Identification of Apurinic/apyrimidinic endonuclease 1 (APE1) as the endoribonuclease that cleaves c-myc mRNA

Abstract

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.

Figure 1.

Purification and identification of proteins that co-purified with endoribonuclease activity. (A) Autoradiograph of an endoribonuclease assay in gel filtration fractions (elution volume 20–76) using 350 fmol 5′ ³²P-labeled CRD c-myc RNA as substrate. The endoribonuclease assay was performed for 5 min at 37°C under the standard condition as described in the ‘Materials and Methods’ section. Filled arrow indicates undigested substrate RNA and unfilled arrow indicates endonucleolytic decay products. (B) Silver-stained SDS–PAGE gel of pooled samples from gel filtration in (A). (C) Coomasie blue-stained SDS–PAGE gel of pooled samples from three separate gel filtration columns. Arrows indicate protein bands that were excised and subjected to LC-MS/MS protein identification analysis.

Figure 2.

Mapping the cleavage sites generated by the purified 17-kDa and 35-kDa native endoribonucleases. (A) Three hundred and fifty femtomole 5′ -labeled c-myc CRD RNA were subjected to post-heparin-Sepharose purified enzyme (lanes 3 and 9), 30–40-kDa fraction purified enzyme (lanes 4–6 and 12 and 13) and 10–20-kDa fraction purified enzyme (lanes 10 and 11) from gel filtration column under the standard endoribonuclease assay. The radiolabeled RNA was also subjected to alkaline hydrolysis (lanes 7 and 14), bovine pancreatic RNase A (1 U) (lane 8), and RNase T1 digestion (1 U) (lane 1) as described previously (30,31). Samples were run on a 12% polyacrylamide/7 M urea gel. The amount of 30–40-kDa fraction purified enzyme used were 0.75 U (lane 4), 0.6 U (lane 5), 0.4 U (lane 6), 0.2 U (lane 12) and 0.1 U (lane 13). The amount of 10–20-kDa fraction purified enzyme used were 3 U (lane 10) and 1 U (lane 11). Asterisks indicate the 1751UA dinucleotide preferentially cleaved by the 30–40-kDa fraction enzyme. Arrows on the left indicate guanosine cleavage sites generated by RNase T1 and the numbers indicate position of nucleotide sequence. Numbers on the right indicate the cleavage sites generated by the endoribonucleases. (B) Secondary structure of c-myc CRD RNA and the cleavage sites generated by the 35-kDa endoribonuclease.

Figure 3.

Presence of RNase1 and APE1 in partially purified native enzyme. Western blot analysis of pooled samples from gel filtration (lanes 3–8) as detected by RNase A (upper panel) or APE1 (lower panel) antibody. Lane 1 contains 5 μg of recombinant bovine pancreatic RNase A.

Figure 4.

Immuno-depletion of the 35-kDa endoribonuclease activity using APE1 or syntaxin18 antibody. (A) Partially purified 35-kDa native enzyme was subjected to Seize X Protein A spin column which has been cross-linked with anti-APE1 antibody. Four microliters of washed (lanes 6 and 7) and eluted samples (lanes 8–10) were tested against 350 fmol 5′-labeled c-myc CRD RNA as described in ‘Materials and Methods’ section. Lane 1 contains the RNA only without any treatment with proteins. Lanes 2 and 3 contain 2 U and 3 U, respectively, of partially purified native enzyme from heparin-Sepharose column. Lanes 4 and 5 are 0.75 U and 1 U, respectively, of pre-loaded partially purified 30–40-kDa fraction native enzyme from gel filtration. Filled arrow indicates the intact c-myc CRD RNA and the decay products are shown with a bracket and unfilled arrow. (B) Western blot analysis of samples from (A) as detected using anti-APE1 antibody. Lane 1 contains 0.5 μg recombinant APE1 and lane 2 has the partially purified 30–40-kDa fraction native enzyme. FT is flow-through from the spin column. (C) As in (A), partially purified 35-kDa native enzyme was subjected to spin column which has been cross-linked with anti-syntaxin18 antibody. Lane 1 contains 2 U of partially purified native enzyme from heparin-Sepharose column. Four microliters of flow through (lane 2), washed (lane 3) and eluted samples (lanes 4 and 5) were tested against 5′-labeled c-myc CRD RNA as described in ‘Materials and methods’ section.

Figure 5.

