Structurally distinct elements mediate internal ribosome entry within the 5′-noncoding region of a voltage-gated potassium channel mRNA
Structurally distinct elements mediate internal ribosome entry within the 5′-noncoding region of a voltage-gated potassium channel mRNA
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DOI：
10.1074/jbc.m405885200
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发表时间：
2004-11-12
影响因子：
4.8
通讯作者：
Semler, BL
中科院分区：
文献类型：
--
作者：
Jang, GM;Leong, LEC;Semler, BL
与编码小鼠Kv1.4（电压门控钾通道Shaker相关亚家族的成员）的mRNA种类的1.2-kb 5 '非编码区（5'-NCR）相似，已证明介导源自脑、心脏的细胞中的内部核糖体进入。骨骼肌，已知表达Kv1.4 mRNA种类的组织。我们还发现，Kv1.4 5 '-NCR的上游类似于1.0 kb和下游类似于0.2 kb独立地介导内部核糖体进入;然而，单独地，这些序列在介导内部核糖体进入方面不如在完整的（和连续的）5'-NCR中一起时有效。利用酶结构探测，预测在存在和不存在类似于1.0 kb的上游的情况下，3 '-最相似的0.2 kb形成三个不同的茎环结构（茎环X、Y和Z）和两个确定的单链区域（环Psi和Omega）。虽然系统性删除3 '端最相似的0.2kb序列导致表达的明显变化，但酶结构探测表明局部RNA折叠没有完全改变。结构探测分析强烈建议茎环X和下游的聚嘧啶道之间的相互作用，然而，相反的活动变化时，观察到这两个区域内的序列被独立删除。此外，与表达的正向和负向变化相关的缺失改变了茎环X内的RNA酶切割，表明该结构可能是一个不可或缺的元素。因此，这些发现表明Kv1.4表达是通过许多不同RNA区域之间的复杂相互作用介导的。
The similar to1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream similar to1.0 kb and the downstream similar to0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete ( and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most similar to0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream similar to1.0 kb. Although the systematic deletion of sequences within the 3'-most similar to0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.