AbstractIn plants, cytoplasmic mRNA decay is critical for posttranscriptionally controlling gene
expression and for maintaining cellular RNA homeostasis. Arabidopsis DCP1-ASSOCIATED NYN
ENDORIBONUCLEASE 1 (DNE1) is a cytoplasmic mRNA decay factor that interacts with proteins
involved in mRNA decapping and nonsense-mediated mRNA decay (NMD). There is limited
information on the functional role of DNE1 in RNA turnover, and the identities of its
endogenous targets are unknown. In this study, we utilized RNA degradome approaches to
globally investigate DNE1 substrates. Monophosphorylated 5′ ends, produced by DNE1, should
accumulate in mutants lacking the cytoplasmic exoribonuclease XRN4, but be absent from
DNE1 and XRN4 double mutants. In seedlings, we identified over 200 such transcripts, most
of which reflect cleavage within coding regions. While most DNE1 targets were
NMD-insensitive, some were upstream ORF (uORF)-containing and NMD-sensitive transcripts,
indicating that this endoribonuclease is required for turnover of a diverse set of mRNAs.
Transgenic plants expressingDNE1cDNA with an active-site mutation in
the endoribonuclease domain abolished thein plantacleavage of
transcripts, demonstrating that DNE1 endoribonuclease activity is required for cleavage.
Our work provides key insights into the identity of DNE1 substrates and enhances our
understanding of DNE1-mediated mRNA decay.