Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

Abstract

Certain thalassemic human 0-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.Pre-mRNA splicing takes place within a large ribonucleoprotein complex termed the spliceosome. The specificity and accuracy of splicing are determined by the interactions of small nuclear ribonucleoprotein particles and protein components of the spliceosome with a number of pre-mRNA sequence elements in pre-mRNA, such as the branch point sequence, the polypyrimidine tract, and the 3' and 5' splice sites (reviewed in references 15, 21, and 29). In addition, exon sequences seem to contribute to the specificity of splicing (references 35, 41, and 49 and references therein). However, besides identification of a regulatory element in the female-specific exon of the doublesex pre-mRNA (17,19,30) and characterization of purinerich motifs in exons from some other spliced transcripts (7,12,27,45,47,49,51), the involvement of exon sequences in splicing remains unclear.In this work, we have used antisense 2'-O-methyl oligoribonucleotides (see reference 43 for a review) to study the function of several intron and exon sequences in pre-mRNA splicing. This approach stems from our recent report which showed that the binding of 2'-O-methyl oligoribonucleotides to the branch point or aberrant splice sites leads to the restoration of correct in vitro splicing of mutated P-globin premRNAs identified in individuals with various forms of 3-thalassemia (11). These oligonucleotides form strong duplexes with RNA which are resistant to RNase H and RNA unwinding activities. In consequence, they remain stably associated with the complementary regions in RNA, efficiently inhibiting the function of the targeted sequences. Antisense 2'-O-methyl oligoribonucleotides were originally used as sequence-specific probes to study the structure of small nuclear ribonucleoproteins and their interactions w...

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

“…Our data are consistent with evidence that antisense oligonucleotides directed against the anchoring site can perturb (72) or block splicing (18). Surprisingly, however, mutations in the anchoring site had no apparent effect on cis-splicing of an Adeno mRNA precursor (18).…”

“…One attractive scenario involves the SF3a and SF3b components of the large SF3 protein complex that is tightly associated with U2 snRNP. SF3 proteins are highly conserved from yeast to humans, and are essential for splicing (18,(42)(43)(44)(45)(46)48,49,72). SF3a associates primarily with the 3′ half of U2 snRNA, and SF3b with the 5′ half including U2 stem-loops Ib and IIa (73).…”

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+…”

Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.

“…As the SF3a/SF3b proteins are components of U2 snRNP, our data indicate that these proteins function, at least in part, to anchor U2 snRNP to the pre-mRNA in the A complex. Significantly, in another study (Dominski and Kole 1994), a 2'O methyl oligonucleotide complementary to a sequence corresponding in position to our anchoring site was shown to switch branch-site selection in a ~3-globin pre-mRNA containing duplicated BPSs. All of our experiments were carried out using adenovims major late (AdML) premRNA.…”