The RNA Helicases AtMTR4 and HEN2 Target Specific Subsets of Nuclear Transcripts for Degradation by the Nuclear Exosome in Arabidopsis thaliana

Abstract

The RNA exosome is the major 3′-5′ RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively.

<i>Arabidopsis</i> AtRRP44 has ribonuclease activity that is required to complement the growth defect of yeast <i>rrp44</i> Mutant

“…Based on available co-immunoprecipitation data showing the direct interaction between the exosome core and AtRRP44 (Lange et al 2014) and our results, we speculate that AtRRP44 endows plant RNA exosome with the catalytic activity. Arabidopsis Col-0 transformants expressing AtRRP44-GUS fusion proteins driven by the AtRRP44 native promoter region showed strong signals especially in organs associated with active turnover (such as shoot and root apical meristems as well as lateral root primordia), indicating that AtRRP44 has pleiotropic functions in plant development through RNA degradation and processing.…”

“…In A. thaliana, well known exosome core components and cofactors, AtRRP44 and AtRRP6Ls, have been determined (Chekanova et al 2007;Kumakura et al 2013;Lange et al 2009Lange et al , 2014. Rrp44/Dis3 proteins in human and yeast have ribonuclease activity and are the catalytic centers of the exosome (Dziembowski et al 2006;Liu et al 2006;Staals et al 2010).…”

“…In plants, Lange et al (2014) reported that AtRRP44 also can interact with the exosome core. To determine the importance of the AtRRP44 PIN domain, we tested whether the atrrp44ΔPIN mutant could complement the scrrp44 mutant.…”

“…Arabidopsis AtRRP44, which has been found to be associated with the RNA exosome and to be able to complement growth defects of yeast rrp44/dis3 mutants (Kumakura et al 2013;Lange et al 2014), was investigated for its RNA degradation activity. In general, Rrp44/Dis3 has two important domains, a pilT N domain (PIN) and an RNase B (RNB) domain.…”

“…Association between nine exosome core components and the cofactor AtRRP44 were detected by a combination of immunoprecipitation and mass spectrometry analysis (Lange et al 2014). Of the nine exosome core components in Arabidopsis, the roles of AtRRP4, AtRRP41, AtRRP41L/MTR3, and AtRRP45B/ CER7 have been investigated mainly by genetic analysis (Chekanova et al 2000(Chekanova et al , 2007Hooker et al 2007;Lam et al 2012;Yang et al 2012).…”