Date Thesis Awarded

5-2010

Document Type

Honors Thesis

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Lizabeth Allison

Committee Member

Patty Zwollo

Committee Member

Diane C. Shakes

Committee Member

Rowan Lockwood

Abstract

The thyroid hormone receptor α1 (TRα) is a nuclear receptor for the thyroid hormone, and acts to either activate or repress transcription of thyroid hormone-responsive genes. While TRα carries out its function as a transctiption factor in the nucleus, TRα actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of this shuttling activity, and to its role as a nuclear transcription factor, is the process by which TRα is imported into the nucleus. Previous research has shown that in mammalian cells, TRα's nuclear import follows a signal-mediated pathway that is temperature and energydependent, and can be mediated in vitro by importins α1 and β. This thesis research investigated which importins are able to mediate nuclear import of TRα in HeLa (human) cells in vivo. RNA interference (RNAi) was used to knock down the expression of individual importins in the cells, after which the subcellular localization of TRα was examined for any changes. RNAi knockdown was validated using reverse transcriptase real-time PCR (RT-qPCR). Total RNA was purified from RNAi-treated cells, and was shown to be of high quality through UV spectrophotometry and electrophoresis. This RNA was then reverse-transcribed into cDNA and analyzed by qPCR. RT-qPCR was used to evaluate eight importins (Imps β, α1, α2, α3, α4, α5, α6, and Ipo 7) for knockdown of their corresponding mRNA levels following transfection with importin-specific RNAi. The data show that RNAi knockdown was validated, with seven out of the eight importins showing knockdown at levels of 70% or higher. RNAi import assays were used to directly evaluate the role of seven individual importins (Imps β, α1, α2, α3, α4, α5 and Ipo 7) in mediating TRα's nuclear import. The qPCR results confirmed that importin-specific short hairpin RNA (shRNA) molecules effectively reduced the level of those importins in the cell. Following RNAi treatment, cells were examined by fluorescence microscopy to evaluate the subcellular localization of TRα. A shift from the normal, primarily nuclear localization of TRα to a more whole-cell distribution was seen as evidence of the RNAi-targeted importin mediating TRα import. Knockdown of Ipo 7 resulted in the most significant cytoplasmic shift of TRα, suggesting that TRα's nuclear import is mediated primarily by Ipo 7. Knockdown of Imp β caused the second most significant shift in TRα distribution, indicating that Imp β also plays a substantial role in TRα's nuclear import. The importin β isoforms varied in the extent to which they were able to mediate TRα import, with the data indicating that Imp α1 is the main adaptor importin acting with Imp β to import TRα into the nucleus. Imp α3 also appears to play a lesser role in TRα's import. Taken together, the data presented in this thesis research suggest that TRα utilizes multiple import pathways, with import facilitated primarily by Ipo 7 and Imp β/Imp α1, and potentially Impβ/Imp α3 to a lesser extent.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Comments

Thesis is part of Honors ETD pilot project, 2008-2013. Migrated from Dspace in 2016.

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