Figure 7

Loss of TROVE2 and/or SSB via small RNA interference increases poly(A) + Y1 RNA, rRNAs, and U1 RNA, misprocessed 18S and 28S rRNA, and alters mRNA length. (A) Selective siRNA-mediated knockdown of TROVE2 or SSB in THP-1 cells or Jurkat T cells. Transcript levels of TROVE2 and SSB were determined by quantitative PCR using oligo d(T) for cDNA synthesis. Results are normalized to cells transfected with a non-specific scrambled siRNA negative control after normalization to transcript levels of GAPDH. (B) Poly(A) + Y1 RNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis. Results normalized to CTRL (transfection of scrambled siRNA). (C) As in (B) except levels of 18S and 28S rRNAs were determined. (D) As in (B) except levels of poly(A) + U1 snRNA were determined. (E, F) Transcript levels of misprocessed rRNAs in THP-1 (E) and Jurkat T cells (F) (-90, -60, -30 bp) were determined and are expressed relative to cells transfected with a scrambled siRNA as described in Figure 2. (G) PCR strategy to test altered lengths of MBP, CSF1R, and NFATC1 transcripts. (H) As in (A) except transcript levels of the MBP intron, CSF1R exons, and NFATC1 short to long mRNA isoform ratios were determined by quantitative PCR and normalized to CTRL. Each experiment was performed a minimum of three times, results are expressed as mean ± S.D *P <0.05.