Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region.
High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products. HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed. We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases. The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1. Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome. Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality.
Lehner
Ben
B
Functional Genomics Group, MRC Rosalind Franklin Centre for Genomics Research, Hinxton, Cambridge, United Kingdom.
Semple
Jennifer I
JI
Brown
Stephanie E
SE
Counsell
Damian
D
Campbell
R Duncan
RD
Sanderson
Christopher M
CM
D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D008285:Major Histocompatibility Complex / Q000235:genetics*; D010865:Pilot Projects; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism
D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025801:Ubiquitin