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. 2011 Jun;10(6):M110.003350.
doi: 10.1074/mcp.M110.003350. Epub 2011 Mar 28.

5'TRU: identification and analysis of translationally regulative 5'untranslated regions in amino acid starved yeast cells

Nicole Rachfall  1 , Isabelle Heinemeyer, Burkhard Morgenstern, Oliver Valerius, Gerhard H Braus
Affiliations

5'TRU: identification and analysis of translationally regulative 5'untranslated regions in amino acid starved yeast cells

Nicole Rachfall et al. Mol Cell Proteomics. 2011 Jun.

Abstract

We describe a method to identify and analyze translationally regulative 5'UTRs (5'TRU) in Saccharomyces cerevisiae. Two-dimensional analyses of (35)S-methionine metabolically labeled cells revealed 13 genes and proteins, whose protein biosynthesis is post-transcriptionally up-regulated on amino acid starvation. The 5'UTRs of the respective mRNAs were further investigated. A plasmid-based reporter-testing system was developed to analyze their capability to influence translation dependent on amino acid availability. Most of the 13 candidate 5'UTRs are able to enhance translation independently of amino acids. Two 5'UTRs generally repressed translation, and the 5'UTRs of ENO1, FBA1, and TPI1 specifically up-regulated translation when cells were starved for amino acids. The TPI1-5'UTR exhibited the strongest effect in the testing system, which is consistent with elevated Tpi1p-levels in amino acid starved cells. Bioinformatical analyses support that an unstructured A-rich 5' leader is beneficial for efficient translation when amino acids are scarce. Accordingly, the TPI1-5'UTR was shown to contain an A-rich tract in proximity to the mRNA-initiation codon, required for its amino acid dependent regulatory function.

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Figures

Fig. 1.
Fig. 1.
De novo proteome of the soluble cell fraction obtained under non-starvation (w/o 3AT) and aa-starvation (10 mm 3AT). Autoradiographies corresponding to two-dimensional-PAGE (supplemental Fig. S2) of 35S-methionine radioactively labeled proteins. The numbers indicate excised protein-spots with an enhanced intensity under aa-starvation conditions.
Fig. 2.
Fig. 2.
Comparison of proteome and transcriptome data generated to monitor effects of aa-starvation conditions. All candidates listed were found to be up-regulated upon aa-starvation in the proteome analyses (see supplemental Table S2 for corresponding protein names/functions and supplemental Table S3 for data on protein sequence identification). The transcriptome data used in this comparison has been obtained under similar conditions (a27). Transcriptome as well as proteome changes induced by aa-starvation conditions are displayed as the quotient of spot-intensity under aa-starvation to spot-intensity under nonstarvation conditions (+3AT/-3AT). To clearly illustrate up- and down-regulation, transcriptome and proteome changes are visualized logarithmically in a horizontal histogram. The reproducibility for each candidate is expressed as frequency, describing the number by which a protein has been identified as up-regulated in n of five biological replicates. The last column represents the ‘confidence factor’ which is composed of the fold change (+3AT/-3AT) of the proteome relative to that of the transcriptome and the frequency of each proteome candidate.
Fig. 3.
Fig. 3.
Reporter-testing system and β-galactosidase assays displaying the effects of candidate-5′UTRs on lacZ-reporter activity dependent on aa-availability. A,The reporter-testing vector is a 2 μm yeast-E. coli shuttle vector carrying selectable marker genes as well as the constitutive PGK1-promoter with a defined transcription start site (TSS) and a lacZ-reporter-gene. Arbitrary 5′UTR sequences can be inserted in between promoter and reporter gene. Possible incorporated translationally regulative elements are depicted in the lacZ-mRNA-5′UTR such as hairpin structures, upstream open reading frames (uORFs) and internal ribosome entry sites (IRES). The three observed effects on lacZ-expression upon introduction of candidate 5′UTR sequences are illustrated in: B, reduced expression w/o significant 3AT effect; C, enhanced expression w/o significant 3AT effect; and D, enhanced expression w/additional positive 3AT effect. β-galactosidase activities were normalized to respective plasmid copy numbers (supplemental Fig. S5A) and are displayed relative to the testing vector without 5′UTR under the respective condition.
Fig. 4.
Fig. 4.
Steady-state protein- and mRNA-amount of candidates determined under non-starvation (-3AT) and aa-starvation (+3AT) conditions. A, myc3-tagged versions of the candidate-proteins Eno1p and Fba1p were hybridized to anti-myc antibody, whereas Tpi1p was detected by anti-Tpi1 antibody. For quantification the total intensity of all spots marked by crosshairs under the respective condition was determined. The illustrated change in de novo biosynthesis was previously determined by two-dimensional-PAGE of metabolically labeled protein extracts and autoradiography analysis (see Figs. 1 and 2). B, Northern hybridizations against ENO1-, FBA1- and TPI1-mRNA were quantified and normalized against ACT1 as loading control. The adjacent graphs, respectively, illustrate the fold changes relative to the signal strengths under non-starvation conditions. Cells were grown to exponential phase before further incubation for 90 min in absence (-3AT) or presence (+3AT) of 10 mm 3AT.
Fig. 5.
Fig. 5.
Evaluation of artificially generated 5′UTRs. A, DNA-fragments were constructed and expressed via reporter-testing system, resulting in the schematically illustrated mRNA-5′UTR sequences. The corresponding minimal free energies (MFEs) were determined with RNAFOLD. For the TPI1- and AHP1-5′UTRs the last 25 nt and altered bases are highlighted. The respective adenine-contents are displayed for full length 5′UTRs and the last 25 nt. B, The β-galactosidase assays display the influence on lacZ-expression exhibited by the respective 5′UTR inserted in the reporter-testing vector under non-starvation (-3AT) and aa-starvation conditions (+3AT). The data was normalized to the plasmid copy numbers for each construct (supplemental Fig. S5B) and are displayed relative to the testing vector without 5′UTR under the respective condition.
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