Related Experiment Video
Updated: Jul 12, 2025
Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences
Published on: February 11, 2019
Screening of Tnfaip1-Interacting Proteins in Zebrafish Embryonic cDNA Libraries Using a Yeast Two-Hybrid System
Shulan Huang1, Hongning Zhang1, Wen Chen1
1State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
Abstract:
TNFAIP1 regulates cellular biological functions, including DNA replication, DNA repair, and cell cycle, by binding to target proteins. Identification of Tnfaip1-interacting proteins contributes to the understanding of the molecular regulatory mechanisms of their biological functions. In this study, 48 hpf, 72 hpf, and 96 hpf wild-type zebrafish embryo mRNAs were used to construct yeast cDNA library. The library titer was 1.12 × 107 CFU/mL, the recombination rate was 100%, and the average length of the inserted fragments was greater than 1000 bp. A total of 43 potential interacting proteins of Tnfaip1 were identified using zebrafish Tnfaip1 as a bait protein. Utilizing GO functional annotation and KEGG signaling pathway analysis, we found that these interacting proteins are mainly involved in translation, protein catabolic process, ribosome assembly, cytoskeleton formation, amino acid metabolism, and PPAR signaling pathway. Further yeast spotting analyses identified four interacting proteins of Tnfaip1, namely, Ubxn7, Tubb4b, Rpl10, and Ybx1. The Tnfaip1-interacting proteins, screened from zebrafish embryo cDNA in this study, increased our understanding of the network of Tnfaip1-interacting proteins during the earliest embryo development and provided a molecular foundation for the future exploration of tnfaip1's biological functions.
Insights
Tumor necrosis factor-alpha-induced protein 1 (TNFAIP1) interacts with proteins regulating DNA replication and repair. This study identified 43 TNFAIP1-interacting proteins in zebrafish embryos, advancing understanding of early development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- TNFAIP1 plays a crucial role in regulating fundamental cellular processes such as DNA replication, repair, and the cell cycle through protein interactions.
- Understanding the proteins that interact with TNFAIP1 is essential for elucidating its molecular regulatory mechanisms and biological functions.
Purpose of the Study:
- To identify novel TNFAIP1-interacting proteins in early zebrafish development.
- To analyze the functional roles of these interacting proteins using bioinformatics tools.
- To provide a molecular basis for further investigation into TNFAIP1's functions during embryogenesis.
Main Methods:
- Construction of a yeast cDNA library from wild-type zebrafish embryo mRNAs at 48, 72, and 96 hours post-fertilization.
- Yeast two-hybrid screening using zebrafish TNFAIP1 as bait to identify interacting proteins.
- Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
- Yeast spotting assays to validate specific interactions.
Main Results:
- A yeast cDNA library with a titer of 1.12 × 10^7 CFU/mL and an average insert length >1000 bp was successfully constructed.
- A total of 43 potential TNFAIP1-interacting proteins were identified.
- Bioinformatic analysis revealed that these proteins are primarily involved in translation, protein catabolism, ribosome assembly, cytoskeleton formation, amino acid metabolism, and the PPAR signaling pathway.
- Four specific interacting proteins (Ubxn7, Tubb4b, Rpl10, and Ybx1) were confirmed through yeast spotting assays.
Conclusions:
- This study successfully identified and characterized a network of TNFAIP1-interacting proteins during early zebrafish embryonic development.
- The findings enhance the understanding of TNFAIP1's regulatory roles in fundamental biological processes.
- The identified interacting proteins provide a molecular foundation for future research on TNFAIP1's functions in development and disease.
