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

Chow Hwee Lee,Peter Leeds,Jeffrey Ross

doi:10.1074/jbc.273.39.25261

Chow Hwee Lee, Peter Leeds + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.39.25261

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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 approximately 39-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.

Highlights

This paper focuses on a ribosome-associated protein with the properties of a c-myc mRNA-degrading ribonuclease
Identification and Purification of a Polysomal, c-myc mRNA-degrading Endonuclease—The existence of a polysomal c-myc mRNA-degrading endonuclease was revealed in previous studies using a cell-free mRNA decay assay [9]
Two additional findings strengthened the connections among an endonuclease, the CRD of c-myc mRNA, and c-myc mRNA stability. (i) The CRD is a major determinant of c-myc mRNA expression and stability in vivo (7, 8, 10 –12). (ii) c-myc mRNA is cleaved endonucleolytically in at least some cells [16, 17]

Summary

EXPERIMENTAL PROCEDURES

Preparation of Radiolabeled Transcripts—The subcloning of the cmyc DNA fragments corresponding to all or part of the CRD of c-myc mRNA has been described [9, 13]. Three hundred mg of RSW protein were loaded at a flow rate of 1 ml/min onto a 2.5 ϫ 12 cm phosphocellulose column (Sigma) equilibrated with 0.05 M KCl, 2 mM DTT, 10% (v/v) glycerol, 25 mM potassium phosphate, pH 6.0. After washing the column until the A280 returned to base line, proteins were eluted with a linear gradient from 0.1 to 1.0 M KCl in Buffer B. The sample was loaded at a flow rate of 1.2 ml/min onto a pre-packed 5 ml heparin-Sepharose column (Amersham Pharmacia Biotech) equilibrated with 0.1 M KCl, 2 mM DTT, 10% (v/v) glycerol, 25 mM potassium phosphate, pH 7.4. The column was washed until the A280 returned to base line, and bound proteins were eluted with a linear gradient from 0.1 to 1.0 M KCl in the same buffer. A separate 10 –30% glycerol gradient was centrifuged in the same manner and contained the following proteins as molecular mass standards: ␤-galactosidase (116.4 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and lysozyme (14.3 kDa)

Ribosomal salt wash

RESULTS

DISCUSSION