Co-fitness analysis.

<p>Variation of fitness profiles in Cluster 1 (<b>A</b>) and Cluster 2 (<b>B</b>). Rows represent individual ncRNA deletion strains. Columns represent the eight growth conditions analysed (B>P: comparison between batch and pool; L>E: comparison between late and early steady state; C-Lim: carbon-limited medium; N-Lim: nitrogen-limited medium). Colour bar represents Log2 fold change between batch and pool or late and early steady state. Haplo-insufficiency is shown in blue, and haplo-proficiency is shown in bright red. Data can be seen in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s012" target="_blank">S12</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s013" target="_blank">S13</a> Tables</b>.</p

Haploid deletion strain phenotypic screen.

<p>Scatter plot of the normalized colony size values for each of the haploid ncRNA deletion strains growing on YPD plates at 30°C. Any strains falling outside the grey shaded area are significantly different than the wild-type strain (p < 0.05). Data can be seen in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s014" target="_blank">S14</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s015" target="_blank">S15</a> Tables</b>.</p

Analysis of early steady state to late steady state fitness changes.

<p>(<b>A</b>) Comparison of fitness between the early steady state (ESS) and late steady state (LSS) stages under the three indicated carbon-limited conditions. Haplo-proficient deletion strains have positive Log2 fold change and haplo-insufficient deletion strains have negative Log2 fold change. (<b>B</b>) Comparison of fitness between the early steady state (ESS) and late steady state (LSS) stages under the three indicated nitrogen-limited conditions. Haplo-proficient deletion strains have positive Log2 fold change and haplo-insufficient deletion strains have negative Log2 fold change. Any strains falling outside the grey shaded area have a significant fitness difference (p < 0.05). Graphs where individual points can be identified are found in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s004" target="_blank">S4</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s009" target="_blank">S9</a> Tables</b>.</p

Analysis of an essential ncRNA.

<p>(<b>A</b>) Overlap of the ncRNA SUT527 with the 3’ UTR of <i>SEC4</i>. (<b>B</b>) qRT-PCR analysis of SUT527 and <i>SEC4</i> RNA levels in a strain with SUT527 under control of the tetO7 element. Grey bars represent the relative expression of SUT527 and <i>SEC4</i> in YPD media (-) Doxycycline. Black bars represent the relative expression of <i>SUT527</i> and <i>SEC4</i> in YPD (+) Doxycycline. Error bars (SD) are from three technical replicates from three independent biological replicates. Relative normalized expression was calculated using <i>ACT1</i>. P values were calculated using the Welch two sample t-test. SUT527: <i>p</i> = 0.02, <i>SEC4</i>: <i>p</i> = 0.03. (<b>C</b>) Primer walking of cDNA isolated from cells expressing (-DOX) or not expressing (+DOX) SUT527 to assess 3’ UTR formation. Top panels depict the locations and number of the different back primers used with a common forward primer for the <i>SEC4</i> and <i>TUB2</i> RNAs. (<b>D</b>) <i>SEC4</i> mRNA was localized by FISH in the presence and absence of SUT527 expression. When SUT527 was expressed in YPD (-) DOX, 32% of the <i>SEC4</i> mRNA was localized to the cell membrane. The absence of SUT527 expression in YPD (+) DOX decreased localisation of <i>SEC4</i> mRNA to 13%. (<b>E</b>) SUT527 localization was determined by FISH. Under normal growth with YPD 33% of SUT527 was observed in foci at the cell surface similar to the localization of <i>SEC4</i>. (<b>F</b>) Three representative images of SEC4 (red) and SUT527 (green) localized together in the same cells. Nuclei are stained with DAPI (blue). Scale bars, 1μm.</p

Diagram of competition experiment and analysis of the pool to batch fitness changes.

