Xenopus laevis RNA-binding protein A is a ubiquitously expressed, double-stranded RNA-binding protein that is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs. X. laevis RNA-binding protein A contains three copies of the double-stranded RNA-binding domain (dsRBD) in tandem arrangement. Two of them, xl1 and xl2, belong to the type A group of dsRBDs that show strong homologies to the entire length of a defined consensus sequence. The xl3 domain, in contrast, is a type B dsRBD which only matches the basic C-terminal end of the dsRBD consensus sequence. Here we show that only xl2 but neither xl1 nor xl3 are able to bind double-stranded RNA substrates in vitro, suggesting that different dsRBD copies have varying RNA binding activities. By fine mapping mutagenesis of the isolated xl2 domain, we identified at least two central aromatic amino acids and a C-terminal alpha-helix that are indispensable for dsRNA binding. Furthermore, we show that different charge distributions within the C-terminal alpha-helices of xl1 and xl2 seem responsible for the different RNA binding behaviors of these two dsRBDs. Analyses of the RNA binding properties of constructs containing various combinations of different dsRBDs reveal that type A dsRBDs exhibit a cooperative binding effect, whereas type B dsRBDs show a rather low binding activity, thus contributing only to a minor extent to a stable RNA-protein interaction.

To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase-coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 10(5) molecules/microg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 10(5) and 1.8 x 10(5) molecules/microg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 10(4) molecules/microg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., beta-naphthoflavone (beta-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.