Figure 1.
Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants.
A. Venn diagram showing the overlap of alternative splice site usage between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events (both PTC-generating and non-PTC-generating). B. Venn diagram showing the overlap of alternative splicing events between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events resulting in a potential PTC. C. Venn diagram showing the overlap of alternative splicing events between the upf1Δ, upf2Δ, and upf3Δ strains for PTC-generating splicing events. D. Distributions of intron-containing gene transcripts showing alternative splicing events (red) or no alternative splicing events (blue) according to their overall abundance in RPKM. Transcripts for which the abundance was higher than 2,300 RPKM were grouped in the final bin. E. Sequence logo analysis of 5′- and 3′- splice sites for all normal and alternative splicing events detected by RNA-Seq in wild-type and NMD mutant strains.
Figure 2.
Spliced species produced from the SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 genes.
Species labeled with an asterisk are subject to NMD. Species labeled with two asterisks are predicted to be subject to NMD but were not observed to do so in subsequent experiments. The alternative 3′-SS of SRC1 is located 4 nt upstream from the annotated 3′-SS. The alternative 3′-SS of RPL22B is located 64 nt downstream from the annotated 3′-SS. The alternative 3′-SS of TAN1 are located 6 nt upstream and 7 nt downstream from the annotated 3′-SS. The alternative 3′-SS of TFC3 is located 17 nt downstream from the annotated 3′-SS. The alternative 5′ and 3′-SS of GPI15 are located 36 nt downstream and 14 nt upstream, respectively, from the annotated 5′ and 3′-SS. The alternative 3′-SS of GCR1 are located 5 nt upstream (GUAUGG); 51 nt downstream (GUAUGG) and 627 nt downstream from the annotated 5′SS. The alternative 3′-SS of GCR1 are located 40 nt upstream (AUG) and 17 nt downstream (CAG) from the annotated 3′-SS.
Figure 3.
RT-PCR analysis of alternatively spliced products for SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 in wild-type, NMD and various splicing mutants.
The unspliced (US) species is also shown on top. The middle portions of the gel where no species were visible have been removed. In all cases, RT-PCR was performed with a Cy3-labeled primer. The labeling of the different alternatively spliced forms is according to the nomenclature shown in Figure 2.
Figure 4.
RT-PCR analysis of alternatively spliced products under stress conditions.
A. Analysis of RPL22B in various stress conditions. Shown are the RT-PCR products obtained from the wild-type or upf1Δ mutant strain after growth in the following conditions: SDC, synthetic define complete medium at 30°C; -AA, 10 minutes in SDC medium at 30°C lacking amino acid (-AA); 25°C, log phase at 25°C in YPD; H.S, 20 minutes at 42°C in YPD; YPD: log phase at 30°C in YPD; LiCl, incubation with 300 mM LiCl in YPD at 30°C for 10 minutes; RAP control, see Materials and Methods; RAP, treatment with Rapamycin for 20 minutes. B. RT-PCR analysis of GCR1 alternative splicing in heat-shock conditions. Labeling of the different species is similar to that of Figures 2 and 3.
Figure 5.
Effects of mutations of the RPL22B alternative 5′ splice site on RPL22B splicing and expression in normal and stress conditions.
A. Organization of the RPL22B precursor, with the normal and alternative 5′-splice sites. Shown are the mutations to the consensus sequence (CS) GUAUGU or the deletion that entirely removes the GUUUGU sequence. B. Analysis of the effect of these mutations on RPL22B splicing and expression at normal temperatures (25°C) or after a 20 min heat shock at 42°C. N, natural 5′-splice site (GUUUGU); CS, consensus sequence (GUAUGU); Δ = deletion of the alternative 5′-splice site. Top panel: RT-PCR analysis. Bottom panel: northern blot analysis. US, *AS-5′, and S indicate the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. For the northern blot, SCR1 was used as a loading control. C. Analysis of the effect of the RPL22B alternative splice site consensus mutation on RPL22B expression during amino acid starvation. Shown is a northern blot of RNA samples extracted from the indicated strains grown at 30°C in normal synthetic define complete (SDC) medium with amino acid (+) or in SDC medium lacking amino acid (−) for 10 minutes. Strains contained either the natural GUUUGU sequence at the alternative 5′-splice site of RPL22B, or the consensus GUaUGU sequence. The nucleotide mutated is highlighted in lower case. Labeling of the different species is similar to that of panel B. SCR1 was used as a loading control.
Figure 6.
Replacement of RPL22B promoter by the GAL promoter results in a decrease in alternative 5′-splice site usage.
Shown are the products generated when growing the indicated strains (wild-type or upf1Δ that contained the natural RPL22B promoter or the GAL promoter upstream RPL22B) in glucose (YPD) or galactose (YPGal)-containing media. Top panel, RT-PCR analysis. US, *AS 5′, and S indicate the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. Bottom Panel, Northern blot analysis. The labeling of the different species is similar to that of the top panel. SCR1 was used as a loading control.