Fig. 5. SMG6 endonucleolytic cleavage is inactivated in SMG5-SMG7 depleted cells.

a Schematic overview of the triosephosphate isomerase (TPI) reporter constructs and their functional elements. The PTC160-containing reporter is subjected to SMG6-mediated endonucleolytic cleavage during NMD. The resulting decay intermediate (3′ fragment) is rapidly degraded by XRN1, until the presence of an xrRNA blocks the processivity of XRN1, ultimately resulting in the accumulation of meta-stable xrFrag molecules. b Northern blot analysis of TPI reporter, 3′ fragments (indicated with red triangles), xrFrag, and 7SL endogenous control. Ethidium bromide-stained 28S and 18S rRNAs are shown as additional controls. Quantification results (normalized to 7SL control) are shown as data points and mean (n = 3 biologically independent samples). c Schematic depiction of NOP56 maturation and the consequences of alternative splicing for inducing NMD. Upon inclusion of the snoRD86 sequence in the mature NOP56 mRNA by alternative splice site usage, a PTC is introduced resulting in endonucleolytic cleavage by SMG6. XRN1-mediated degradation of the decay intermediate is hindered by snoRD86, resulting in the accumulation of this meta-stable cleavage product. d Northern blot analysis of endogenous NOP56 (n = 3 biologically independent samples). Different transcript isoforms and fragments are indicated.