a, Summary of the pipeline used for the identification of intron functions.b, Validation of the BSP PCR assay to detect variation in cell number. Intron-deletion cells were diluted with wild-type cells and the BSP PCR was performed on each dilution; the amplicon amount was plotted as function of dilution. The mean value of two independent experiments is presented and the s.d. is indicated by error bars. The correlation value (Spearman correlation coefficient) is indicated. This figure is related to Fig. 1.

a–f, The growth curves of wild-type cells and ∆i strains of genes with unrelated functions were determined in minimal medium with dextrose (a,f) or in minimal medium with low dextrose (b–e). The experiments were repeated independently three times with similar results. The expression profile of each host gene was determined by RT–qPCR in wild-type (dark grey) and ∆i (light grey) cells in log phase or stationary phase of growth, and is presented in the form of a bar graph. The gene name is indicated in each panel. The mean value of three (two for wild type) biologically independent strains is presented and the s.d. is indicated by error bars. This figure is related to Figs. 2,3.

Source Data

a, Introns that accumulate in the stationary phase (increased), and those that did not increase or decrease (not increased), are indicated. Intron abundance was considered to be increased when the transcripts per million (TPM) of introns at 48 h increased by more than 1.5× the TPM detected in log phase.b, The ratio of spliced and unspliced mRNA was calculated in cells that lack theMMS2 orYSF3 intron, in the log phase and stationary phase of growth; the per cent of introns is shown.c, The number of introns that accumulate or decrease upon the deletion of eitherMMS2 orYSF3 introns (or with both deletions) is indicated, for the log phase and stationary phase of growth. The pie charts shown are a descriptive representation of the data obtained by RNA sequencing, and the data that were validated using RT–qPCR (for example, see Fig. 3c and Supplementary Table 7).d, Intron deletion increases the splicing of RPGs. The intron accumulation of eight RPGs that display enhanced splicing upon the deletion ofMMS2 orYSF3 introns is shown. Relative intron accumulation was determined between Δi and wild-type strains in stationary phase of growth as described inb.e,MMS2 introns were deleted in wild-type cells, and in cells that express temperature-sensitive alleles of the splicing factorsPRP4 orPRP11 or express the RPG transcription factorIFH1 from an inducible promoter; these cells were tested for growth in low-dextrose medium at the semi-permissive temperature. The relative growth was calculated by subtracting the optical density at 660 nm (OD_660 nm) after 96 h of growth of the Δi or double-mutant strains from that of the wild-type or the single-mutant strains, respectively. The growth assays were repeated independently three times with similar results. Differences between groups were calculated using a two-sidedt-test assuming unequal variances. **P = 0.0044. This figure is related to Figs. 3,4.

Source Data

Growth profiles of ∆i cells that carry different plasmids or mutations.a,b, Expression of the host gene is not required for growth under starvation conditions. Cells that lack introns were transformed with plasmids that express the host gene of the respective intron (mms2∆i + pMMS2 orysf3Δi + pYSF3) and the growth profile was compared to wild-type (WT + p-empty) or ∆i strains (mms2∆i + p-empty orysf3Δi + p-empty) containing empty plasmids. The experiments were repeated independently nine times with similar results.c,d, Stop-codon and branch-point mutations increase intron abundance. The splicing index (calculated by dividing the amount of unspliced over total RNA, ×100) was detected using end-point PCR, and the relative abundance of cDNA (dark grey), intronic RNA (grey) and exonic RNA (light grey) was determined using RT–qPCR. The average value of three (two for wild type) biologically independent replicates is presented and the s.d. is indicated by error bars; forc, differences between wild type andmms2∆i were calculated using two-sidedt-test. *P = 0.013, **P < 0.0072, ***P = 9.8 × 10⁻⁸.e,f, Increasing the number of introns and not the host cDNA inhibits cell maintenance under starvation conditions. Growth profiles of wild-type cells transformed with empty plasmids (p-empty), plasmids expressingMMS2 RNA (p-MMS2),MMS2 RNA carrying a stop codon (p-MMS2-stop) and plasmid expressingMMS2 cDNA (p-mms2∆i). The experiments were repeated independently four times with similar results. The position of the log phase and stationary phase of the growth are indicated on the growth curves. This figure is related to Fig. 3.

