Fig 2: Xrn1-cKO mice exhibit metabolic disorders with hyperglycemia, hyperinsulinemia, and hyperleptinemia(A) Levels of circulating blood glucose (left), serum insulin (middle) and serum leptin (right) in 5-week male control (white, n = 5–8) and Xrn1-cKO (red, n = 5–6) mice.(B and C) (C) To test leptin sensitivity, 5-week-old Xrn1flox/flox (black, n = 5) and Xrn1-cKO (red, n = 5) mice were intraperitoneally administered with leptin (5 µg/g body weight) every day for 5 days (blue arrows), daily body weight (B) and food intake (C) were measured for 11 days. ?body weight (%) is shown as the percentage of daily weight gain normalized by daily body weight.(D) Fed (left) and fasting (right) blood glucose levels in 5-, 7- and 9-week-old control (black, n = 5) and Xrn1-cKO (red, n = 5) mice are shown.(E) Intraperitoneal glucose tolerance test (IPGTT) using 5-week-old control (black, n = 5) and Xrn1-cKO (red, n = 5) mice. Areas under the curves (AUC) of IPGTT are shown on the right side.(F) Insulin tolerance test (ITT) using 5-week-old control (black, n = 5) and Xrn1-cKO (red, n = 5) mice. Values at 0 min are set as 100%. Areas under the curves (AUC) of ITT are shown on the right side. Data represent means ± SEM. Two-way ANOVA followed by Bonferroni post hoc test for fed and fasting blood glucose, IPGTT and ITT. Unpaired Student’s t test for AUC, *p < 0.05; **p < 0.01; and ***p < 0.001. ns represents “not significant.”

Fig 3: Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. (A–D) A PCR-based approach was used to quantify 3' fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A)+ RNA was extracted. 3' Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets (A, B, C, D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). (A) PDRG1, SURF6, and MED10 transcripts with a long 3' UTR. (B) IFRD1 and BAG1 transcripts with an uORF. (C) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. (D) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.

Fig 4: Xrn1-cKO mice exhibit upregulated appetite and energy homeostasis-related genes in the hypothalamus(A) Quantitative PCR (qPCR) analysis of the indicated mRNA levels in the hypothalamus of 10- to 14-week-old control (Xrn1flox/flox: white, n = 5; Camk2a-Cre: gray, n = 10) and Xrn1-cKO mice (red, n = 5). The value of the average of Xrn1flox/flox mice was set as 1. Data represent means ± SEM. One-way ANOVA, Tukey post hoc test, *p < 0.05; **p < 0.01; and ***p < 0.001.(B) qPCR analysis of the indicated pre-mRNAs in the hypothalamus of 10- to 14-week-old control (Xrn1flox/flox: white, n = 5; Camk2a-Cre: gray, n = 10) and Xrn1-cKO mice (red, n = 5). The average value of Xrn1flox/flox mice was set as 1. Data represent means ± SEM. One-way ANOVA, Tukey post hoc test, *p < 0.05; **p < 0.01; and ***p < 0.001.(C) Protein expression of XRN1, LERP, INSR, STAT3, FOXO1, SOCS3, PTP1B, TCPTP, and a-Tubulin in 12-week-old control (Xrn1flox/flox: white, n = 3; Camk2a-Cre: gray, n = 3) and Xrn1-cKO mice (red, n = 3).(D) Quantitative analysis of protein bands in panel 5C. Band intensities were measured using NIH ImageJ. Values of the average of Xrn1flox/flox mice were set as 1. Data represent means ± SEM. One-way ANOVA, Tukey post hoc test, *p < 0.05; **p < 0.01; and ***p < 0.001.(E) Immunofluorescence analysis of AgRP (red) and nuclei by counterstaining with DAPI (blue) in the ARC of Xrn1-cKO and control (Xrn1flox/flox) mice at 12 weeks old.(F) Quantification of the AgRP signal in the ARC of Xrn1-cKO (red) and control (white) mice.

Fig 5: Effect of XRN1 depletion on wild-type and C3U HCV RNA abundances, replication, and viral protein expression.(A) Huh7.5 cells were transfected with non-targeting (NT) and XRN1 siRNAs one day prior and twenty-four hours after transfection with wild-type and C3U H77.S3/GLuc RNAs. Viral RNA replication, measured by luciferase activity in the supernatants of transfected cells, was monitored for up to 72 hours (*p<0.05 and **p<0.005). (B) Efficiency of XRN1 knockdown and its effects on HCV core viral protein levels were monitored by Western blot analysis three days post RNA transfection. (C) Cells were treated as in (A) and HCV RNA abundances were monitored by Northern blot analysis. (D) Quantification of viral RNA levels following XRN1 depletion normalized to actin. The data are representative of three independent replicates. (E) HCV RNA stability was monitored following the addition of a 5’ non-methylated guanosine cap analog to wild-type and C3U replication defective viral RNAs at the indicated time points.