The dependence of splicing efficiency on the length of 3' exon

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[clphenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcriptionpolymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.The development of cell-free splicing systems has led to rapid and continuing progress in understanding biochemical mechanisms involved in intron removal and in the definition of the spliceosomal machinery. However, the exact requirements for recognition of splices sites and the rules governing splice site selection remain unclear. Analysis of consensus sequences provided the first definition of splice sites (49). Most efforts to move beyond that initial step have focused on site-directed mutagenesis studies (for reviews, see references 4 and 31). In these experiments, the effects of splice site mutations are typically assayed by using cell-free splicing extracts (in. vitro) or after transient transfection of cultured mammalian cells or injection of RNA directly into frog oocytes (in vivo).Conflicting results between these in vitro and in vivo assays for the effect of mutations on splicing have frequently been reported (2,38,39,45,83), raising the possibility that artifacts are being introduced in one system or the other. Moreover, recent experiments suggesting that the splicing machinery may be compartmentalized within t...

Section: Resultsmentioning

confidence: 99%

Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Chen¹,

Chasin²

1993

“…Although it has been known that both the sequences (6,12,14,35,41,49,51) and the lengths (2,9,13,14,34,44,48,51) of exons are important determinants in the mechanism of splice site selection, the details of their involvement are not well understood. We have presented here a method for probing exon sequences with 2'-O-methyl oligoribonucleotides in search of the signals required for splice site selection.…”

Section: Methodsmentioning

confidence: 99%

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

Domiński¹,

Kole²

1994

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...

“…Furthermore, in certain instances, including physiological alternative splicing, the context of 5' and 3' splice sites, i.e., the sequences of adjacent exons, seemed to affect splice site selection (12,38). The role of exons in splicing was previously suggested by the results from this (14, 15) and other (33,52) laboratories which showed that monointronic transcripts containing a short downstream exon were spliced in vitro very inefficiently.…”

mentioning

confidence: 90%

Selection of splice sites in pre-mRNAs with short internal exons.

Domiński¹,

Kole²

1991

219

182

Model pre-mRNAs containing two introns and three exons, derived from the human I8-globin gene, were used to study the effects of internal exon length on splice site selection. Splicing was assayed in vitro in HeLa nuclear extracts and in vivo during transient expression in transfected HeLa cells. For substrates with internal exons 87, 104, and 171 nucleotides in length, in vitro splicing proceeded via a regular splicing pathway, in which all three exons were included in the spliced product. Primary transcripts with internal exons containing 23, 29, and 33 nucleotides were spliced by an alternative pathway, in which the first exon was joined directly to the third one. The internal exon was missing from the spliced product and together with two flanking introns was included in a large lariat structure. The same patterns of splicing were retained when transcripts containing 171-, 33-, and 29-nucleotide-long internal exons were spliced in vivo. A transcript containing a 51-nucleotide-long exon was spliced in vitro via both pathways but in vivo generated only a correctly spliced product. Skipping of short internal exons was reversed both in vitro and in vivo when purines in the upstream polypyrimidine tract were replaced by pyrimidines. The changes in the polypyrimidine tract achieved by these substitutions led in vitro to complete (transcripts containing 28 pyrimidines in a row) or partial (transcripts containing 15 pyrimidines in a row) restoration of a regular splicing pathway. Splicing in vivo of these transcripts led exclusively to the spliced product containing all three exons. These results suggest that a balance between the length of the uninterrupted polypyrimidine tract and the length of the exon is an important determinant of the relative strength of the splice sites, ensuring correct splicing patterns of multiintron pre-mRNAs.There are four sequence elements within introns of mammalian pre-mRNAs which are required for efficient splicing. These are the conserved sequence at the 5' splice junction, the invariant AG dinucleotide at the 3' splice junction, a weakly conserved sequence at the site of lariat formation, called the branch point sequence, and a pyrimidine-rich region of variable length between the branch point and the 3' splice junction, referred to as the polypyrimidine [poly(Y)] tract (for reviews see references 20, 32, 43). The importance of these elements has been shown experimentally in a number of studies employing pre-mRNAs containing point mutations or deletions within existing sequences (1,37,41,49,51,53). Although important, the four sequence elements seem insufficient to account for the high specificity of the splicing reaction. Similar sequence motifs, termed cryptic splice sites, are present in many regions throughout premRNAs and yet are not recognized under normal conditions (31), whereas in transcripts bearing point mutations or deletions within natural elements, such sites can be activated (1,37,50,53). Furthermore, in multi-intron primary transcripts, the mechanism of splice selec...