The third RNA recognition motif of Drosophila ELAV protein has a role in multimerization

Abstract

ELAV is a neuron-specific RNA-binding protein in Drosophila that is required for development and maintenance of neurons. ELAV regulates alternative splicing of Neuroglian and erect wing (ewg) transcripts, and has been shown to form a multimeric complex on the last ewg intron. The protein has three RNA recognition motifs (RRM1, 2 and 3) with a hinge region between RRM2 and 3. In this study, we used the yeast two-hybrid system to determine the multimerization domain of ELAV. Using deletion constructs, we mapped an interaction activity to a region containing most of RRM3. We found three conserved short sequences in RRM3 that were essential for the interaction, and also sufficient to give the interaction activity to RRM2 when introduced into it. In our in vivo functional assay, a mutation in one of the three sequences showed reduced activity in splicing regulation, underlining the functional importance of multimerization. However, RRM2 with the three RRM3 interaction sequences did not function as RRM3 in vivo, which suggested that multimerization is not the only function of RRM3. Our results are consistent with a model in which RRM3 serves as a bi-functional domain that interacts with both RNA and protein.

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

“…56 Multimerization of HuR protein is then dependent on RRM3 motif and more specifically on its well-conserved W261. 51 In fact, the whole W261-T271 stretch is highly conserved among Hu proteins as it was described in earlier studies. 25 Our structural studies on HuR RRM3 also demonstrate that the monomer/ dimer exchange of HuR RRM3 takes place even in absence of RNA, in contrast to previous reports suggesting that RNA promotes HuR FL multimerization.…”

“…Thus, we propose that RRM3 dimerization involves its a 1 -helix and the loop a 1-b 2 , both placed at the opposite side of the RNA-binding platform, as previously proposed for homologous proteins such as ELAV in Drosophila upon mutating W419 (W261 in HuR). 51 This suggestion is supported by MD computations explaining the different stability of 2 possible dimeric structures and the effects of the W261E mutation to prevent dimerization. There are other instances of RRM-RRM interactions taking place through their a-helices.…”

“…Notably, the oligomerization of ELAVa homolog to HuR in Drosophila -requires its RRM3 domain. 51 Moreover, W419 in ELAV (the residue corresponding to W261 in HuR) seems to be essential for ELAV oligomerization. Thus, we hypothesized that the W261-T271 stretch forms a dimerization epitope that involves the C-terminal end of helix a 1 .…”

“…50 Besides its role in RNA recognition, RRM3 is also responsible for the formation of HuR multimers, 34 as occurs with the homologous Drosophila ELAV protein. 51 In fact, mutations in the RRM3 domain of Drosophila ELAV protein lead to a temperature-sensitive phenotype by impairing HuR oligomerization, highlighting the crucial role of this RRM module. 52 Given the multifunctional relevance of the HuR RRM3 domain in RNA recognition and modification as well as in HuR oligomerization, we set out to investigate the molecular structure of this motif, which has been hampered by its low solubility when recombinantly produced in large quantities for structural studies.…”

Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1

“…S1) thus potentially capable of accommodating dozens of HuR molecules on a single Circ-PABPN1 molecule. If one then considers that each HuR site on CircPABPN1 can allow binding of HuR multimers 32,33 and that a fraction of cellular HuR may be phosphorylated at residues that inhibit HuR binding to RNA, CircPABPN1 might be able to associate with a sizeable pool of cytoplasmic HuR.…”