Two Different RNA Binding Activities for the AU-Rich Element and the Poly(A) Sequence of the Mouse Neuronal Protein mHuC

Abstract

HuC is one of the RNA binding proteins which are suggested to play important roles in neuronal differentiation and maintenance. We cloned and sequenced cDNAs encoding a mouse protein which is homologous to human HuC (hHuC). The longest cDNA encodes a 367 amino acid protein with three RNA recognition motifs (RRMs) and displays 96% identity to hHuC. Northern blot analysis showed that two different mRNAs, of 5.3 and 4.3 kb, for mouse HuC (mHuC) are expressed specifically in brain tissue. Comparison of cDNA sequences with the corresponding genomic sequence revealed that alternative 3' splice site selection generates two closely related mHuC isoforms. Iterative in vitro RNA selection and binding analyses showed that both HuC isoforms can bind with almost identical specificity to sequences similar to the AU-rich element (ARE), which is involved in the regulation of mRNA stability. Functional domain mapping using mHuC deletion mutants showed that the first RRM binds to ARE, that the second RRM has no RNA binding activity by itself, but facilitates ARE binding by the first RRM and that the third RRM has specific binding activity for the poly(A) sequence.

Xenopus Cold-inducible RNA-binding Protein 2 Interacts with ElrA, the Xenopus Homolog of HuR, and Inhibits Deadenylation of Specific mRNAs

“…Previous studies showed that RRM1 and RRM2 are sufficient for binding to the ARE (35,36), whereas RRM3 is capable of binding to poly(A) in a filter binding assay (37). By deletion mutant analysis, it has been shown that RRM3 of HuR is necessary for stabilization (21) and that the hinge region contains the nucleocytoplasmic shuttling signal (38).…”

Characterization of the Interaction between Neuronal RNA-binding Protein HuD and AU-rich RNA

“…In vitro selection had previously been carried out for HuB and HuC (19,26,56). Three rounds of binding selection with HuB, using an RNA carrying a randomized region of 25 nucleotides, had yielded predominantly RNAs that contained U 3-5 -tracts (19) and binding selection using a 3ЈUTR library had selected RNAs that contained the sequence Pu(U/A)U 3 AU 3 (U/A)Pu (where Pu indicates a purine) (56).…”

“…Three rounds of binding selection with HuB, using an RNA carrying a randomized region of 25 nucleotides, had yielded predominantly RNAs that contained U 3-5 -tracts (19) and binding selection using a 3ЈUTR library had selected RNAs that contained the sequence Pu(U/A)U 3 AU 3 (U/A)Pu (where Pu indicates a purine) (56). A similar iterative selection of HuC-binding RNAs indicated that HuC binds to RNAs carrying the sequence Pu(U) 2-5 (Pu) 1-2 (U) [2][3][4][5] Pu (26).…”

Cytoplasmic localization is required for the mammalian ELAV‐like protein HuD to induce neuronal differentiation

“…3). Previous in vitro studies on Hu proteins have shown that RBDs 1 and 3 are required for binding to AREs and the poly(A) tail, respectively (Abe et al 1996b;Chung et al 1996;Ma et al 1997). Therefore, the binding of HuD to some speci®c ARE-containing mRNAs and also possibly the export of the mRNAs to the cytoplasm by shuttling may be involved in the induction of neurite outgrowth.…”