Identification of Apurinic/apyrimidinic endonuclease 1 (APE1) as the endoribonuclease that cleaves c-myc mRNA.
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 (32)P-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.
Barnes
Tavish
T
Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, British Columbia V2N 4Z9, Canada.
Kim
Wan-Cheol
WC
Mantha
Anil K
AK
Kim
Sang-Eun
SE
Izumi
Tadahide
T
Mitra
Sankar
S
Lee
Chow H
CH
D000818:Animals; D000911:Antibodies, Monoclonal; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000201:enzymology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000145:classification
D000911:Antibodies, Monoclonal; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004722:Endoribonucleases; D012259:Ribonuclease, Pancreatic; C085108:APEX1 protein, human; C470319:Apex1 protein, rat; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase