Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Ja'Laquan Mitchell - Dartmouth College
Co-Author(s): Johnny J. He and Khalid A. Timani, Department of Cell Biology and Immunology, UNT Health Science Center at Fort Worth, TX
Background: HIV-1 Tat-interacting protein of 110 kDa (Tip110) is a 963-amino acid, nuclear-RNA-binding protein that plays important roles in pre-mRNA splicing, spliceosome assembly, regulation of viral gene expression, and tumor development. Our previous mass spectroscopy-based-proteomic study showed that hnRNP-U, which is a member of the heterogeneous nuclear ribonucleoproteins, is present in the Tip110 complex. HnRNP-U plays roles in gene expression, pre-mRNA processing, regulation of transcription, and alternative splicing.
Purpose: The purpose of this work is to confirm the possible interaction between Tip110 and hnRNP-U and its proposed functional mechanisms. Methods: Human kidney fibroblast (293T) cells were used in this study. Immunoprecipitation and immunofluorescence analysis were employed to identify the Tip110-hnRNP-U interaction. Western blotting was utilized to study whether Tip110 regulates hnRNP-U protein expression.
Results: From our investigation, we confirmed that Tip110 complexed with hnRNP-U. Immunofluorescence assay demonstrated that hnRNP-U co-localized with Tip110 in the nucleus. In addition, the increasing concentration of Tip110 in 293T transfected cells revealed an increased in hnRNP-U protein expression. To confirm that hnRNP-U can in fact shuttle between the nucleus and cytoplasm, we used actinomycin D to prevent hnRNP-U from entering the nucleus. Results further showed that Tip110 expression may affect the translocation of hnRNP-U from the nucleus to the cytoplasm. Conclusion: Tip110 and hnRNP-U proteins interacted with each other and Tip110 regulates hnRNP-U expression levels. A future study will include seeing this interaction invivo.
Funder Acknowledgement(s): Funded by the Department of Health and Human Services, National Institute of Health, National Heart, Lung and Blood Institute, SMART Grant 4R25HL007786-24 to Jamboor K. Vishwanatha.
Faculty Advisor: Khalid Timani, email@example.com
Role: I did everything from the western blotting and precipitation, to the immunofluorescence assays. The co authors are my PI and Mentor. They taught me the skills in order to run the experiment, but they did not do/find any of the research I will be discussing on my poster.