The RNA-Binding and RNA-Melting Activities of the Multifunctional Protein Nucleobindin 1

Abstract

Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca²⁺-binding superfamily. NUCB1 interacts with Galpha_i3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGAUAU loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro.

Keywords: EF-hand motif; RNA-binding activity; RNA-melting; microRNA; nucleobindin 1.

Figure 1

Secondary structures of (a) E-box RNA transcript (sequence corresponding to the E-box is outlined); (b) SRP19 mRNA; (c) Picornovirus mRNA IRES element; (d) Human tRNA^Gly. The RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi, accessed on 22 June 2021) was used to predict structures of the RNA fragments in (a–c).

Figure 2

Chemical probing of the RNAs alone and in the presence of NUCB1. (a–c) Representative gels showing structure probing of E-box RNA (a), SRP19 mRNA (b), and miR-200a 3p RNA (c) using DMS, CMCT, and kethoxal (KET) in the presence and absence of NUCB1. Unmodified RNAs alone or in the complex with NUCB1 were used as controls. U, A, C, and G are sequencing lanes generated by reverse transcription in the presence of ddATP, ddTTP, ddGTP, and ddCTP, respectively. Nucleotides are numbered according to their position in an RNA transcript. The triangles on the right side of the gels indicate increased base reactivity upon NUCB1 addition, whereas the inverted triangles indicate the opposite. Secondary structure models of the used RNA fragments: E-box RNA (d), SRP19 mRNA (e), and miR-200a 3p RNA (f). Nucleotides are numbered as in (a–c). Blue lines indicate regions whose reactivity changed in the presence of NUCB1.

Figure 3

Schematic representation of NUCB1 and its truncated forms.

Figure 4

Molecular beacon melting assay. (a) Scheme of the MB-miR melting experiment. (b) Melting curves of MB-miR alone (grey curve) or after addition of proteins (colored curves). Error bars for each curve indicate the standard deviation.