Figure 1.

Discovery of HuR as a trans-acting post-transcriptional regulatory factor for Msi1. (A) A schematic of the MSI1 mRNA with the 3′ untranslated region shown in the inset. The nucleotide tracts bolded, italicized, and underlined indicates the putative HuR binding sites. (B) Immunoprecipitation of RNA:HuR protein complexes identifies Musashi1 mRNA as a binding partner for HuR. HuR ribonucleoprotein complexes were pulled down with an anti-HuR specific antibody. The RNA recovered was reverse transcribed and qPCR was performed on the complementary DNA. Primers were used for GAPDH, PTMA, and MSI1 mRNAs. GAPDH mRNA is used as a negative control, and PTMA mRNA is a positive control. (C) Diagram of the mRNA fragments used in biotin pulldown experiment. Three fragments were made, one for the CR and two for each half of the 3′ UTR. (D) Biotin pulldown assay confirms HuR interaction with MSI1 mRNA. Biotinylated fragments of the MSI1 mRNA were in vitro transcribed. Three fragments of the mRNA were analyzed, one for the coding region and two for each half of the 3′ untranslated region. The GAPDH 3′ UTR was included as a negative control. After incubation of the fragments with streptavidin beads and biotinylated RNA, the protein was recovered by pulldown analysis and immunoblotted for HuR. A beads only control was included to account for any nonspecific interaction and a ‘lysate’ was included as a positive control for the immunoblot. (E) Influence of HuR on Msi1 protein expression is dependent on the 3′ untranslated region. The Msi1 3′ untranslated region was cloned downstream of a PEST-destablized luciferase gene (luc2P). The luciferase construct was cotransfected with an TAP-tagged HuR expression vector (denoted as TAP-HuR) in HeLa cervical adenocarcinoma cells. An empty vector (denoted as TAP) was utilized as a negative control. Data were analyzed with Student’s t-test and is presented as the mean ± standard error of the mean. Experiment was performed in triplicate.