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.

Background-Synthetic, not natural, progestagen may negate the favorable effects of estrogen. Nonetheless, observational studies report no differences in risk for clinical cardiovascular events between users of unopposed estrogen and users of estrogen combined with synthetic progestin. Methods and Results-In a double-blind study, we randomly assigned 20 healthy postmenopausal women to micronized progesterone (MP) 200 mg or medroxyprogesterone acetate (MPA) 10 mg for 10 days with conjugated equine estrogen (CEE) 0.625 mg for 25 days and the remaining 5 days off cyclically during 2 months, followed by crossover to the alternate therapy. CEEϩMP and CEEϩMPA significantly improved the percent flow-mediated dilator response to hyperemia relative to baseline measurements (Pϭ0.004 by ANOVA) by a similar degree (Pϭ0.863).

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Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, H309S), which target amino acid residues comprising the abasic DNA endonuclease active site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic site single-stranded RNA (AP-ssRNA) cleavage activity, with concurrent loss of AP site cleavage activity on dsDNA and ssDNA. The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis, and not RNA binding. Interestingly, both endoribonuclease and ssRNA AP site cleavage activity of wild-type APE1 was present in the absence of Mg2+, while ssDNA AP site cleavage required Mg2+ (optimally at 0.5 to 2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA-cleavage by WT APE1[comment: I think I would leave this result out of the abstract), consistent with APE1 leaving a 3′-PO42− group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, AP-ssRNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.