Intrinsic differences between authentic and cryptic 5' splice sites

Abstract

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5' splice sites (5'ss), we systematically analyzed a set of 76 cryptic 5'ss derived from 46 human genes. These cryptic 5'ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5'ss. Statistical analysis of the strengths of the 5'ss using the Shapiro and Senapathy matrix revealed that authentic 5'ss have significantly higher score values than cryptic 5'ss, which in turn have higher values than the mutant ones. beta-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the beta-globin authentic and cryptic 5'ss, but not their surrounding context, determine the correct 5'ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5'ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5'ss-disrupting mutations at the mRNA level.

Figure 1

Diagram of a portion of the human porphobilinogen deaminase (PBGD) gene, spanning exons 10–11. Gray boxes and uppercase letters represent exons 10 and 11, and lowercase letters and line represent intron 10. The two possible splicing patterns, by use of an authentic 5′ss (A arrow) or a cryptic 5′ss (C arrow) in exon 10 are represented above and below the sequences, respectively. The latter pathway is only seen when the authentic 5′ss is disrupted by a mutation (M arrow), such as G to T transversion at position –1 (39). Also shown are two pseudo 5′ss (P arrows), i.e. sequences that match the 5′ss consensus but are not functional in either the wild-type or mutant contexts.

Figure 2

Density plot of the distribution of distances between authentic and cryptic 5′ss. The R statistical package (http://cran.r-project.org) was used to fit a kernel density plot to the distances between authentic and cryptic 5′ss. The y-axis shows the density of cryptic 5′ss, and the x-axis, the distance from the cryptic to the authentic site at position +1. (a) Positive and negative numbers correspond to cryptic splice sites located in the downstream intron or the upstream exon, respectively. The number of occurrences for each distance is shown by the blue bars at the bottom of the display, and the corresponding scale is shown on the right side. (b) Density of distances for exonic (blue) or intronic (red) cryptic 5′ss. In this case, only the absolute distances are shown.

Figure 3

Average S&S consensus values for five types of 5′ss. The different 5′ss categories are: authentic (A), mutant (M), cryptic (C), pseudo (P) and alternative (AS). (A) Average score (y-axis) of each category. (B) Average of the score differences between pairs of 5′ss of each category associated with the same exons.

Figure 4

Analysis of cryptic 5′ss activation in human β-globin. (A) Cryptic 5′ss in the human β-globin gene. The diagram shows the first two exons (gray boxes) and first intron (horizontal thin line) of HBB. The sequence around the first intron 5′ss is shown below the diagram. Vertical lines represent the cryptic 5′ss, whose GT dinucleotides are underlined. The arrows indicate the cleavage/ligation sites. The phase, or position of the intron within a codon, is given in Roman numerals, and the number in parenthesis is the relative position of the cryptic 5′ss relative to the authentic 5′ss at +1. The numbers above each splice site are their S&S consensus values. The authentic (+1) and main cryptic (–16) 5′ss are shown in red. The cryptic 5′ss at +13 was mutated in all the constructs (see Materials and Methods), and the cryptic 5′ss at –38 is used very inefficiently. Three different β-thalassemia mutations (16) are shown below the sequence, with the position and nucleotide substitution indicated in each case. (B) In vitro splicing analysis of β-globin substrates. Labeled pre-mRNAs were spliced in HeLa cell nuclear extract, and the products were analyzed by electrophoresis on a denaturing polyacrylamide gel and autoradiography. Each construct is shown as a vertical diagram above each lane, with the exons as light-blue boxes and the intron as a line. The horizontal line across exon 1 shows the position of the main cryptic 5′ss at position –16. The small circles indicate the authentic (green) or mutant (red) 5′ss, and the open red ‘do not’ symbol denotes extensive mutation of the authentic 5′ss. The mobilities of the pre-mRNA and spliced mRNA bands are indicated by the diagrams on the left. Lane 1, the authentic 5′ss at +1 was mutated from CAG/GTTGGT to CAG/AACCCG; lane 2, the cryptic 5′ss at –16 was mutated to a duplicate copy of the authentic site at +1; lane 3, the authentic site at +1 was mutated to a duplicate copy of the cryptic 5′ss at –16; lane 4, the positions of the authentic site at +1 and the cryptic site at –16 were swapped; lane 5, wild-type pre-mRNA; lane 6, IVS1-G1A thalassemia mutation.