Figure 1: Characterization of xrRNA elements enabling the detection of mRNA degradation intermediates.

(a) Depicted are the general mRNA degradation pathways leading either directly (decapping and endocleavage) or indirectly (deadenylation) to 5′–3′ decay executed by XRN1. The presence of a stable XRN1-resistant RNA structure (xrRNA) prevents XRN1 from further progression and thus protects the remaining RNA fragment (xrFrag) from degradation from the 5′ end. (b) The DNA sequence of the xrRNA element used in reporter constructs is shown with annotations of sequence motifs. (c,e) Schematic representation of the TPI reporter mRNA. The TPI gene is depicted as blue boxes representing single exons (exon numbers indicated). The positions of the normal stop codons (stop) and premature translation termination codons are shown. Northern blot probe binding sites in the 3′ UTR are depicted as grey boxes and single xrRNA structures 1 and 2 are shown in red and purple. The difference between full xrRNA and 1+2 is the presence or absence of a short spacer region (indicated in b). (e) The 60 bp elements with varying GC content were derived from the RAB7A 3′ UTR, the 4MS2 binding sites are identical to those used in tethering experiments (Fig. 5) and the stem–loop structure is used in other reporters to block translation initiation (Fig. 5). (d,f) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated reporter constructs. Co-transfected LacZ served as control mRNA. (f) Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to the TPI reporter without insert. The ratio of xrFrag to reporter mRNA levels is indicated below the graph.