Structurally Distinct Elements Mediate Internal Ribosome Entry within the 5′-Noncoding Region of a Voltage-gated Potassium Channel mRNA*

The ∼1.2-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 ∼1.0 kb and the downstream ∼0.2 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 ∼0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Ψ and Ω) in the presence and absence of the upstream ∼1.0 kb. Although the systematic deletion of sequences within the 3′-most ∼0.2 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.