Abstract
α-complex protein 2 (α-CP2) is known as an RNA-binding protein that interacts in a sequence-specific manner with single-stranded polycytosine [poly(C)]. This protein is involved in various post-transcriptional regulations, such as mRNA stabilization and translational regulation. In this study, the full-length mouse α-CP2 gene was expressed in an insoluble form with an N-terminal histidine tag in Escherichia coli and purified for homogeneity using affinity column chromatography. Its identity was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Recombinant α-CP2 was expressed and refolded. The protein folding conditions for denatured α-CP2 were optimized. DNA and RNA electrophoretic mobility shift assays demonstrated that the recombinant α-CP2 is capable of binding to both single-stranded DNA and RNA poly(C) sequences. Furthermore, plasmids expressing α-CP2 activated the expression of a luciferase reporter when co-transfected with a single-stranded (pGL-SS) construct containing a poly(C) sequence. To our knowledge, this study demonstrates for the first time that α-CP2 functions as a transcriptional activator by binding to a single-stranded poly(C) sequence.
Highlights
The polycytosine [poly(C)]-binding proteins (PCBPs) are characterized by heterogeneous nuclear ribonucleoproteinKey words: α-complex protein 2, post‐transcriptional regulation, transcriptional regulation, electrophoretic mobility shift assays, poly(C) sequence, transcriptional activatorK homology (KH) domains and high affinity sequence-specific interactions with polycytosine [poly(C)] nucleic acid sequences
The common feature of all PCBPs is the presence of three heterogeneous nuclear ribonucleoprotein (hnRNP) KH domains [6]
The KH domain of PCBPs consists of three α-helices and β-strands arranged as follows: β1-α1-α2-β2-β3-α3 [7]
Summary
The polycytosine [poly(C)]-binding proteins (PCBPs) are characterized by heterogeneous nuclear ribonucleoprotein (hnRNP)K homology (KH) domains and high affinity sequence-specific interactions with polycytosine [poly(C)] nucleic acid sequences. Five evolutionarily related PCBPs have been identified: PCBP1-4 and hnRNP K [1]. These PCBPs belong to one of two subgroups: hnRNP K or the α-complex proteins (α-CPs or PCBP1-4) [2]. The common feature of all PCBPs is the presence of three hnRNP KH domains [6]. These are RNA-binding modules that are approximately 70 amino acids in length. The Gly-X-X-Gly loop is located between α1 and α2, and the variable loop is located between β2 and β3 These KH domain sequences are conserved in PCBPs [7,8]. PCBP3 is more divergent, and PCBP4 is the most distantly related (52% divergence from α-CP2) [5,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
