Glisovic Tina, Ben-David Yaacov, Lang Matti A, Raffalli-Mathieu Françoise
Division of Pharmaceutical Biochemistry, Uppsala University, Uppsala Biomedical Centre, Box 578, SE-751 23 Uppsala, Sweden.
FEBS Lett. 2003 Jan 30;535(1-3):147-52. doi: 10.1016/s0014-5793(02)03893-0.
Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.
我们之前的证据表明，不均一核核糖核蛋白（hnRNP）A1通过与CYP2A5 mRNA的3'非翻译区（UTR）相互作用，参与Cyp2a5基因的调控。然而，这种相互作用的确切作用尚不清楚。本研究的目的是进一步深入了解Cyp2a5的调控过程。为此，将CYP2A5的3'UTR与荧光素酶mRNA的编码区融合。含有全长3'UTR或缺失先前描述的71个核苷酸（nt）区域（hnRNP A1主要结合位点）的3'UTR的荧光素酶重组体，在表达或缺乏hnRNP A1的细胞中瞬时表达。在mRNA和酶活性水平上检测荧光素酶重组体的表达。结果表明，携带CYP2A5全长3'UTR的重组体的高表达需要hnRNP A1的存在。hnRNP A1主要结合位点的缺失显著改变了表达模式：缺失突变体的mRNA水平和荧光素酶活性与hnRNP A1无关。这些结果确凿地证明，CYP2A5 mRNA 3'UTR中的71 nt区域可以赋予基因hnRNP A1依赖性调控。此外，RNA水平和荧光素酶活性的比较表明，hnRNP A1结合位点侧翼的区域可以调节CYP2A5 mRNA的翻译。这些结果与一个模型一致，即hnRNP A1与CYP2A5 mRNA 3'UTR中71 nt假定发夹环区域的结合可能通过保护mRNA不被降解而上调mRNA水平。
