Abstract
Transcription of genes coding for the small nuclear RNAs (snRNAs) is dependent upon a unique transcription factor known as the small nuclear RNA-activating protein complex (SNAPc). SNAPc binds to an essential proximal sequence element located about 40-65 base pairs upstream of the snRNA transcription start site. In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct polypeptides (DmSNAP190, DmSNAP50, and DmSNAP43) that are stably associated with each other and bind to the DNA as a complex. We have used mutational analysis to identify domains within each subunit that are involved in complex formation with the other two subunits in vivo. We have also identified domains in each subunit required for sequence-specific DNA binding. With one exception, domains required for subunit-subunit interactions lie in the most evolutionarily conserved regions of the proteins. However, DNA binding by DmSNAPc is dependent not only upon the conserved regions but is also highly dependent upon domains outside the conserved regions. Comparison with functional domains identified in human SNAPc indicates many parallels but also reveals significant differences in this ancient yet rapidly evolving system.
Highlights
In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct polypeptide chains that are orthologous to HsSNAP190, HsSNAP50, and HsSNAP43 [13, 14]
Because it seemed likely that deletion of this most conserved region of DmSNAP190 would be highly detrimental to DmSNAPc activity, we focused our studies on the non-conserved regions
D, same as C except FLAG affinity purification was carried out under high-salt conditions (0.35 M NaCl). These results indicated that the C-terminal domain of DmSNAP190 is essential for DmSNAPc to bind efficiently to the PSEA, even though it is not required for assembly with either DmSNAP43 or DmSNAP50 (Fig. 2, C and D)
Summary
In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct polypeptide chains that are orthologous to HsSNAP190, HsSNAP50, and HsSNAP43 [13, 14]. A homologous complex (tSNAPc) is required for transcription of the spliced leader snRNA in trypanosomes (16 –18) This indicates that a SNAP-like complex arose very early in eukaryotic evolution and continues to be essential for snRNA transcription in widely divergent contemporary eukaryotes. We report a mutational analysis of the three subunits of DmSNAPc from the fruit fly D. melanogaster to identify functional domains within each of the subunits. We have identified domains within each DmSNAP polypeptide that are essential for sequence-specific binding to the PSEA (the fly equivalent of the PSE) but are dispensable for assembly with the other two subunits. Our mutational analysis of DmSNAPc expands on the knowledge obtained in the human system and in some cases reveals surprising differences in the localization of functional domains for complex assembly and DNA binding of the different metazoan SNAP complexes
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