Conserved Residues at the Mtr4 C-Terminus Coordinate Helicase Activity and Exosome Interactions.

Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions.

RNA degradome analysis reveals DNE1 endoribonuclease is required for the turnover of diverse mRNA substrates in Arabidopsis.

In 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 expressing DNE1 cDNA with an active-site mutation in the endoribonuclease domain abolished the in planta cleavage 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.