Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences

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.

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.

Figure 2. Degradation of NMD substrates is traceable by xrFrag analysis.

(a) Depiction of the β-globin reporter mRNAs as in Fig. 1. (b) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Co-transfected LacZ served as control mRNA. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and for each knockdown condition the PTC values were normalized to the WT. The ratio of xrFrag to reporter mRNA levels is indicated below the graph. (c) Western blot analysis of siRNA knockdown efficiency using the indicated antibodies. Tubulin served as loading control. (d–f) Total RNA was extracted from stable HeLa Flp-In T-REx cells expressing the indicated reporter RNA and analysed by northern blotting. (e,f) The cells were transfected with the indicated siRNA 72 h before induction of expression. 7SL RNA served as endogenous control RNA. Unless indicated otherwise (f), reporter mRNA expression was induced for 24 h with 1 μg ml⁻¹ doxycycline (Dox). Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to the WT control (+Dox for d; Luc or UPF1 knockdown for e; 12 h after Dox for f). The ratio of xrFrag to reporter mRNA levels is indicated below the graph.

Figure 3. Kinetics of xrFrag accumulation and degradation.

(a) Schematic representation of the TPI reporter mRNA as in Fig. 1. The NMD-activating long 3′ UTR fragment of SMG5 is indicated. (b–d) Northern blot analysis of RNA samples derived from HeLa stable cell lines expressing the indicated reporter constructs as described in Fig. 2. Mean values of reporter, xrFrag and 3′ fragment signal±s.d. (n=3) were quantified and normalized to the 7SL endogenous control. (b) 2 h after induction of transcription by doxycycline (Dox), actinomycin D (5 μg ml⁻¹) was added and the cells harvested at the indicated time points. (c) Transcription was induced for 4 h with Dox and reporter mRNAs were chased for the indicated time with actinomycin D. (d) Induction of reporter transcription was performed for the indicated time with Dox.

Figure 4. Tracing distinct degradation pathways employed during NMD by monitoring xrFrags.

(a) Model showing the recruitment of NMD-specific decay-inducing factors to activated UPF1 and their contribution to xrFrag accumulation. (b) Western blot analysis of knockdown efficiencies was performed with the indicated antibodies, tubulin served as loading control. (c–e) Northern blots of RNA samples extracted from stable HeLa cell lines transfected with the indicated siRNAs and expressing the indicated reporter constructs. Endogenous 7SL served as control RNA. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and for each knockdown condition the PTC values were normalized to the WT. The ratio of xrFrag to reporter mRNA levels is indicated below the graph.

Figure 5. Characterization of mRNA decay induced by direct protein tethering via xrFrag analysis.

(a,e) TPI and NanoLuc tethering reporter mRNAs with 4MS2 binding sites are depicted as in Fig. 1. (e) The position of the 5′ stem–loop inhibiting ribosome scanning is indicated. (b–d, g,h) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated tethering and reporter constructs. Mean values±s.d. (n=3) for reporter and xrFrag levels were quantified and normalized to tethered GST, which served as control. The ratio of xrFrag to reporter mRNA levels is indicated below the graph. Western blots show the expression levels of the MS2V5-tagged constructs with GFP serving as transfection control. Unspecific bands are indicated with asterisks. (f) Translational efficiency (mean±s.d., n=3) was measured by dual luciferase assay and compared for NanoLuc reporter with or without the 5′ stem–loop. Expression of the NanoLuc mRNAs is shown by northern blotting, co-transfected LacZ served as control.

Figure 6. Isolated ARE and miRNA decay elements induce deadenylation-dependent mRNA degradation.

(a,e) Schematic representation of TPI reporter mRNAs depicted as in Fig. 1, containing (a) the decay-inducing AREs from c-fos and GM–CSF in the 3′ UTR or (e) WT or mutated miRE for let-7 and miR-21. (b,c, f,g) Hela Flp-In T-REx cells expressing the indicated constructs were harvested and RNA was extracted and analysed by northern blotting. (c,g) Knockdown was performed by transfecting the cells with the indicated siRNAs. Expression of reporter mRNAs was induced with 1 μg ml⁻¹ doxycycline (+Dox) for 24 h. Endogenous 7SL RNA levels are shown as a control. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to the WT control (+Dox for b and f; per knockdown condition for c and g). The ratio of xrFrag to reporter mRNA levels is indicated below the graph. (d) Semi-quantitative PCR analysis of the CNOT1 and GAPDH (control) expression levels in siRNA treated cells.

Figure 7. The degradation induced by TNF-α and IL6 3′ UTRs is translation dependent.

(a) TPI reporter mRNAs with RAB7A (control), TNF-α or IL6 (decay-inducing) 3′ UTRs are represented as in Fig. 1, with the inserted sequences shown as coloured boxes. (b) Northern blot analysis of RNA samples derived from HeLa cells transiently transfected with the indicated reporter constructs and LacZ as control. (c–f) HeLa Flp-In T-REx cells expressing the indicated reporter RNA were harvested, total RNA extracted and analysed by northern blotting. 7SL RNA serves as endogenous control RNA. Unless indicated otherwise (c,d), reporter mRNA expression was induced for 24 h with 1 μg ml⁻¹ doxycycline (Dox). Cycloheximide treatment together with doxycycline induction was performed for 8 h with 100 μg ml⁻¹ of cycloheximide. Puromycin (Puro) treatment together with doxycycline induction was performed for 4 h with 20 μg ml⁻¹ of puromycin. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to RAB7A control (+Dox for c; 12 h after Dox for d; with or without cycloheximide for e; with or without Puro for f). The ratio of xrFrag to reporter mRNA levels is indicated below the graph.

Figure 8. The degradation induced by TNF-α and IL6 3′ UTRs involves endocleavage.

(a,d) TPI reporter mRNAs with xrRNA-framed RAB7A (control), TNF-α or IL6 (decay-inducing) 3′ UTRs are represented as in Fig. 1, with the inserted sequences shown as coloured boxes. Dual xrRNA elements (xrA and xrB) are indicated. (d) Deletion mutants are shown containing the stem–loop, marked as a hairpin, or the cleavage site, marked by a schematic endonuclease. (b,c; e,f) HeLa Flp-In T-REx cells expressing the indicated reporter RNA were harvested, total RNA extracted and analysed by northern blotting. 7SL RNA serves as endogenous control RNA. The reporter mRNA expression was induced for 24 h with 1 μg ml⁻¹ doxycycline (Dox). Knockdown was performed by transfecting the indicated siRNA. (c,e) Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to RAB7A control. The ratio of xrFrag to reporter mRNA levels is indicated below the graph.