Abstract
Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3′ untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3′ UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3′ UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3′ UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3′ UTR that is required for KRAS 3′ UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3′ UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3′ UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression.
Highlights
Post-transcriptional gene regulation by RNAbinding proteins (RBPs) and non-coding RNAs, such as microRNAs, is critical for normal eukaryotic development and physiology [1,2,3]
Of the many RBPs that bind to the KRAS 3′ untranslated region (UTR), AGO2 [22, 23], HuR [4, 22, 24], IGF2BP1/2/3 [23], PUM2 [23], EWSR1 [25], TAF15 [25], FUS [25], and TIA1 [26] have been previously implicated in cancer
The psiCHECK-2 dual-luciferase vector (pKRAS) expression increased following knock-down of HuR and Dicer individually in HeLa cells relative to its expression with si-Neg (Figure 1C). These findings indicate that HuR and potentially miRNAs regulate KRAS expression, at least partially through its 3′ UTR
Summary
Post-transcriptional gene regulation by RNAbinding proteins (RBPs) and non-coding RNAs, such as microRNAs (miRNAs), is critical for normal eukaryotic development and physiology [1,2,3]. The RNA-binding protein HuR (ELAVL1) can compete with a variety of miRNAs for sequence specific motifs in target mRNAs [4], or it can act cooperatively to recruit a let-7 miRNA to repress gene expression [5]. In another example, the PUM1 RBP can alter the secondary structure of its target RNA, thereby allowing miR-221 and miR-222 to access their complementary sites [6]. 3′ UTRs can mediate post-transcriptional gene regulation by acting as venues to coordinate interactions among various transregulatory factors and cis-acting 3′ UTR elements
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