Figure 6.

SRSF1 binds to the 7SK RNA and activates transcription of the viral promoter. (A) SRSF1 transcription activation is dependent on TAR. HEK293 cells were transfected with the reporter constructs pLTR-Xm-LR and pLTR-XTm-LR, which carries a deletion of the TAR sequence, the control pEGFP, the SRSF1 expression constructs, control or SRSF1-specific siRNAs I the absence of Tat. Luciferase activity and mRNA expression were assayed 48 h after transfection. (B) SRSF1 and Tat bind overlapping sequences within the 5′ hairpin of the 7SK RNA. RAC assays were set up with bait RNAs containing the wild-type (5hpWT) and mutated (5hpM) sequences of the 5′ hairpin of the 7SK RNA. The RNA substrates were then incubated with 100 ng of recombinant Tat or purified SRSF1 in separate reactions. (C) HEK293 cells were transfected with the viral clone pMtat(–), a control plasmid, the SRSF1 expression clone or SRSF1 siRNA. ChIP assays were performed 48 h after transfection, with antibodies for the indicated proteins and the GFP-tagged Tat. Values represent the relative enrichment in ChIP signal to the TAR and ORF relative to the control IgG, and are the average of three independent PCRs from two independent ChIP experiments.