The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

Abstract

We have shown previously that truncation of the human ,3-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence.Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit j3-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.All pre-mRNAs that contain introns have a number of specific sequence elements that are essential for accurate and efficient splicing. These elements appear to be contained within the intron and include the 5' and 3' splice sites, the branch point near the 3' splice site, and the polypyrimidine region. The importance of these sequences has been shown by (i) their high degree of sequence conservation between pre-mRNAs (18), (ii) the occurrence of mutations in each of the elements which severely alter or completely inactivate normal splicing (for recent reviews see references 9, 21, and 27), and (iii) the observation that these sequences specifically bind ribonucleoprotein particles (RNPs) shown to be involved in pre-mRNA splicing (reviewed in reference 16).It is likely that there are additional sequence elements in pre-mRNA involved in the regulation of splicing that have not yet been characterized. We have shown previously that efficient removal of intron 1 of the human ,B-globin premRNA can be dramatically reduced in vitro by truncating the transcript so that only 14 nucleotides of the second exon remain. This transcript is able to undergo the first step in splicing, i.e., cleavage at the 5' splice site and lariat formation, but is unable to efficiently generate the final spliced product or the released lariat intron (7). Similar observations have also been reported by Ruskin and Green (24). Reed and Maniatis (23) reported that different exon sequences can affect the efficiency of splice site selection when tested in an in vitro cis-competition assay. Also, examples of mutations affecting splicing at a distance are the skipping of exon 5 in the DHFR gene in Chinese hamster ovary cells (17) or skipping of exon 2 when transcripts with an altered 5' splice site in intron 2 of the rabbit ,-globin gene are spliced in vitro (1). Since alternatively spliced mRNAs are frequently generated by exon skipping, it seems plausible that this important process is controlled by sequences distant from a given intron, possibly via the interaction of a specific trans-acting factor (for a review of alternative splicing, see reference 15).All these results suggest that spl...

Sensitivity of splice sites to antisense oligonucleotides in vivo

“…-745; data not shown, Senapathy et al+, 1990) or in terms of the number of base-pairs formed with U1 snRNA (IVS2-705 . -654 ϭ -745; Table 2)+ Since the variable part of the internal exons does not seem to contain typical splicing enhancers or silencers (Fig+ 1B), it seems likely that it is the length of the aberrant exon that modulates its recognition+ The effects of exon length on splicing efficiency and exon inclusion both in vitro and in vivo have been recognized previously (Reed & Maniatis, 1986;Furdon & Kole, 1988;Dominski & Kole, 1991;Sterner & Berget, 1993)+ Nevertheless, the fact that aberrant exons IVS2-654 and IVS2-705, which in vivo are both fully included into the spliced product, differ in their respective sensitivity to the antisense oligonucleotides at the 59 splice site by 29-fold was surprising+ Note that a trace of correctly spliced product is detectable in untreated IVS2-705 but not in IVS2-654 HeLa cells (cf+ lanes 1 in Figs+ 3 and 2, respectively)+ Thus the efficiency of exon inclusion and its sensitivity to oligonucleotides are in qualitative if not quantitative agreement+ A similar correlation was observed in vitro in cell-free extracts (Dominski & Kole, 1993, 1994a) Accordingly, the differences in sensitivity to the oligonucleotides are even more striking for IVS2-654 and -745 exons, the latter being partly included in the aberrant b-globin mRNA in the absence of the oligonucleotide (Fig+ 4, lane 1 and Table 2)+ The fact that consensus mutations at the 59 splice sites in IVS2-654 and IVS2-705 mutants affect sensitivity of the 39 splice site to ON-39cr oligonucleotide are consistent with the concept of exon bridging (Kuo et al+, 1991;Hertel et al+, 1997) inherent in the exon definition model (Robberson et al+, 1990)+ In this interpretation, the ability of ON-39cr oligonucleotide to inhibit aberrant splicing of IVS2-705con but not of IVS2-654con premRNA points to differences in bridging and recognition of the two exons flanked by the same 39 splice site upstream and almost identical consensus 59 splice site downstream (Table 2)+ Whether the kinetic or steric hindrance models are responsible for the differences in the ability of the oligonucleotides to correct splicing of mutant pre-mRNAs, it appears that the oligonucleotides compete with the splicing factors for the target sequences and that the interactions of the latter with pre-mRNAs vary significantly+ Although the experiments did not identify the competing factors, they did indicate that antisense oligonucleotides may provide useful tools for modifying and probing the interactions of the spliceosomes with the pre-mRNAs+…”

Mutation Detection in the and Alleles of the Sodium Channel

“…The insertion is flanked by a 12-bp repeat, a characteristic feature that is believed to originate from staggered breaks in genomic DNA at the site of L1 integration (Hutchison et al, 1983). Other polypyrimidine insertions interfere with splice site cleavage (Furdon and Kole, 1988). The splice sites flanking exons 2 and 3 in med genomic DNA were identical with the wild type.…”

“…The 180-bp L1 insertion in exon 2 of Scn8a results in an exon length of 300 bp, which is within the normal range. Skipping of exon 2 in the med transcript is therefore more likely to be related to the poly(U) sequence of the L1 element, since insertion of polypyrimidine tracts inhibits splicing (Furdon and Kole, 1988).…”

U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection.

“…To minimize the effect of sequence context on relative splice site strength (Pikielny and Rosbash 1985;Reed and Maniatis 1986;Furdon and Kole 1988;Nelson and Green 1988), the duplications were constructed so that both 5'-splice sites, separated by 75 nucleotides, would be situated in the same local sequence environment. The fact that there was no difference in the choice of the two sites indicates the absence of any obvious proximity or polarity features of the selection system.…”