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. 2006 Sep 1;2(9):e138.
doi: 10.1371/journal.pgen.0020138. Epub 2006 Jul 20.

Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

Keren Borensztajn  1 , Marie-Laure Sobrier, Philippe Duquesnoy, Anne-Marie Fischer, Jacqueline Tapon-Bretaudière, Serge Amselem
Affiliations

Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

Keren Borensztajn et al. PLoS Genet. .

Abstract

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic Representation of the Human F7 Gene and Wild-Type and Mutant F7 Minigenes Cloned into pTracer-CMV Vector
Sizes of exons (E, open boxes) and introns (I, horizontal lines) are indicated. Primers Pa, Pb, Pe, Ph, and Pm are represented by horizontal arrows indicating their respective positions in the minigene sequence. Inset indicates the last three nucleotides of exon 7 (capital letters) and the first ten nucleotides of IVS7 (in lowercase characters) of the wild-type F7 gene. The invariant gt dinucleotide is indicated in underlined bold letters. A vertical arrow indicates the nucleotide change in the mutant sequence.
Figure 2
Figure 2. Assessment of the Ability of the IVS7 Pseudo-Sites to Be Activated
(A) Schematic representation of the constructs used in this experiment. Top: Organization of the wild-type F7 IVS7 proximal region (pF7wt); exons and introns are represented by open boxes and thin lines, respectively. The first 37-bp monomer, which spans the exon7–IVS7 boundary, is repeated within IVS7. The monomers are represented by a thick grey line. Each pseudo-splice site is represented by a grey box, and is separated from the next one by 28 bp. Bottom: Schematic representation of the various F7 minigenes carrying a T-to-A transition located at the main dinucleotide of the consensus donor splice site and involving several consecutive 37-bp monomer elements found in IVS7. Mutations are represented by a black cross. (B) RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 3
Figure 3. Splice Site Selection in the Context of Functional 5′ Pseudo-Sites, Separated by One or Several Inactivated 5′ Splice Sites
(A) Schematic representation of the constructs used in this experiment (see legend to Figure 2A for the meaning of each symbol). (B) RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 4
Figure 4. The Identified Scanning Mechanism for Splice Site Selection Is Not Cell Type–Dependent
(A) Schematic representation of the constructs used in this experiment (see legend to Figure 2A for the meaning of each symbol). (B) RT-PCR amplification of F7 transcripts isolated from COS-7 and HeLa cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 5
Figure 5. Splicing of a Series of Mutants with Increased Complementarity of the IVS7 5′ Pseudo-Sites to U1snRNA
(A) Splicing of F7 transcripts generated from F7 minigenes with a CV of 85 or 82.2. (B) Splicing of F7 transcripts generated from F7 minigenes with a CV of 91. (A and B) Top: Wild-type (middle line in [A], lower line in [B]) and mutated (lower and upper lines in [A], upper line in [B]) 5′ pseudo-sites in the F7 minigene. Dots show the location of the introduced mutations. Suffixes of the constructs' names and the resulting CVs are indicated. Middle: Schematic representation of the constructs transfected in CHO cells. Improved splice sites are represented by a hatched square (see legend to Figure 2A for the meaning of each symbol). Bottom: RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 6
Figure 6. Splicing of a Series of Mutants with IVS7 5′ Pseudo-Sites Perfectly Matching U1snRNA
Top: Wild-type (lower line) and mutated (upper line) 5′ pseudo-sites in the F7 minigene. Dots show the location of the introduced mutations. Suffix of the constructs' names and the resulting CV of 100 are indicated. Middle: Schematic representation of the constructs transfected in CHO cells. Splice sites matching the consensus are represented by a hatched square (see legend to Figure 2A for the meaning of each symbol). Bottom: RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 7
Figure 7. Search for Putative Intronic Sequences Regulating Splice Site Recognition
(A) Top: Schematic representation of the constructs used in this experiment with deletions involving one or several 37-bp monomers in the context of pF7wt or pF7m (see legend to Figure 2A for the meaning of each symbol). Bottom: RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure. (B) Top: Schematic representation of the constructs used in this experiment with insertions of several 37-bp monomers in the context of pF7wt or pF7m (see legend to Figure 2A for the meaning of each symbol). Bottom: RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 8
Figure 8. Role of G Triplets in IVS7 Splice Site Recognition
Top: Wild-type (middle line) and mutated (lower and upper lines) 5′ pseudo-sites in the F7 minigene. Dots show the location of the introduced mutations. Suffixes of the constructs' names and the resulting CVs are indicated. Middle: Schematic representation of the constructs transfected in CHO cells. Mutated splice sites are represented by a hatched box (see legend to Figure 2A for the meaning of each symbol). Bottom: RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
Figure 9
Figure 9. Effect on the Splice Site Selection of Exon 7 Sequences with Increased Complementarity to U1snRNA
(A) Splicing of F7 transcripts generated from F7 minigenes harbouring an exonic cryptic site at −113 with a CV of 85. (B) Splicing of F7 transcripts generated from F7 minigenes harbouring an exonic cryptic site at −54 with a CV of 72 or 89. (A and B) Top: Wild-type (lower line in [A], middle line in [B]) and mutated (upper lines in [A], lower and upper line in [B]) 5′ pseudo-sites in the F7 minigene. Dots show the location of the introduced mutations. Suffixes of the constructs' names and the resulting CVs are indicated. Middle: Schematic representation of the constructs transfected in CHO cells. Improved splice sites are represented by a hatched square (see legend to Figure 2A for the meaning of each symbol). Bottom: RT-PCR amplification of F7 transcripts isolated from CHO cells transfected with the above-mentioned constructs. The products corresponding to the different PCR fragments, as identified by sequencing, are schematically represented on both sides of the figure.
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