Genetic and biochemical characterization ofDrosophilaSnipper: A promiscuous member of the metazoan 3′hExo/ERI-1 family of 3′ to 5′ exonucleases

Abstract

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 39hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 39hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 39-59 exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 39 overhangs of 2-5 nucleotides (nt) in the presence of Mg 2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 39 overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 39hExo or ERI-1.

Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5′ to 3′ and 3′ to 5′

“…Despite the fact that the 3ЈhExo can potentially degrade the 3Ј end of histone mRNA, we find no evidence for a role of this enzyme in histone mRNA degradation in mammalian cells (Fig. 1) or Drosophila (Kupsco et al 2006). In contrast, knocking down components of both the 5Ј-to-3Ј decay pathway or the exosome resulted in a reduced rate of degradation of histone mRNA when DNA replication was inhibited (Figs.…”

EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates

“…4A). There is no known ortholog of 3 ′ hExo in Drosophila (Kupsco et al 2006), and the enzyme that does the trimming is unclear. Both samples also had a substantial number of 1-to 2-nt nontemplated tails, resulting from further trimming of the mRNA followed by nucleotide addition, suggesting that a similar reaction can occur on histone mRNAs in Drosophila and mammalian cells to maintain the length of the mRNA.…”

Structure-specific nucleic acid recognition by L-motifs and their diverse roles in expression and regulation of the genome

“…Another intriguing question is: does SLBP play a direct role in DNA metabolism, especially in DNA replication? Although SLBP does not bind B-form DNA (137), it is conceivable that it may recognize branched non-B DNA intermediates during replication. Such a speculative model is intriguing because SLBP is expressed only during S-phase of the cell cycle, and is one of two cell cycle regulated factors involved in histone mRNA metabolism whose expression is correlated with that of histone mRNA (138).…”