Aim: To estimate HuR protective activity against proteasome-associated ribonuclease for c-myc and c-fos mRNAs.
Introduction: Proteasome-associated proteins are attractive targets for multiple myeloma treatment. One of them is HuR protein known to selectively bind ARE-containing mRNAs and protect them from degradation. HuR is supposed to play a role in cancerogenesis since its expression is elevated in many cancer types and it stabilizes a lot of mRNAs encoding proteins involved in oncogenesis. Previously, it was shown that proteasome in addition to its main function – protein degradation – may act as a selective RNase. Moreover, HuR and proteasome have common targets – c-myc and c-fos protooncogene mRNAs.
Methods: HuR-GST fusion protein has been cloned, expressed and purified by affinity chromatography. Fragments of c-myc and c-fos were cloned and mRNAs have been transcribed in vitro. Proteasomes have been isolated from К562 cell line (human proerytroleykemia) and Im-9 cells (human multiple myeloma). mRNAs were treated by proteasomes in presence and absence of HuR. The estimation of mRNA cleavage was held by gel-electrophoresis.
Results: GST-HuR has specifically bound ARE-containing fragments of c-myc and c-fos mRNAs. Proteasomes extracted from Im-9 and K562 cells cleaved target mRNAs in absence of HuR. It was shown that HuR prevents degradation of c-fos mRNA by proteasomal endoribonuclease, whereas c-myc mRNA was cleaved in the same conditions. GST protein didn’t bind with target mRNAs and didn’t affect proteasome cleavage activity.
Conclusion: HuR protects c-fos mRNA from proteasome ribonuclease cleavage in vitro, but can’t prevent c-myc mRNA degradation. HuR and proteasome compete with each other for manifestation of their opposite activities. Thus, a new mechanism of regulation of proto-oncogenes expression was observed. However, the functional role of this process in vivo should be evaluated in further studies.1–4