Abstract
The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5′–3′ mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3′ UTRs of cytokine mRNAs reveals the contribution of 5′–3′ decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses.
Highlights
The turnover of messenger RNAs is a key regulatory step of gene expression in eukaryotic cells
A potential alternative to the knockdown of XRN1 is the incorporation of viral XRN1-resistant RNA sequence (xrRNA) elements in reporter messenger RNAs (mRNAs) (Fig. 1a), which were previously reported to block the processively degrading XRN1 upstream of the xrRNA structure[23]
We found decreased levels of the premature termination codons (PTCs)-containing mRNA and increased xrFragB compared with the wild type (WT)
Summary
The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses. A well-studied example is the degradation of transcripts containing premature termination codons (PTCs) by the nonsense-mediated mRNA decay pathway (NMD)[6,7]. Further degradation of the mRNA occurs via the cytoplasmic Lsm1-7-Pat[1] complex in combination with the eIF4E-binding protein 4E-T, which recruit decapping factors to the 50 end of the deadenylated mRNA12,13. The deadenylated mRNA may be eliminated by the cytoplasmic exosome, a multi-protein complex, via its [30–50] exonucleolytic subunit DIS3L SMG6 is recruited to PTC-containing mRNAs and cleaves them in the vicinity of the termination codon[21]
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