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.

The c-myc mRNA coding region determinant-binding protein (CRD-BP) has high affinity for the coding region determinant (CRD) of c-myc mRNA. Such affinity is believed to protect c-myc CRD from endonucleolytic attack. We have recently purified a mammalian endoribonuclease which can cleave within the c-myc CRD in vitro. The availability of this purified endonuclease has made it possible to directly test the interaction between CRD-BP and the endonuclease in regulating c-myc CRD RNA cleavage. In this study, we have identified the coding region of MDR-1 RNA as a new target for CRD-BP. CRD-BP has the same affinity for c-myc CRD nts 1705–1886 and MDR-1 RNA nts 746–962 with Kd of 500 nM. The concentration-dependent affinity of CRD-BP to these transcripts correlated with the concentration-dependent blocking of endonuclease-mediated cleavage by CRD-BP. In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage. Finally, we have identified RNA sequences required for CRD-BP binding. These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.

3Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is known to be involved in base excision DNA repair, acting as the major abasic endonuclease; the protein also functions as a redox coactivator of several transcription factors that regulate gene expression. Recent findings highlight a novel role for APE1 in RNA metabolism. The new findings are as follows: (i) APE1 interacts with rRNA and ribosome processing protein NPM1 within the nucleolus; (ii) APE1 interacts with proteins involved in ribosome assembly (i.e., RLA0, RSSA) and RNA maturation (i.e., PRP19, MEP50) within the cytoplasm; (iii) APE1 cleaves abasic RNA; and (iv) APE1 cleaves a specific coding region of c-myc mRNA in vitro and influences c-myc mRNA level and half-life in cells. Such findings on the role of APE1 in the posttranscriptional control of gene expression could explain its ability to influence diverse biological processes and its relocalization to cytoplasmic compartments in some tissues and tumors. In addition, we propose that APE1 serves as a "cleansing" factor for oxidatively damaged abasic RNA, establishing a novel connection between DNA and RNA surveillance mechanisms. In this review, we introduce questions and speculations concerning the role of APE1 in RNA metabolism and discuss the implications of these findings in a broader evolutionary context.There is an emerging body of evidence that links DNA repair proteins to specific aspects of RNA metabolism. Currently, little is known about how cells cope with damaged RNA, either modified or oxidized, but it is clear that such RNA can impair protein synthesis, thus affecting cell function and viability. Specific surveillance mechanisms are therefore needed to remove damaged molecules from the RNA pool to guarantee the biological integrity of cells.The idea that quality control mechanisms might exist to repair RNA was brought to the forefront with the identification of the biochemical activities of the mammalian AlkB homologs. In particular, it was discovered that AlkB (from Escherichia coli) and the human homolog hABH3, besides being able to directly reverse alkylation damage on DNA bases, are able to demethylate damaged bases on RNA, thus playing a key role in the repair of specific RNA lesions (1, 30). While repair mechanisms have been demonstrated for alkylated RNA, such activity is not yet known for oxidatively damaged RNA. However, their existence seems improbable, in the absence of direct reversal strategies, due to the lack of a template for accurate repair, as in the case of double-stranded DNA. Since, under oxidative stress conditions, oxidation of RNA can occur up to a 10-to 20-fold-higher extent than oxidation of DNA (24), the question is: how is oxidized RNA specifically removed or repaired?The recent findings of Berquist et al. (7), Barnes et al. (4), and Vascotto et al. (39) highlight a novel "moonlighting" role for the repair apurinic/apyrimidinic (AP) endonuclease 1 (APE1) in RNA metabolism, both as a possible "cleansing" factor for damaged abasic ...

The multidrug resistance (MDR) phenotype exhibited by cancer cells is believed to be the major barriers to successful chemotherapy in cancer patients. The major form of MDR phenotype is contributed by a group of ATP-binding cassette (ABC) drug transporters which include P-glycoprotein, multidrug resistance-associated protein 1, and breast cancer resistance protein. There has been intense search for compounds which can act to reverse MDR phenotype in cultured cells, in animal models, and ultimately in patients. The ongoing search for MDR modulators, compounds that act directly on the ABC transporter proteins to block their activity, has led to three generations of drugs. Some of the third-generation MDR modulators have demonstrated encouraging results compared to earlier generation MDR modulators in clinical trials. These modulators are less toxic and they do not affect the pharmacokinetics of anti-cancer drugs. Significant numbers of natural products have also been identified for their effectiveness in reversing MDR in a manner similar to the MDR modulators. Other MDR reversing strategies that have been studied quite extensively are also reviewed and discussed in this chapter. These include strategies aimed at destroying mRNAs for ABC drug transporters, approaches in inhibiting transcription of ABC transporter genes, and blocking of ABC transporter activity using antibodies. This review summarizes the development of reversing agents for ABC drug transporters up to the end of 2008, and provides an optimistic view of what we have achieved and where we could go from here.