Background: Coding region determinant-binding protein (CRD-BP) interacts physically with oncogenic mRNAs. Results: Point mutation in the K-homology (KH) domains of CRD-BP abolishes its RNA-binding ability. Conclusion: Two KH domains of CRD-BP are required for efficient binding to oncogenic mRNAs and for granule formation in zebrafish embryos. Significance: Learning how the KH domains interact with mRNAs is crucial for understanding oncogenic function of CRD-BP.

Edited by Xiao-Fan WangMicroRNAs are essential in many cellular processes. The ability to detect microRNAs is important for understanding its function and biogenesis. This study is aimed at using a molecular beacon to detect miR-430 in developing zebrafish embryos as a proof of principle. miR-430 is crucial for the clearance of maternal mRNA during maternal zygotic transition in embryonic development. Despite its known function, the temporal and spatial expression of miR-430 remains unclear. We used various imaging techniques, including laser scanning confocal microscopy, spinning disk, and lightsheet microscopy, to study the localization of miR-430 and any developmental defects possibly caused by the molecular beacon. Our results show that miR-430 is expressed early in development and is localized in distinct cytoplasmic granules where its target mRNA can be detected. We also show that the designed molecular beacon can inhibit the function of miR-430 and cause developmental defect in the brain, notochord, heart, and kidney, depending on the delivery site within the embryo, suggesting that miR-430 plays a diverse role in embryonic morphogenesis. When compared with morpholino, molecular beacon is 2 orders of magnitude more potent in inhibiting miR-430. Thus, our results reveal that in addition to being used as a valuable tool for the detection of microRNAs in vivo, molecular beacons can also be employed to inhibit microRNAs in a specific manner. MicroRNAs (miRNAs)2 are a class of short, non-coding RNAs involved in the regulation of gene expression at the posttranscriptional level. MiRNAs are implicated in many diseases and are essential in many cellular processes including development (1). Understanding the spatial expression of these small RNAs inside the cell can provide important information about their molecular function. The use of in situ hybridization to study intracellular localization of miRNAs in fixed cells has technical limitations because of the small size and low quantities of the RNA (2). In addition, an unusual high background, possibly contributed by nonspecific binding to DNA, can make interpretation of results difficult (3). Live imaging of miRNAs has been attempted in many studies, but success remains questionable, mainly because of challenges in the delivery of miRNA sensors to the appropriate compartment inside the cell. Most sensors were introduced into cells using a lipid-based delivery system, which may bias the delivery of the sensors to the endocytic pathway of the cell destined for degradation. In fact, the pattern of miRNA expression in punctate structures shown by these studies differs substantially from in situ hybridization experiments (3-10). Among the many types of sensors used in tracking miRNAs, molecular beacons hold the greatest promise because of their high specificity toward small sequences of RNA and low signal to noise ratio (11,12). Furthermore, molecular beacons are able to provide real time signals without relying on other indirect and complicated detection systems suc...

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