not inhibit tubulin synthesis. Vinblastine also disrupts microtubules but leads to the aggregation of tubulin into large paracrystals with an apparent decrease in the concentration of free tubulin. This drug does not inhibit tubulin production but rather leads to a measurable enhancement of tubulin synthesis.