Abstract
EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations. In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1. EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor. Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.
Highlights
Chromosomal abnormalities such as deletions, inversions, or translocations are common genetic mechanisms to induce mutations that contribute to tumorigenesis
We identified U1C, one of three U1 small nuclear ribonucleoprotein1-specific proteins. snRNPs play an essential role in splicing through a large complex known as the spliceosome
U1C Interacts with the N Terminus of EWS—We performed a yeast two-hybrid screen to identify proteins that interact with the amino-terminal region of EWS found in several tumorspecific chromosomal translocations
Summary
Chromosomal abnormalities such as deletions, inversions, or translocations are common genetic mechanisms to induce mutations that contribute to tumorigenesis. Similar fusion proteins between EWS or the related proteins TLS or TAFII68 with other transcription factor DNA binding domains have been observed in many other tumor types [7,8,9,10,11,12,13,14,15,16,17]. Plasmids—To generate t-EWS/LexA as bait for the yeast two-hybrid screen, a fragment corresponding to the tumor-associated portion of EWS (t-EWS, amino acids 1–264) was PCR-amplified from HL-60 cDNA and cloned into a modification of the yeast expression vector pSD.08 [38] to allow in-frame fusion with the DNA binding domain of LexA
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