2013 Rustbelt RNA Meeting
mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human β-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5’-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA with known 5’ end sequence. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5’-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process, full-length nonsense-containing β-globin mRNA was increased and the Δ169 decay intermediate was decreased in cells knocked down for SMG6. This effect was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these conditions altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage.
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Keywords: nonsense-mediated mRNA decay, SMG6, globin