The Rho family GTPases function as key molecular switches, controlling a variety of actin-dependent cellular processes, such as the establishment of cell polarity, cell morphogenesis, and movement in diverse eukaryotic organisms. A novel subfamily of Rho GTPases, Rop, has been identified in plants. Protein gel blot and RNA gel blot hybridization analyses indicated that one of these plant Rho GTPases, Rop1, is expressed predominantly in the male gametophyte (pollen and pollen tubes). Cell fractionation analysis of pollen tubes showed that Rop is partitioned into soluble and particulate fractions. The particulate Rop could be solubilized with detergents but not with salts, indicating that it is tightly bound to membranes. The membrane association appears to result from membrane anchoring via a geranylgeranyl group because an in vitro isoprenylation assay demonstrated that Rop1Ps is geranylgeranylated. Subcellular localization, using indirect immunofluorescence and confocal microscopy, showed that Rop is highly concentrated in the cortical region of the tube apex and in the periphery of the generative cell. The cortical Rop protein at the apex forms a gradient with decreasing concentration from tip to base and appears to be associated with the plasma membrane. These results suggest that the apical Rop GTPase may be involved in the signaling mechanism that controls the actin-dependent tip growth of pollen tubes. Localization of the Rop GTPase to the periphery of the generative cell is analogous to that of myosin, suggesting that the Rop GTPase plays an important role in the modulation of an actomyosin motor system involved in the movement of the generative cell.
Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000-fold by the 5′ SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3′ LTR and 3′ untranslated sequence between env and the LTR did not support Rev-independent gag expression. Quantitative RNase protection assays indicate that the SNV 5′ RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5′ RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.
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