<p>(<b>A</b>) The pool of heterozygous deletion strains was grown in batch culture before the switch to continuous culture. Samples were taken at the initial pool stage, the batch stage, early steady state (ESS), mid steady state (MSS) and late steady state (LSS). (<b>B</b>) Comparison of fitness between the pool and batch stages under the three indicated carbon-limited conditions. Haplo-proficient deletion strains have positive Log2 fold change and haplo-insufficient deletion strains have negative Log2 fold change. (<b>C</b>) Comparison of fitness between the pool and batch stages under the three indicated nitrogen-limited conditions. Haplo-proficient deletion strains have positive Log2 fold change and haplo-insufficient deletion strains have negative Log2 fold change. Any strains falling outside the grey shaded area have a significant fitness difference (p < 0.05). Graphs where individual points can be identified are found in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s004" target="_blank">S4</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007253#pgen.1007253.s009" target="_blank">S9</a> Tables</b>.</p

Genome location of ncRNA deletions and RT-PCR quantitation of mRNA levels.

<p>(<b>A</b>) Genome location of CUT248 compared to <i>DPS1</i>. All of CUT248 was deleted. Relative expression of <i>DPS1</i> in the wild type diploid background is represented by grey bars and <i>DPS1</i> expression in the CUT248 diploid heterozygous deletion background is represented by black bars. Cells were grown in either rich media (YPD) or N-limited conditions. <i>DPS1</i>: YPD <i>p</i> = 0.006, N-limited p = <0.01. (<b>B</b>) Genome location of SUT233/CUT707 between the <i>HAP4</i> and <i>KTI12</i> genes. Relative expression of <i>HAP4</i> or <i>KTI12</i> in the wild type diploid background is represented by grey bars and <i>HAP4</i> or <i>KTI12</i> expression in the SUT233/CUT707 diploid heterozygous deletion background is represented by black bars. Cells were grown in either rich media (YPD) or N-limited conditions. <i>HAP4</i>: YPD <i>p</i> = 0.97, N-limited p = <0.01. <i>KTI12</i>: YPD <i>p</i> = 0.05, N-limited p = <0.01. The fold change (2^) in expression, relative to the wild-type was calculated using the ΔΔCт method and <i>ACT1</i> as a reference gene. Error bars were calculated using three independent biological samples. P values were calculated using the Welch two sample t-test.</p

Spot assays for strains containing <i>EMP46</i>, <i>GAL2</i>, <i>EMP46/GAL2</i> or empty pBEVY-GA overexpression plasmids.

<p>The comparative fitness of the identified strain when grown on galactose media or glucose media, which activates and inactivates the GAL1/10 promoter, respectively.</p

Expression of SUT075 in <i>trans</i> rescues the lethal phenotype of a SUT075 deletion and increases <i>PRP3</i> expression.

<p>(<b>A</b>) Haploid spores from dissection of six <i>MAT</i>a<i>/α</i> SUT075<i>Δ</i>/SUT075 diploid tetrads were spotted on SD media lacking uracil and containing 2% galactose. The plasmid expressing the SUT075 ncRNA is selected for using the <i>URA</i> auxotrophic marker. Galactose induces expression of SUT075 present in the plasmid. (<b>B</b>) Haploid spores from dissection of six <i>MAT</i>a<i>/α</i> SUT075<i>Δ</i>/SUT075 diploid tetrads were spotted on SD media lacking uracil containing 2% galactose and 300mg/L G418 disulphate. G418 resistance selects for haploids deleted for SUT075. Spores growing in both panels A and B are considered to contain the G418 resistance SUT075 deletion cassette and the SUT075 ncRNA expressing plasmid. (<b>C</b>) Expression levels of <i>PRP3</i> in the ΔSUT075 and the ΔSUT075 (+ sense SUT075 recovery plasmid) heterozygote diploid strains measured by qRT-PCR. The relative expression of <i>PRP3</i> in the wild-type background is represented by a grey bar and a black bar in the ncRNA deletion strain backgrounds. Using the ΔΔCт method and <i>ACT1</i> as a reference gene, the fold change (2^) in expression, relative to the wild-type was calculated. Error bars are calculated using each of the three independent biological samples. P values calculated using the Welch two sample t-test determine there to be a significant difference (<i>p</i> = 0.02) between the ΔSUT075 and the ΔSUT075 (+ sense SUT075 recovery plasmid) strains.</p