Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue

We report the investigation of an endosperm-specific promoter from the rsus3 gene from rice (Oryza sativa). The promoter was characterized by deletion analysis and transient expression in guar (Cyamopsis tetragonoloba) seed-tissue. Transient expression was monitored by histochemical GUS assay, and quantitative dual reporter assays comprising firefly luciferase as a test reporter, and Renilla luciferase and GUS as reference reporters. These revealed high expression levels of the reporter genes directed by the rsus3 promoter in guar endosperm. Specificity for this tissue in seeds was apparent from a virtual absence of reporter activity in guar cotyledons. Removal of a putative intron region only slightly raised the expression level, whereas duplication of the minimal promoter region, in a tandem-repeat rsus3 promoter construct, retained endosperm specificity in guar, and displayed three times the reporter activity observed with the single copy construct.     

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.     

mRNA quality control: Marking the message for life or death

A protein complex deposited upstream of exon-exon junctions after pre-mRNA splicing may serve a dual role in mRNA quality control by directing mRNA nuclear export and, possibly, serving as a downstream 'mark' for nonsense-mediated decay.     

Analysis of splice donor and acceptor function in a novel<Emphasis Type="Italic"> Ac</Emphasis>-based gene trap construct

In this study, an Ac-based gene trap construct was engineered to increase gene trapping efficiency by an effective use of triple acceptor sites preceding a reporter gene. The target of the engineering process was a synthetic intron preceding the GUS reporter. Two different gene trap constructs were designed. In one construct, three of the sequence elements serving as signals for recognition of an intron 3' boundary were systematically modified to allow for almost optimal acceptor site recognition, while these sequences remained unchanged in the other construct. To compare recognition of the engineered intron with that of the unmodified intron, tester constructs were transiently transformed into barley (Hordeum vulgare L.) tissue and the accuracy and efficiency of splicing was determined by mRNA mapping and reporter-gene expression frequency analysis. By employing this test system, we could show that systematic engineering of the intron sequence elements results in advanced intron recognition, compared to the unmodified intron, and that all three acceptor sites were activated, but with unequal frequency. The impact of our findings on reporter expression in a gene-trap approach is discussed.     

Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells

We have examined how splicing affects the expression of a range of human and nonhuman genes in vertebrate cells. Although our data demonstrate that splicing invariably enhances the level of gene expression, this positive effect is generally moderate. However, in the case of the human beta-globin gene, splicing is essential for significant protein expression. In the absence of introns, 3' end processing is inefficient, and this appears to be causally linked to a significant decrease in the level of both nuclear and cytoplasmic 3' end-processed RNA. In contrast, splicing appears to only modestly enhance nuclear mRNA export. Consistent with this observation, intronless beta-globin gene expression was only partially rescued by the insertion of retroviral nuclear mRNA export elements. Surprisingly, in the case of the highly intron dependent beta-globin gene, the mRNA that did reach the cytoplasm was also only inefficiently translated if it derived from an intronless expression plasmid. Together, these data argue that splicing can increase gene expression by enhancing mRNA 3' end processing, and hence, mRNA production. Moreover, in the case of the highly intron-dependent beta-globin gene, splicing also significantly enhanced the translational utilization of cytoplasmic beta-globin mRNAs.     

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.