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Arabidopsis mRNA decay landscape shaped by XRN 5′-3′ exoribonucleases

ABSTRACT: XRN 5′-3′ exoribonucleases play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of the Arabidopsis wild type and mutants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 but not nuclear XRN2 activity largely altered Arabidopsis mRNA decay profiles. In addition to the primary XRN4 substrates derived from decapping and microRNA-directed slicing, terminating ribosome- and exon junction complex-protected fragments produced from XRN4-mediated cytoplasmic decay also represent the most abundant decay intermediates of Arabidopsis mRNAs. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that polyadenylation cleavage frequently occurs after an adenosine. An increase in decay intermediates with 5′ ends upstream of a consensus motif in the xrn4 mutant suggests an endonucleolytic cleavage mechanism targeting the 3′ untranslated region of many Arabidopsis mRNAs. However, analysis of decay fragments stabilized in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence might rarely result in major decay intermediates. Together, the results of this study reveal major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE205190 | GEO | 2022/09/06

REPOSITORIES: GEO

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Project description:One of the major players controlling RNA decay is the cytoplasmic 5’ to 3’ exoribonuclease, which is conserved among eukaryotic organisms. In Arabidopsis, the 5’ to 3’ exoribonuclease XRN4 is involved in disease resistance, the response to ethylene, RNAi and miRNA-mediated RNA decay. Curiously, XRN4 appears to display selectivity among its substrates because certain 3’ cleavage products formed by miRNA-mediated decay, such as from ARF10 mRNA, accumulate in the xrn4 mutant, whereas others, such as from AGO1, do not. To examine the nature of this selectivity, transcripts that differentially accumulate in xrn4 were identified by combining PARE and Affymetrix arrays. Certain functional categories, such as stamen-associated proteins and hydrolases, were over-represented among transcripts decreased in xrn4, whereas transcripts encoding nuclear-encoded chloroplast-targeted proteins and nucleic acid-binding proteins were over-represented in transcripts increased in xrn4. To ascertain if RNA sequence influences the apparent XRN4 selectivity, a series of chimeric constructs were generated where the miRNA-complementary sites and different portions of the surrounding sequences from AGO1 and ARF10 were interchanged. Analysis of the resulting transgenic plants revealed that the presence of a 150 nucleotide sequence downstream of the ARF10 miRNA-complementary site conferred strong accumulation of the 3’ cleavage products in xrn4. In addition, sequence analysis of differentially accumulating transcripts led to the identification of 27 hexamer motifs that were over-represented in transcripts or miRNA-cleavage products accumulating in xrn4. Taken together, the data indicate that specific mRNA sequences, like those in ARF10, and mRNAs from select functional categories are attractive targets for XRN4-mediated decay. Mixed-stage inflorescences from Col-0 and xrn4-5 plants were harvested. Total RNA was extracted and hybridized to the ATH1 array to determine genes differentially expressed in xrn4-5 compared to Col-0. 2 replicates each.

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Arabidopsis mRNA decay landscape shaped by XRN 5′-3′ exoribonucleases

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