Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D ...Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.展开更多
To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-m...To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound spe-cifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunc-tional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding pro-teins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA pro-moter.展开更多
基金supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 5R01AR037399the UCLA Vector Core (Emmanuelle Faure and Kip Hermann) for vector and viral preparations supported by JCCC/P30 CA016042 and CURE/P30 DK41301
文摘Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.
文摘To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound spe-cifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunc-tional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding pro-teins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA pro-moter.
