Insights into the human cDNA: A descriptive study using library screening in yeast

Abstract

The utilization of human cDNA libraries in yeast genetic screens is an approach that has been used to identify novel gene functions and/or genetic and physical interaction partners through forward genetics using yeast two-hybrid (Y2H) and classical cDNA library screens. Here, we summarize several challenges that have been observed during the implementation of human cDNA library screens in Saccharomyces cerevisiae (budding yeast). Upon the utilization of DNA repair deficient-yeast strains to identify novel genes that rescue the toxic effect of DNA-damage inducing drugs, we have observed a wide range of transcripts that could rescue the strains. However, after several rounds of screening, most of these hits turned out to be false positives, most likely due to spontaneous mutations in the yeast strains that arise as a rescue mechanism due to exposure to toxic DNA damage inducing-drugs. The observed transcripts included mitochondrial hits, non-coding RNAs, truncated cDNAs, and transcription products that resulted from the internal priming of genomic regions. We have also noticed that most cDNA transcripts are not fused with the GAL4 activation domain (GAL4AD), rendering them unsuitable for Y2H screening. Consequently, we utilized Sanger sequencing to screen 282 transcripts obtained from either four different yeast screens or through direct fishing from a human kidney cDNA library. The aim was to gain insights into the different transcription products and to highlight the challenges of cDNA screening approaches in the presence of a significant number of undesired transcription products. In summary, this study describes the challenges encountering human cDNA library screening in yeast as a valuable technique that led to the identification of important molecular mechanisms. The results open research venues to further optimize the process and increase its efficiency.

Keywords: Genetic screens; Saccharomyces cerevisiae; Yeast two-hybrid; cDNA library screens.

Fig. 1

Y2H cDNA library construction and screening procedure. A) The conventional procedure for cDNA library generation. B) Using the cDNA library in Y2H screens to identify protein–protein interactions. C) Using the cDNA library to screen for rescue genes involved in DNA repair. D) Screening randomly selected plasmids from the cDNA library to screen random samples from the library “Created with Biorender.com.”.

Fig. 2

pACT2 sequence. The pACT2 plasmid map shows the transcript cloned between the two restriction sites (EcoR1 and Xho1). P_ADH1; ADH1 promoter, T_ADH1; ADH1 transcription termination signal, NLS; Nuclear localization sequence, HA; HA epitope tag. pACT2-F and pACT2-R primers are used for sequencing purposes. “Created with Biorender.com.”.

Fig. 3

The inefficiency of the reverse transcriptase on large transcripts. “Created with Biorender.com.”.

Fig. 4

mRNA priming in cDNA libraries. A) Normal priming through reverse transcriptase elongation of cDNA after oligo (dT) binding to true poly-A tails. B) Internal priming that leads to the generation of truncated transcripts due to internal A-rich regions and reverse transcriptase stalling. “Created with Biorender.com.”.

Fig. 5

Oligo (dT) primers bind to A-rich regions in genomic DNA “Created with Biorender.com.”.

Fig. 6

Schematic view of mitochondrial transcripts observed in our screens “Created with Biorender.com”.

Fig. 7

Pie chart showing the percentage of analyzed cDNAs and other transcripts in this study.