Small DNA pieces in C. elegans are intermediates of DNA fragmentation during apoptosis

Abstract

While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, approximately 10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3' phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway.

Figure 1. Characterization of small nucleic acid pieces.

(A) Northern analysis of nucleic acid isolated from wildtype embryos (emb) or adults (ad) using the “total RNA” protocol, and hybridization with probes complementary to the 3′ UTR of clec-67 (Materials and Methods; Figure S1). M, RNA decade markers (Ambion). (B) A representative northern blot is shown (n≥3) preformed as in (A) using embryo samples and subjecting “total RNA” to various treatments prior to electrophoresis and northern analysis: D, DNase treatment; R, RNase treatment, NaOH, alkaline hydrolysis. M, 10 bp DNA ladder (Invitrogen). Blots were reprobed for U6 snRNA to confirm specificity of various treatments (bottom panels). In different experiments, U6 snRNA migrated either as a single or double band. (C) A representative northern blot is shown (n≥2) performed as in (A) for embryo “total RNA” using probes for Class I or Class II transposons (see Materials and Methods). Asterisk marks an RNase sensitive band. ImageQuant software was used to calculate the relative radioactivity hybridizing to Class 1 and Class II transposons (1∶4.4); radioactivity in the entire lane, excluding the RNase sensitive band, was evaluated.

Figure 2. Analysis of small DNA pieces in C. elegans apoptosis mutant strains.

Northern analyses as in Figure 1 were performed on “total RNA” isolated from C. elegans embryos of wildtype (N2) animals or those containing mutations in genes that affect the killing or engulfment phase of apoptosis (A) or those implicated in nucleolytic degradation of DNA during apoptosis (B). For each strain, multiple northern analyses (2–5) were performed on multiple, independent RNA preparations (2–5) and representative data are shown. As much as possible, cultures of various strains were maintained at similar developmental stages, but embryos isolated from gravid adults varied slightly in their precise stage. This may explain the variation in levels of small DNA pieces between strains; further, some differences may be due to mutations in apoptosis genes that alter the kinetics of PCD. The blot shows an absence of small DNA pieces in ced-3(n717), and other alleles (n1286, n2888) gave identical results (Figure S4). Blots were reprobed for U6 as a loading control (bottom panels).

Figure 3. Small DNA pieces correlate with TUNEL negative fragments.

Embryos were isolated from gravid adults, of N2 or nuc-1(e1392) genotype, and nucleic acid isolated using the “total RNA” protocol. A representative PhosphorImage (n = 7) shows DNA pieces radiolabeled with TdT and α-³²P-Cordycepin 5′ triphosphate, before (-) or after (+) treatment with CIP. Positions of 10 bp DNA ladder (M) are labeled on the right.