Article Figures & Data
Figures
- FIG. 1.
Enhancer dependency of variable 5′ splice sites for efficient intron-defined splicing. (A) Diagram of modified β-globin minigene templates with variable 5′ splice sites (Table 1), an MS2 hairpin ESE activated by the addition of MS2-RS-9G8, and either a 120-nt or a 360-nt intron. (B) Autoradiogram showing a time course analysis of splicing with a weak 5′ splice site (guG|GUucGUAU; MaxEnt Score = 3.3 [see Table 1 for description]) without enhancer (left) compared to that with the same splice site activated by MS2-RS-9G8 recruitment (right). Pre-mRNA transcript, spliced product, and intermediate lariat bands are indicated. (C and D) Comparison of the splicing rates determined from short intron (C) or long intron (D) pre-mRNAs according to splice site strength (MaxEnt Score range, −9.4 to 10.9). Average rates were determined from multiple repeats with less than 30% difference between experiments. Further comparison of 5′ splice site sequences, strength (MaxEnt Score), and splicing rates can be found in Table 1.
- FIG. 2.
Proximal 5′ splice sites are preferred over a strong distal 5′ splice site. (A) Diagram of a modified β-globin minigene template with an upstream invariable 5′ splice site (CAG|GUAAGUgU), a downstream variable 5′ splice sites, and a short intron. (B and C) Representative autoradiograms showing a time course of the in vitro splicing assay. (B) Gel analysis of splicing with a moderately strong 5′ splice site (Cuc|GUAAGUca; MaxEnt Score = 9.6) without enhancer (left) compared to that with the same splice site activated by MS2-RS-9G8 recruitment (right). (C) Same as panel B except the splice site is stronger (CAG|GUAAGUca; MaxEnt Score = 10.9). Pre-mRNA transcript and proximally and distally spliced product bands are indicated. (D) Comparison of the proximal-to-distal 5′ splice site ratio according to splice site strength with and without MS2-RS-9G8 recruitment. Further comparison of 5′splice site sequences, strength, and splicing rates can be found in Tables 2 and 3. (E) Fractional changes in the proximal-to-distal splice site selection ratio as a consequence of moving competing 5′ splice sites into closer proximity. The bar graph illustrates that reducing the distance between competing 5′ splice sites decreases proximal splice site selection in the context of short or long introns.
- FIG. 3.
MS2-RS-9G8 Recruitment inhibits upstream 5′ splice site. (A) Diagram of the β-globin minigene template with an abolished downstream 5′ splice site. The X indicates the abolished proximal splice site. (B) Representative autoradiogram showing a time course of the in vitro splicing assay. Gel analysis of splicing with the strong upstream 5′ splice site (CAG|GUAAGUgU; MaxEnt Score = 10.9) without MS2-RS-9G8 recruitment (left) compared to that with the same splice site inhibited by MS2-RS-9G8 recruitment (right) is shown. Pre-mRNA transcript and distally spliced product bands are indicated. (C) Graph comparison of the fraction spliced during the time course of the experiment.
- FIG. 4.
Upstream 5′ splice sites activate the downstream variable 5′ splice site. (A) The graph compares splicing rates of various short intron-proximal 5′ splice sites with and without upstream activation according to splice site strength. (B) The graph compares splicing rates of various long intron-proximal 5′ splice sites with and without upstream activation according to splice site strength.
- FIG. 5.
A competing upstream 5′ splice sites increases proximal splicing efficiency. (A) Diagrams of β-globin minigene templates with functional upstream and downstream 5′ splice sites (upper) compared to the isogenic construct with an abolished upstream 5′ splice site (lower). The X indicates the abolished splice site. (B) Representative autoradiogram showing a time course experiment in the absence of MS2-RS-9G8. The wild-type (wt) upstream 5′ splice site (left) is compared to the abolished upstream 5′ splice site (right). Pre-mRNA transcript and proximally spliced product bands are indicated on the right. (C) Representative autoradiogram showing an identical time course experiment in the presence of MS2-RS-9G8. (D) Quantitation of the data shown in panel B in the absence of MS2-RS-9G8.
- FIG. 6.
Distribution of strong and weak potential 5′ splice sites along constitutive exons. The presence of potential 5′ splice sites in 5,000 constitutive exons was measured using the MaxEnt scoring scheme (31), where a 5′ MaxEnt score of >7 represents a strong 5′ splice site and a MaxEnt score of <0 represents a weak 5′ splice site. The y axis shows the two classes of potential 5′ splice sites binned in 10-nt bins. Each bin was normalized to 5′ splice site frequencies observed for the first 10-nt bin closest to the 3′ splice site. The x axis represents the distance of 5′ splice site bins from the 3′ splice site.
Tables
- TABLE 1.
Splicing rates of 5′ splice sites correlate with U1 snRNP potential
Construct Short intron Long intron No. 5′ splice site sequencea ΔGb MaxEnt scorec Avg rated Fold changed MS2-RS-9G8 Fold change − MS2-RS-9G8 + MS2-RS-9G8 − MS2-RS-9G8 + MS2-RS-9G8 0 gua|cUucuaca >0 −26.3 * * * * * * 1 CAa|GUcAagcc 0 −9.4 0.15 0.64 4.3 ** ** ** 2 guG|GUucuaca −0.9 −8.2 * * * 0.007 0.04 6.4 3 gAG|GUucuaca −2.8 −1.6 0.11 0.47 4.1 0.03 0.16 5.2 4 CAG|GUucuaca −3.6 0.7 0.10 0.36 3.5 0.07 0.20 2.7 5 CuG|GUcuGUAU −3.3 3.1 0.14 0.58 4.0 0.14 0.41 3.0 6 guG|GUucGUAU −3.3 3.3 0.25 0.85 3.4 0.08 0.35 4.7 7 guG|GUucGUca −1.6 3.3 0.69 1.13 1.6 0.11 0.23 2.1 8 CAG|GUugGUAU −8.5 8.1 0.37 1.62 4.4 0.24 0.54 2.3 9 Cuc|GUAAGUca −3.5 9.6 0.20 0.66 3.2 ** ** ** 10 CAc|GUAAGUca −4.0 10.2 0.34 1.33 3.9 0.15 0.41 2.8 11 CAG|GUAAGUAg −11.3 10.9 1.10 1.35 1.2 0.37 0.40 1.1 12 CAG|GUAAGUca −9.4 10.9 0.59 0.75 1.3 0.44 0.74 1.7 ↵ a Uppercase letters indicate matches with U1 snRNA, and lowercase letters indicate mismatches with U1 snRNA.
