Requirements for intron-mediated enhancement of gene expression in Arabidopsis

To explore possible mechanisms of intron-mediated enhancement of gene expression, the features of PAT1 intron 1 required to elevate mRNA accumulation were systematically tested in transgenic Arabidopsis. This intron is remarkably resilient, retaining some ability to increase mRNA accumulation when splicing was prevented by mutation of 5' and 3' splice sites, branchpoint sequences, or when intron U-richness was reduced. Enhancement was abolished by simultaneously eliminating branchpoints and the 5' splice site, structures involved in the first two steps of spliceosome assembly. Although this suggests that the splicing machinery is required, intron splicing is clearly not enough to enhance mRNA accumulation. Five other introns were all efficiently spliced but varied widely in their ability to increase mRNA levels. Furthermore, PAT1 intron 1 was spliced but lost the ability to elevate mRNA accumulation when moved to the 3' UTR. These findings demonstrate that splicing per se is neither necessary nor sufficient for an intron to enhance mRNA accumulation, and suggest a mechanism that requires intron recognition by the splicing machinery but also involves nonconserved intron sequences.     

RNA sequences upstream of the 3' splice site repress splicing of mutantyeast ACT1 introns.

A yeast ACT1 intron in which both the first and last intron nucleotides are mutated, the /a-c/ intron, splices 10% as well as wild type. We selected for additional cis-acting mutations that improve the splicing of /a-c/ introns and recovered small deletions upstream of the 3' splice site. For example, deletion of nucleotides -9 and -10 upstream of the 3' splice site increased the splicing activity of the /a-c/ intron to 30% that of the wild-type ACT1 intron. To determine if the increased /a-c/ splicing was due to changes in intron spacing or sequence, we made mutations that mimicked the local sequence of the delta-9, -10 deletion without deleting any nucleotides. These mutants also increased /a-c/ splicing, indicating that the increased splicing activity was due to changes in intron sequence. The delta-9, -10 deletion was not allele specific to the /a-c/ intron, and improved the splicing efficiency of many mutant introns with step II splicing defects. To further define the sequences required for improved splicing of mutant introns, we randomized the region upstream of the ACT1 3' splice site. We found that almost all sequence alterations improved the splicing of the /a-c/ intron. We postulate that this sequence near the 3' end of the intron represses the splicing of mutant introns, perhaps by serving as the binding site for a negative splicing factor.     

Tissue-preferential expression of a rice alpha-tubulin gene, OsTubA1, mediated by the first intron

The genomic clone encoding an alpha-tubulin, OsTubA1, has been isolated from rice (Oryza sativa L.). The gene consists of four exons and three introns. RNA-blot analysis showed that the gene is strongly expressed in actively dividing tissues, including root tips, young leaves, and young flowers. Analysis of chimeric fusions between OsTubA1 and beta-glucuronidase (GUS) revealed that the intron 1 was required for high-level GUS expression in actively dividing tissues, corresponding with normal expression pattern of OsTubA1. Fusion constructs lacking the intron 1 showed more GUS staining in mature tissues rather than young tissues. When the intron 1 was placed at the distal region from 5'-upstream region or at the 3'-untranslated region, no enhancement of GUS expression was observed. Sequential deletions of the OsTubA1 intron 1 brought about a gradual reduction of GUS activity in calli. These results suggest that tissue-preferential expression of the OsTubA1 gene is mediated by the intron 1 and that it may be involved in a mechanism for an efficient RNA splicing that is position dependent.     

Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.

The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human beta-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long- and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene.     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.     

Correlation between splicing sites within an intron and their sequence complementarity with U1 RNA

We have determined the nucleotide sequence of two short introns (respectively 215 and 90 nucleotides) in the chick alpha 2-collagen (type I) gene as well as parts of the adjacent exons. For one of these introns we find that the 5' end of U1 RNA is complementary not only to the two ends of the intron but also to one end of the intron and sequences inside this intron. These complementarities predict three potential internal splicing sites. By S1 mapping experiments we find three discrete RNA precursors in which different portions of this intron have been deleted. The sizes of the deleted segments are in good agreement with the location of the predicted splicing points inside the intron. The DNA sequence indicates that removal of one portion of the intron should still allow the subsequent elimination of the rest of the intron and the correct splicing of the coding segments located at each end of the intron. The new introns created by the first splicing events contain sequences at each end which are also complementary to U1 RNA. Our data indicate that in the intron which we have examined the sequences at the 3' end of the intron are removed before those at the 5' end.     

Sequences within the last intron function in RNA 3''-end formation in cultured cells.

In cultured cells, little if any mRNA accumulates from an intronless version of the human gene for triosephosphate isomerase (TPI), a gene that normally contains six introns. By deleting introns either individually or in combinations, it was demonstrated by Northern (RNA) blot hybridization that while the deletion of a greater number of introns generally results in a lower level of product mRNA, not all introns contribute equally to mRNA formation. For example, intron 1 appeared to be dispensable, at least when the remaining introns are present, but deletion of the last intron, intron 6, reduced the level of product mRNA to 51% of normal. To determine how intron 6 contributes to mRNA formation, partial deletions of intron 6 were constructed and analyzed. Deletion of the lariat and acceptor splice sites or the donor, lariat, and acceptor splice sites, each of which precluded removal of the intron 6 sequences that remained, reduced the level of product mRNA to < 1 or 27% of normal, respectively. As measured by RNase mapping and cDNA sequencing, the decrease in mRNA abundance that was attributable to the complete and partial intron 6 deletions was accompanied by an increase in the abundance of pre-mRNA that lacked a mature 3' end, i.e., that was neither cleaved nor polyadenylated. We infer from these and other data that sequences within the final intron facilitate proper 3'-end formation, possibly through an association with the components of a productive spliceosome.     

UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation

Efficient splicing of higher plant pre-mRNAs depends on AU- or U-rich sequences in introns. Moreover, AU-rich sequences present in 3'-untranslated regions (3'-UTRs) may play a role in 3' end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross-linked to U-rich intron and 3'-UTR sequences in vitro, and associates with nuclear poly(A)(+) RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3'-UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre-mRNAs.     

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.     

RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b (cob) gene, which contains two introns, and a "long form" of the cytochrome oxidase subunit I (coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of coI intron 1, which is excised as a 2.6 X 10(3) base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 degrees C to 37 degrees C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.