Source Data

a, Increasing the number of genes that contain nutrient-independent introns does not affect growth. Growth profile of wild-type cells transformed with empty plasmids or a plasmid expressing theTUB1 gene (which contains an intron that has no effect on cell growth under starvation conditions). The experiments were repeated independently four times with similar results.b–d, Genes that contain nutrient-independent introns do not rescue the intron-deletion phenotype. Growth profile ofmms2∆i,ysf3∆i orglc7∆i cells transformed with plasmid expressing the intron-containingTUB1 gene. Forb–d, the growth assays were repeated independently six times with similar results.e,f, Intron deletions are genetically epistatic. The growth profiles of wild-type (black), single (red) and double (blue) ∆i strains were monitored for 48 h in minimal medium that is low in dextrose. The experiments were repeated independently eight times with similar results. The position of the log phase and stationary phase of the growth are indicated on the growth curves. This figure is related to Figs. 3,4.

Source Data

a,b, Growth profile ofmms2∆i andysf3∆i cells expressingMMS2 orYSF3 introns from a heterologous gene (YFP-MMS2I and YFP-YSF3I).c, Growth profile ofmms2∆i cells transformed with plasmid expressing a version ofMMS2 carrying theYSF3 intron.d, Growth profile ofmms2∆i cells transformed with plasmid expressing a version of theMMS2 gene terminating with a heterologous 3′ UTR and transcription termination sequence (ADH1t).e, Growth profile ofysf3∆i cells transformed with plasmid expressing a version ofMMS2 carrying the exon 2 ofYSF3.f, Growth profile ofmms2∆i cells transformed with plasmid expressing a version ofMMS2 carrying the exon 2 ofTUB1. Fora–f, the experiments were repeated independently six times with similar results. The position of the log phase and stationary phase of the growth are indicated on the growth curves. This figure is related to Figs. 3,4.

Source Data

a, The structure of the 5′ UTR, exon 1 and the intron that affects growth under starvation conditions was calculated using the mfold default setting, and the average of 15 suboptimal structures is presented.b, Structure of the 5′ UTR, exon 1 and the intron of theTUB1 gene, which does not affect growth under starvation conditions.c, Structures of the mutated constructs tested in Fig. 4c. The substitution of the first half (SWAP1) or the second half (SWAP2) of the intron with sequence ofACT1 intron is indicated in red.d, Structure of the mutated constructs tested in Fig. 4e, f. The position of the mutations that disrupt (left) or restore (right) the structure is shown in red. 5′S indicates the position of the 5′ splice site. The 5′ UTR and exon 1 are indicated in pale and dark blue, respectively. This figure is related to Fig. 4.

a, Comparison between the expression profiles of wild-type cells in log (WT LP) and stationary (WT SP) phases of growth.b, GO analysis of genes that are downregulated after the deletion of introns fromMMS2 andYSF3 in the stationary phase of growth. The per cent of genes in each process or activity is indicated in form of a pie chart.c, Comparison between the expression profiles of wild-type and ∆i strains in the log phase.d, Comparison between the expression profiles of ∆i strains in log phase and stationary phase.e, Comparison between the expression profiles of the two ∆i strains in the log phase (left) and stationary phase (right) of growth. Blue, red, grey and black dots indicate the number of genes that are upregulated, downregulated, not affected and upregulated, respectively, in ∆i strains in the stationary phase. Fora,c–e, the mean value from two biologically independent replicates is presented and theP value (t-test; one-sided) of the difference between the different strains and comparison was calculated as described in Methods, and is shown on each graph. This figure is related to Fig. 5.

Source Data

The growth profile of wild-type cells, cells that lack introns or cells that lack both introns and either a component of the TORC1 (tco89∆ ortor1∆) or TORC2 (avo2∆,slm1∆,slm2∆,bit61∆ orbit2∆) pathways was examined as function of the optical density of the culture in minimal medium with low dextrose. Deletions of theYSF3 intron are shown on the left and those ofMMS2 intron are shown on the right. All these experiments were repeated independently three times with similar results. This figure is related to Fig. 6.

Source Data

a, Intron deletions do not alter the expression of TORC1 targets under starvation conditions. The expression profile of 12 regulatory targets of TORC1. The fold change (expressed as log₂(mRNA abundance in SP/mRNA abundance in LP)) in RNA abundance of genes regulated by TORC1 pathway, in the log phase and stationary phase of growth, was determined using RNA sequencing in wild-type cells and cells that lackMMS2 (mms2∆i) orYSF3 (ysf3∆i) introns. The mean value and s.d. from two biologically independent replicates are presented. Genes are up- or downregulated with aP value < 0.001 (t-test; one-sided).b,c, Introns promote cell growth in a PKA-dependent manner. Growth profile of cells that lack different components of the PKA regulatory pathway (TPK1,TPK2 andTPK3) in the presence or the absence ofMMS2 andYSF3 introns. Inb,c, experiments were repeated independently three times with similar results. This figure is related to Fig. 6.

Source Data

This file contains gel source data for Figure 6d

This file contains Supplementary Tables S1-S7.