Endonucleolytic cleavage of the coding region determinant (CRD) of c-myc mRNA appears to play a critical role in regulating c-myc mRNA turnover. Using 32P-labeled c-myc CRD RNA as substrate, we have purified and identified two endoribonucleases from rat liver polysomes that are capable of cleaving the transcript in vitro. A 17-kDa enzyme was identified as RNase1. Apurinic/apyrimidinic (AP) DNA endonuclease 1 (APE1) was identified as the 35-kDa endoribonuclease that preferentially cleaves in between UA and CA dinucleotides of c-myc CRD RNA. APE1 was further confirmed to be the 35-kDa endoribonuclease because: (i) the endoribonuclease activity of the purified 35-kDa native enzyme was specifically immuno-depleted with APE1 monoclonal antibody, and (ii) recombinant human APE1 generated identical RNA cleavage patterns as the native liver enzyme. Studies using E96A and H309N mutants of APE1 suggest that the endoribonuclease activity for c-myc CRD RNA shares the same active center with the AP-DNA endonuclease activity. Transient knockdown of APE1 in HeLa cells led to increased steady-state level of c-myc mRNA and its half-life. We conclude that the ability to cleave RNA dinucleotides is a previously unidentified function of APE1 and it can regulate c-myc mRNA level possibly via its endoribonuclease activity.
|Number of pages||13|
|Journal||Nucleic Acids Research|
|State||Published - 2009|
Bibliographical noteFunding Information:
Natural Sciences & Engineering Research Council (227158); the Canadian Cancer Society [to C.H.L, who is a Research Scientist of the National Cancer Institute of Canada (13546)]; T.B was a recipient of UNBC Graduate Entrance and NSERC Canada Graduate Scholarships; W-C.K was a recipient of Pacific Century, Michael Smith Foundation of Health Research Junior Graduate, and NSERC Canada Graduate Scholarships; S-E.K was a recipient of NSERC Undergraduate Summer Research Award; the United States Public Health Service grants (R01 ES08457, R01 CA53791, and P30 ES06676 to S.M. and A.K.M.); and National Institutes of Health/National Cancer Institute grant (CA98664 to T.I.). Funding for open access charge: Natural Sciences & Engineering Research Council Discovery Grant 227158.
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