RNAi Studies and Tandem Affinity Purification of Trypanosoma brucei Essential Nuclear Factor, a Novel and Essential Trypanosome Protein

Trypanosoma brucei, the single-celled eukaryotic parasite responsible for causing African sleeping sickness, has many unusual biochemical features and processes, including RNA polymerase II (RNAPII)-dependent gene transcription. We have identified a nuclear, trypanosome-specific protein that is associated with the RNAPII transcriptional machinery, and is essential for parasite growth as assessed by RNA interference. This protein, named essential nuclear factor (ENF), contains motifs associated with known transcription factors, but the protein is unique to trypanosomes. The function of this protein is unknown.;In order to look at transcript levels when TbENF is depleted RNA interference cell lines were established, and total RNA was isolated from cells with TbENF and depleted TbENF. Next generation sequencing was used to evaluate the transcript levels in RNAi induced and non-induced cells.. RNAi cell lines were successfully established and TbENF was depleted in RNAi-induced cell cultures. To identify the TbENF-interacting proteins, N and C terminus Tandem Affinity Purification (TAP) tagged TbENF cell lines were generated using an HA-FLAG tag system. The HA-FLAG tagged proteins have been expressed in the parasites, the HA-FLAG appears accessible, and purification conditions have been preliminarily determined for the tagged protein from parasite whole cell extracts.