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Abstract:
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Transcription of protein-coding genes is coordinated with pre-mRNA processing as well as mRNP assembly and export in mammalian cell nuclei. Btf (Bcl-2 associated transcription factor) and TRAP150 (Thyroid Hormone Receptor Associated Protein of 150 kDa or THRAP3) are serine-arginine-rich (SR) proteins that have 39% sequence identity and 66% sequence similarity; however it is not clear if the functions of these two proteins completely overlap. Btf and TRAP150 were previously reported to associate with synthetic affinity-purified in vitro spliced mRNPs, and also as a part of the spliceosome complex, suggesting they are involved in pre- mRNA processing. We used an in situ approach to show that both Btf and TRAP150 are recruited to a constitutively active beta-tropomyosin reporter minigene locus in HeLa cells as well as to the U2OS 2-6-3 inducible reporter gene locus in its transcriptionally active but not inactive state. Upon inhibition of RNA polymerase II, Btf and TRAP150 were absent from the locus, indicating their presence at the locus requires transcription. At the activated locus, both Btf and TRAP150 showed some overlap with reporter RNA and other pre-mRNA processing factors, but showed the most extensive overlap with the exon junction complex protein Magoh. Intriguingly, RNA-FISH with fluorescently tagged oligo-dT probes showed an increase in cytoplasmic polyadenylated RNA in HeLa cells specifically following Btf depletion but not TRAP150 depletion. qRT-PCR revealed an increase of beta- tropomyosin minigene reporter transcripts in the cytoplasm in Btf depleted cells. Our data suggests that modulation of Btf and TRAP150 at transcription sites affects processing and nuclear/cytoplasmic distribution of mRNAs. |