↵ b ΔG values are based on nearest-neighbor contributions (32).
↵ c Rows are organized according to MaxEnt scores, which are values based on modeling the sequences of short sequence motifs that simultaneously account for nonadjacent as well as adjacent dependencies between positions. This method is based on the maximum-entropy principle and generalizes most previous probabilistic models of sequence motifs such as weight matrix models and nonhomogeneous Markov models (31).
↵ d The data show rate and fold activation comparisons of the single 5′ splice sites in the context of intron versus exon definition, with and without MS2-RS-9G8 recruitment. Average rates and fold activation were determined from multiple repeats with less than 30% difference between experiments. *, value not different from background. **, not tested in this context.
- TABLE 2.
In the context of competing splice sites, proximal splice site selection is the predominant splice pattern
Construct Short intron Long Intron No. 5′ splice site sequencea MaxEnt scoreb Avg ratioc Fold changec Avg ratio Fold Change − MS2-RS-9G8 + MS2-RS-9G8 − MS2-RS-9G8 + MS2-RS-9G8 0 gua|cUucuaca −26.3 * * * * * * 1 CAa|GUcAagcc −9.4 7.4 10 1.4 2.6 3.7 1.4 2 guG|GUucuaca −8.2 1.4 1.7 1.2 5.3 8.8 1.7 3 gAG|GUucuaca −1.6 3.2 3.9 1.2 ** ** ** 4 CAG|GUucuaca 0.7 5.8 10 1.7 ** ** ** 5 CuG|GUcuGUAU 3.1 8.7 18 2.1 4.9 9.4 1.9 6 guG|GUucGUAU 3.3 12 15 1.2 ** ** ** 7 guG|GUucGUca 3.3 5.1 6.6 1.3 2.7 5.3 2.0 8 CAG|GUugGUAU 8.1 33 68 2.1 11 13 1.2 9 Cuc|GUAAGUca 9.6 5.7 24 4.2 ** ** ** 10 CAc|GUAAGUca 10.2 4.6 5.8 1.3 5.9 4.9 0.8 11 CAG|GUAAGUAg 10.9 32 35 1.1 7.9 9.4 1.2 12 CAG|GUAAGUca 10.9 24 38 1.6 ** ** ** Distal CAG|GUAAGUgU 10.9 NA NA NA NA NA NA ↵ a Uppercase letters indicate matches with U1 snRNA, and lowercase letters indicate mismatches with U1 snRNA.
↵ b Rows are organized according to MaxEnt scores (31).
↵ c Average ratios of proximal/distal usage and fold activation were determined from multiple repeats with less than 30% difference between experiments. *, value not different from background. **, not tested in this context. NA, not applicable.
- TABLE 3.
Rates of proximal 5′ splice site usage in the presence of a competing upstream 5′ splice site
Construct Short intron Long intron No. 5′ splice site sequencea MaxEnt scoreb Avg ratec Fold changec Avg rate Fold change − MS2-RS-9G8 + MS2-RS-9G8 − MS2-RS-9G8 + MS2-RS-9G8 0 gua|cUucuaca −26.3 * * * * * * 1 CAa|GUcAagcc −9.4 0.91 0.92 1.0 0.62 0.74 1.2 2 guG|GUucuaca −8.2 0.001 0.002 1.1 0.40 0.83 2.1 3 gAG|GUucuaca −1.6 0.17 0.45 2.6 ** ** ** 4 CAG|GUucuaca 0.7 0.40 0.58 1.5 0.21 0.34 1.6 5 CuG|GUcuGUAU 3.1 0.88 0.98 1.1 0.33 0.60 1.8 6 guG|GUucGUAU 3.3 1.0 1.1 1.1 0.41 0.77 1.9 7 guG|GUucGUca 3.3 0.92 0.98 1.1 0.50 0.84 1.7 8 CAG|GUugGUAU 8.1 1.1 1.1 1.0 0.71 0.81 1.1 9 Cuc|GUAAGUca 9.6 0.82 1.00 1.2 ** ** ** 10 CAc|GUAAGUca 10.2 0.84 0.64 0.8 0.84 0.56 0.7 11 CAG|GUAAGUAg 10.9 0.82 0.83 1.0 0.26 0.21 0.8 12 CAG|GUAAGUca 10.9 1.0 1.0 1.0 ** ** ** Distal CAG|GUAAGUgU 10.9 0.61 0.06 −10.3 ** ** ** ↵ a Uppercase letters indicate matches with U1 snRNA, and lowercase letters indicate mismatches with U1 snRNA.
↵ b Rows are organized according to MaxEnt scores (31).
↵ c The data show rate and fold activation comparisons of each 5′ splice site in the context of intron versus exon definition, upstream 5′ splice site competition, and with and without MS2-RS-9G8 recruitment. Average rates and fold activation were determined from multiple repeats with less than 30% difference between experiments. *, value not different from background. **, not tested in this context.