Endoribonuclease activity of recombinant APE1 on c-myc CRD RNA. (A) Left panel, Coomasie blue-stained SDS–PAGE gel of recombinant human APE1 at 5 μg (lane 2) and 1 μg (lane 3). Lane 1 is molecular weight marker. Right panel, western blot analysis of 3 μg of recombinant human APE1 generated by us (lane 1) or in Dr. Hickson's laboratory (lane 2), as detected by specific monoclonal antibody against APE1. (B) Left panel, 350 fmol 5′ -labeled c-myc CRD RNA were treated with 0.1 U of the partially purified 35-kDa native enzyme for 5 min (lanes 2 and 3), or with 0.05 μg of purified recombinant human APE1 generated in Dr. Hickson's laboratory (lane 4). Lane 1 had no protein added. Right panel, 350 fmol 5′ -labeled c-myc CRD RNA were treated with 0.1 μg of purified and renatured recombinant human APE1 for 5 min (lane 2), 10 min (lane 3) and 20 min (lane 4), or with 0.1 U of the partially purified 35-kDa native enzyme for 5 min (lane 5), 10 min (lane 6) and 20 min (lane 7) under the standard endoribonuclease assay. Lane 1 had no protein added. Samples were run on 8% polyarylamide/7 M urea gel. Numbers on the right indicate cleavage sites generated by the enzymes.

Figure 6.

Endoribonuclease activity of recombinant APE1 polypeptide. (A) Left panel, 350 fmol 5′ -labeled c-myc CRD RNA were treated with the purified and renatured wild-type APE1 (lanes 2 and 9), H309N (lanes 3–5) or E96A (lanes 6–8) mutant APE1 for 5 min at 37°C at the amount indicated under the standard endoribonuclease assay containing 20 μl reaction. Lanes 1 and 10, no protein added. Right panel, 350 fmol 5′-labeled c-myc CRD RNA were treated with the purified and renatured wild-type recombinant APE1 (lane 2) or N-terminus truncated APE1, ND42 (lane 3). Lane 1, no protein added. Samples were run on 8% polyarylamide/7 M urea gel. Numbers on the right indicate cleavage sites generated by the enzymes. (B) 5′-labeled c-myc CRD RNA were treated with purified recombinant human APE1 (lanes 2 and 3), HADHSC (lanes 4 and 5) or annexin3 (lanes 6 and 7) for 5 min at 37°C at the amount indicated under the standard endoribonuclease assay.

Figure 7.

APE1 is an endoribonuclease. c-myc CRD RNA corresponding to nts 1730–1766 was either 5′-labeled with ³²P-γ-ATP (A) or uniformly labeled with ³²P-α-UTP (B) before subjecting to 0.1 μg purified recombinant human APE1 for 2 min (lane 2) or 5 min (lane 3) under the standard endoribonuclease assay. Samples were run on 8% polyarylamide/7 M urea gel. The secondary structure of RNA is on the right of each figure and arrows indicate the cleavage sites generated by APE1. Asterisks on the structure in (B) indicate radiolabeled uridine. Schematic RNA secondary structures on the right represent 5′fragment of cleavage products while those on the left in (B) represent 3′fragment of cleavage products. (C) 5′-labeled oligonucleotide corresponding to nts 1742–1757 of c-myc CRD was treated for 5 min with increasing concentrations (0.08–0.8 μg) of recombinant APE1 (lanes 3–9) or with 2 U partially purified native APE1 from heparin sepharose column (lane 2). Samples were run on 12% polyarylamide/7 M urea gel. The secondary structure of the oligo is on the right panel and the arrow indicates the RNA cleavage site generated by APE1. Schematic secondary structures on the right represent the full-length oligo (top) or 5′fragment of the cleavage product (bottom).

Figure 8.

APE1 knockdown increases c-myc mRNA level in HeLa cells. (A) Western blot analysis of APE1 and β-actin protein levels in HeLa cells at 24 and 48 h after transfection with 20 μM Control-dsRNAi or 20 μM APE1-dsRNAi. (B) APE1 and β-actin mRNA levels in HeLa cells transfected with 20 μM APE1-dsRNAi or 20 μM Control-dsRNAi for 24 and 48 h were analyzed by quantitative real-time RT-PCR (top panel). C_T values obtained in triplicates from separate transfection experiments were used in the comparative C_T method calculation as described in ‘Materials and Methods’ section. Data are mean ± SD of three independent experiments (*, P < 0.05). The APE1/β-actin mRNA ratios are presented as a percentage relative to Control-dsRNAi (top panel). The same RNA samples were analyzed for c-myc mRNA levels and the c-myc/β-actin mRNA ratios are presented as a percentage relative to Control-dsRNAi (bottom panel).

Figure 9.

Effect of APE1 knockdown on c-myc mRNA decay in HeLa cells. Twenty-four hours after plating at 1.0 × 10⁵ cells/well, cells were transfected with either 60 nM Control-dsRNAi or 60 nM APE1-dsRNAi. After a further 30 h incubation, cells were subjected to transcriptional inhibition by the addition of 200 µM DRB. Total RNA was then extracted from cells after 20, 40 and 60 min of further incubation. c-myc and β–actin mRNA levels were then quantified as described in Figure 8. After normalizing the levels of c-myc mRNA to β-actin mRNA, c-myc/β-actin mRNA ratios at different time points were expressed as percentage to that at 0 min. Data are the mean ± SD of two independent experiments. Linear regression analysis was performed to compare the two treatment groups and statistically significant difference was found in the slope (P < 0.05).