In vivo cooperation between introns during pre-mRNA processing.

Abstract

In higher eukaryotes the large number of introns present in most genes implies that the pre-mRNA processing machinery should be efficient and accurate. Although this could be achieved at the level of each intron, an attractive alternative would be that interactions between introns improve the performance of this machinery. In this study we tested this hypothesis by comparing the processing of transcripts of the tumor necrosis factor I~ gene, which differ only by their number of introns. We took advantage of the ordered splicing of the three introns present in this gene to design constructs that should generate, as primary transcripts, molecules that are normally produced by splicing. We established that the apparent splicing rate of intron 3 is increased 2.5-and 3.5-fold by the presence of one or two other introns on the primary transcript, respectively. Similarly, the apparent splicing rate of intron 2 is increased by the presence of intron 1. As these effects involve the splice sites of the upstream intron, these observations support the existence of cooperative interactions between introns during pre-mRNA processing.[Key Words: Pre-mRNA processing; splicing; cooperation between introns; tumor necrosis factor 6]

Regulation of mRNA splicing and transport by the tyrosine kinase activity of src

“…This gene constitutes a convenient reporter for premRNA processing studies (Neel et al, 1993(Neel et al, , 1995Weil et al, 1990) because of its short size, moderate level of splicing complexity (three introns which are sequentially removed from the polyadenylated primary transcript), ine cient splicing of intron 3 and the export to the cytoplasm of intron 3 containing precursors as well as fully mature mRNA ( Figure 1b). RNase protection assays can be used to unambiguously identify the di erent RNA species and reliably quantitate their accumulation.…”

“…The apparent splicing rate of intron 3 was determined as indicated in Materials and methods (see also Neel et al, 1993Neel et al, , 1995 and these results are presented in Figure 3b. The wt src gene and both Y527F and E378G activating mutations induced a 3 ± 4-fold reduction of this rate while the kinase inactive mutant had no e ect.…”

“…Moreover, the results obtained with the kinase inactive mutant indicate that these modi®cations of pre-mRNA maturation as well as the transactivation of the CMV promoter require the tyrosine kinase activity of src. (Neel et al, 1993(Neel et al, , 1995. The four nuclear species are designated by N3, N2, N1 and N0 according to the number of introns they contain.…”

“…Separation of the nuclear and cytoplasmic fractions was achieved by NP40 lysis as described (Neel et al, 1993). RNA was puri®ed by addition of guanidinium thiocyanate and centrifugation over a cesium chloride cushion.…”

Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes

“…11 First, introns can act co-operatively in enhancing the levels of mRNA pro- duced 12 -an effect that would require the presence of at least two introns. Second, genomic/cDNA hybrid genes usually contain authentic introns that are considerably larger than the minimal sequences required for efficient splicing and they can therefore represent an unnecessary load for gene delivery systems with limited capacity for foreign DNA.…”