Purification and Characterization of a Polysome-associated Endoribonuclease That Degrades c-myc mRNA in Vitro

Abstract

The regulation of mRNA half-lives is determined by multiple factors, including the activity of the messenger RNases (mRNases) responsible for destroying mRNA molecules. Previously, we used cell-free mRNA decay assays to identify a polysome-associated endonuclease that cleaves c-myc mRNA within the coding region. A similar activity has been solubilized and partially purified from a high salt extract of adult rat liver polysomes. Based on a correlation between protein and enzyme activity, the endonuclease is tentatively identified as a ϳ39-kDa protein. It cleaves the coding region stability determinant of c-myc mRNA with considerable specificity. Cleavages occur predominantly in an A-rich segment of the RNA. The endonuclease is resistant to RNase A inhibitors, sensitive to vanadyl ribonucleoside complex, and dependent on magnesium. In these and other respects, the soluble enzyme we have purified resembles the polysome-associated c-myc mRNase.

Targeted Knockdown of the RNA-binding Protein CRD-BP Promotes Cell Proliferation via an Insulin-like Growth Factor II-dependent Pathway in Human K562 Leukemia Cells

“…CRD-BP is thought to be a c-myc mRNA-binding protein that stabilizes c-myc mRNA, based upon results with a cell-free mRNA decay system (13,37). However, our estimates of molecule numbers revealed ϳ400,000 CRD-BP protein molecules and ϳ50 c-myc mRNA molecules per K562 cytoplasm.…”

“…Therefore, CRD-BP may regulate cell proliferation in a cell typespecific manner. CRD-BP binds to c-myc, IGF-II, ␤-actin mRNAs, and to H19 RNA, and it affects RNA localization, turnover, and translation (13,19,37,38,46,47). Our results show that among those targets, neither c-myc, ␤-actin, nor H19 RNA appears to be responsible for the proliferative effect of CRD-BP knockdown.…”

A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: A novel mechanism of oncogene de-regulation

“…Thus suggesting that control of translation is important in regulating cellular levels of c-myc in addition to the other mechanisms that have been described. These include alterations in rates of transcription of the mRNA, and the stability of both the mRNA and protein (Ross, 1995;Lee et al, 1998;LuÈ scher and Eisenmann, 1988;Shindo et al, 1993). The number of levels that c-myc expression is controlled at is probably a re¯ection of the diverse cellular processes in which this proto-oncogene is thought to function e.g.…”

Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3′-untranslated region by the mRNA endonuclease polysomal ribonuclease 1

“…F or many mRNAs, cytoplasmic stability is determined by endonuclease cleavage within the body of the message (1)(2)(3)(4)(5)(6). Endonuclease-catalyzed decay is likely to be regulated by RNAbinding proteins interacting with cis-acting mRNA stability elements to block cleavage by site-selective mRNA endonucleases (3,(7)(8)(9).…”