Genome-Wide Analysis of Translation in Replicatively Aged Yeast

Abstract

Protein synthesis is an essential process that affects major cellular functions including growth, energy production, cell signaling, and enzymatic reactions. However, how it is impacted by aging and how the translation of specific proteins is changed during the aging process remain understudied. Although yeast is a widely used model for studying eukaryotic aging, analysis of age-related translational changes using ribosome profiling in this organism has been challenging due to the need for isolating large quantities of old cells. Here, we provide a detailed protocol for genome-wide analysis of protein synthesis using ribosome profiling in replicatively aged yeast. By combining genetic enrichment of old cells with the biotin affinity purification step, this method allows large-scale isolation of aged cells sufficient for generating ribosome profiling libraries. We also describe a strategy for normalization of samples using a spike-in with worm lysates that permits quantitative comparison of absolute translation levels between young and old cells.

Keywords: Aging; Mother enrichment program; Protein translation; Ribo-Seq; Saccharomyces cerevisiae; Spike-in; Yeast.

Fig. 1

Overview of the Ribo-Seq protocol for analysis of translation in replicatively aged yeast cells. (a) Yeast cells are biotinylated and are grown in the presence of estradiol for 2 hrs (YNG) or 30 hrs (OLD), which induces the Mother Enrichment Program (MEP) preventing division of daughter cells. (b) Biotinylated mother cells are then separated using magnetic cell sorting enabling isolation of large quantities of old cells sufficient for generating ribosome profiling libraries. The age of yeast cells that have been isolated using the MEP and magnetic sorting is determined by counting the number of “bud scars” stained with calcofluor dye. The population of OLD cells obtained after sorting on average contains ~11–13 more bud scars per cell compared to YNG cells. (c) An equal amount of the worm lysate spike-in control (1%) is added into each sample proportional to the number of A₂₆₀ units in yeast lysates allowing normalization of translation changes in aged and young cells.

Fig. 2

Sucrose gradient fractionation of the yeast lysates prepared from varying quantities of old cells. Cell lysates containing the indicated number of A₂₆₀ units (a) or varying numbers of cells (b) were digested with RNase I and fractionated using 10–50% sucrose gradients. In our experience, we were able to collect the monosome peak and successfully prepare Ribo-Seq libraries from as low as 50×10⁶ cells.

Fig. 3

Representative footprint and fragmented mRNA size selection gels after T4 polynucleotide kinase treatment. (a) Footprint fragments prepared from YNG and OLD yeast samples according to our protocol were separated on 15% polyacrylamide TBU-urea gel. 28 nt and 32 nt RNA oligonucleotides are used to guide the size of the gel slice that should be excised. (b) For fragmented mRNA samples, cut the gel slice around 50–70 nt. 63-mer RNA oligo is used as a control.

Fig. 4

Representative size selection gels used to isolate footprint and mRNA samples after reverse transcription. (a) Footprint samples obtained after reverse transcription were separated on 10% polyacrylamide TBU-urea gel. A mixture of RT primer and 128 nt marker oligonucleotide is used as a control. Cut the gel slice around 128 nt (upper band), which corresponds to the reverse transcription product. (b) For mRNA samples, cut just above the 128 nt marker. The size of the excised product should be around 150–170 nt.

Fig. 5

Representative size selection gels used to isolate PCR amplified sequencing libraries. Following PCR amplification with varying number of cycles, the samples were separated on non-denaturing 8% polyacrylamide TBE gels. Select the desired number of cycles, in which the PCR products form a single, but bright enough band. Avoid lanes with high background. Cut the band ~150 bp for footprint libraries, and 170–190 bp for mRNA libraries.