Figure 2. Ars2 regulates expression of numerous mRNAs and associates with replication-dependent histone mRNAs.
(A) RNA used for the miRNA array in Figure 1b was reverse-transcribed and hybridized to Affymetrix GeneChip® Human Gene 1.0ST Arrays. The number of mRNAs decreased at least 2-fold (log2) following transfection of all three siRNAs targeting Ars2 or DGCR8 are depicted by Venn Diagram.
(B) The number of mRNAs increased at least 2-fold (log2) following transfection of all three siRNAs Ars2 or DGCR8 are depicted by Venn Diagram.
(C) To confirm microarray results, HeLa cells were transfected independently with control siRNAs (c1, c2, c3) or three siRNAs to Ars2 (a1, a2, a3) and the indicated mRNA transcript levels were measured by TaqMan®-based qPCR. Bars represent relative quantification using the ΔΔCt method normalized to c1 +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control.
(D) To determine if Ars2-containing protein complexes could associate with genes found to increase after Ars2 depletion, paraformaldehyde crosslinking followed by immunoprecipitation (PFA-CLIP) with a monoclonal antibody to Ars2 (2G10) or control antibody (SP/0) was performed. RNA isolated from the precipitates was reverse transcribed with random hexamer primers and used for qPCR with TaqMan® primer/probe sets to the indicated genes. Bars represent relative quantification using the ΔΔCt method normalized to control antibody CLIP +/- 95% confidence interval of three replicates. Non-specifically bound 18S rRNA was used as an endogenous control.