Probing a role for TRAP150 in gene regulation

by Potabathula, Divya

Potabathula, Divya. M.S., Biological Sciences, Wright State University. 2009. Probing a Role for TRAP150 in Gene Regulation. The mammalian cell nucleus is a highly organized cellular compartment. The nucleus is surrounded by the nuclear envelope and contains distinct domains such as nucleoli, nuclear speckles and Cajal bodies that may reflect nuclear functions such as transcription and RNA processing. Nuclear speckles are regions in the nucleus that are enriched in splicing factors and other RNA processing factors, as well as some transcription factors and the large subunit of RNA polymerase II. Mass spectrometry analysis of purified nuclear speckles revealed 146 nuclear speckle proteins of which 33 are novel nuclear speckle components. One such novel nuclear speckle protein is thyroid hormone receptor associated protein (TRAP150/THRAP3). Immunofluorescence of TRAP150 in Hela cells shows that it localizes predominantly in a diffuse distribution throughout the nucleoplasm that resembles the localization of transcription factors. Phylogenetics showed that TRAP150 and Btf (another novel speckle protein) are members of the same gene family. The amino acid sequence alignment of TRAP150 and Btf showed that they share 39.5% sequence identity and 62% similarity. High sequence similarity was observed at the C-termini, suggesting a second functional domain of unknown function. TRAP150 and Btf associate with affinity-purified in vitro-spliced human mRNPs. Hence, I hypothesized that TRAP150 might have a role in transcription and/or pre-mRNA processing. Inhibition of RNA polymerase II transcription caused extensive enrichment of TRAP150 in large, rounded nuclear speckles. I observed the association of TRAP150 and Btf with a reporter gene locus in vivo. Results showed that TRAP150 and Btf associate more frequently with the activated reporter locus than with the inactive locus. TRAP150 and Btf also localized in the vicinity of the reporter RNA. These results are all consistent with a role for TRAP150 and Btf in gene expression.