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The Important Importins: Nuclear Import of the Thyroid Hormone Receptor alpha

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Author: Parente, Laura Elizabeth
Advisor: Allison, Lizabeth
Committee Members: Zwollo, Patty; Shakes, Diane C.; Lockwood, Rowan
Issued Date: 2010-05-13
Subjects: Importins
Thryoid hormone receptor
URI: http://hdl.handle.net/10288/2009
Description: The thyroid hormone receptor ?1 (TR?) is a nuclear receptor for the thyroid hormone, and acts to either activate or repress transcription of thyroid hormone-responsive genes. While TR? carries out its function as a transctiption factor in the nucleus, TR? 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 TR? is imported into the nucleus. Previous research has shown that in mammalian cells, TR?�s nuclear import follows a signal-mediated pathway that is temperature and energydependent, and can be mediated in vitro by importins ?1 and ?. This thesis research investigated which importins are able to mediate nuclear import of TR? 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 TR? 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 ?, ?1, ?2, ?3, ?4, ?5, ?6, 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 ?, ?1, ?2, ?3, ?4, ?5 and Ipo 7) in mediating TR?�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 TR?. A shift from the normal, primarily nuclear localization of TR? to a more whole-cell distribution was seen as evidence of the RNAi-targeted importin mediating TR? import. Knockdown of Ipo 7 resulted in the most significant cytoplasmic shift of TR?, suggesting that TR?�s nuclear import is mediated primarily by Ipo 7. Knockdown of Imp ? caused the second most significant shift in TR? distribution, indicating that Imp ? also plays a substantial role in TR?�s nuclear import. The importin ? isoforms varied in the extent to which they were able to mediate TR? import, with the data indicating that Imp ?1 is the main adaptor importin acting with Imp ? to import TR? into the nucleus. Imp ?3 also appears to play a lesser role in TR?�s import. Taken together, the data presented in this thesis research suggest that TR? utilizes multiple import pathways, with import facilitated primarily by Ipo 7 and Imp ?/Imp ?1, and potentially Imp?/Imp ?3 to a lesser extent.
Degree: Bachelors of Science in Biology

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