Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

Abstract

Nonsense-mediated mRNA decay (NMD) is traditionally portrayed as a quality-control mechanism that degrades mRNAs with truncated open reading frames (ORFs). However, it is meanwhile clear that NMD also contributes to the post-transcriptional gene regulation of numerous physiological mRNAs. To identify endogenous NMD substrate mRNAs and analyze the features that render them sensitive to NMD, we performed transcriptome profiling of human cells depleted of the NMD factors UPF1, SMG6, or SMG7. It revealed that mRNAs up-regulated by NMD abrogation had a greater median 3'-UTR length compared with that of the human mRNAome and were also enriched for 3'-UTR introns and uORFs. Intriguingly, most mRNAs coding for NMD factors were among the NMD-sensitive transcripts, implying that the NMD process is autoregulated. These mRNAs all possess long 3' UTRs, and some of them harbor uORFs. Using reporter gene assays, we demonstrated that the long 3' UTRs of UPF1, SMG5, and SMG7 mRNAs are the main NMD-inducing features of these mRNAs, suggesting that long 3' UTRs might be a frequent trigger of NMD.

FIGURE 1.

Venn diagrams illustrating the genes differentially expressed in UPF1, SMG6, and SMG7 knockdowns (>1.5-fold, false discovery rate ≤ 0.05). (A) Up-regulated sets of genes. (B) Down-regulated sets of genes. The total number of differentially expressed genes in a specific knockdown is depicted in parentheses below the respective NMD factor, and the percentage values indicate the genes that were differentially expressed only in one of the three knockdown conditions.

FIGURE 2.

Analysis of NMD-inducing features among the genes on the microarray. (A) The median 3′-UTR length is increased in genes up-regulated by UPF1, SMG6, and SMG7 knockdown (nts, nucleotides). (B) Genes harboring uORF(s) are enriched among the populations up-regulated upon UPF1 and SMG6 depletion. (C) The percentage of the genes that harbor at least one intron >50 nt downstream from the termination codon is elevated among the genes up-regulated by UPF1, SMG6, and SMG7 knockdown. The number of transcripts in each group is indicated in parentheses.

FIGURE 3.

Autoregulation of NMD factors. The levels of mRNAs are normalized to β-actin, GAPDH mRNA, or 18S rRNA levels. (A) Transcripts encoding UPF2, SMG1, SMG5, SMG6, and SMG7 are up-regulated in UPF1 KD conditions. The average values and standard deviations of three biological replicates represent changes of mRNA upon UPF1 knockdown and were determined by RT-qPCR. (B) UPF1 mRNA is up-regulated when NMD is abrogated by knockdown of other NMD factors. Average values and standard deviations of two independent experiments represent the change of UPF1 mRNA levels upon KD of the indicated factors. (C) UPF1 protein levels increase when NMD is abrogated by knockdown of other NMD factors. The quantification of UPF1 protein amounts, relative to no KD control and normalized to β-actin levels, is displayed below the Western blots.

FIGURE 4.

Schematic illustration of UTR reporter constructs used in Figures 5 and 6. (A) Mini mu wild-type (wt) reporter with a 288-bp-long 3′ UTR. (B) Reporters with either UPF1 (1834 bp), SMG5 (1483 bp), or SMG7 (2381 bp) full-length 3′ UTRs. (C) Reporters with truncated UPF1 (770 bp) or SMG7 (773 bp) 3′ UTRs. The length of these 3′-UTR fragments is similar to the average human 3′-UTR length. (D) UPF1 (255 bp) or SMG7 (252 bp) 3′-UTR fragments corresponding to the wt reporter's 3′-UTR length. (E) The inverted sequence of the UPF1 (1834 bp) or SMG7 (2381) full-length 3′ UTR. (F) Reporter with the wt 5′ UTR of SMG5. (G) Reporter with a SMG5 5′ UTR in which the start codon of the uORF was mutated (gAUG to gUGG). The constructs are not drawn to scale.

FIGURE 5.

Untranslated regions of UPF1, SMG7, and SMG5 are sufficient to trigger NMD. The levels of mRNAs are normalized to cotransfected wild-type (wt) β-globin reporter levels. Asterisks indicate that the P-value of the fold change (Student's t-test) is <0.05 compared with the fold induction of the mu wt 3′-UTR reporter. Full-length but not truncated sequences of the UPF1 3′ UTR (A) and of the SMG7 3′ UTR (B) convert the mini mu reporter gene into a strong NMD substrate. Average values and standard deviations of four independent experiments are shown. (C) The 3′ UTR of SMG5 converts mini mu reporter to a strong NMD substrate (average of three experiments).

FIGURE 6.

Reduced half-lives of reporter mRNAs with 3′ UTRs of UPF1, SMG5, and SMG7. To determine the decay kinetics, the tet-off system was used. Relative mRNA levels were measured by RT-qPCR 0, 2, 4, and 8 h after stopping the transcription of the reporter gene by addition of doxycycline. Average values and standard deviations of three independent experiments are shown. The levels of mRNAs are normalized to cotransfected wild-type (wt) β-globin reporter levels.

FIGURE 7.

RNA immunoprecipitation with anti-UPF1 antibody. Fold enrichment of indicated mRNAs in the UPF1 immunoprecipitate fraction relative to negative control (GFP IP fraction) is shown. Relative mRNA levels were determined by RT-qPCR and were normalized to input levels of mRNA. Asterisks signify <0.05 P-value (Student's t-test) for enrichment compared with the enrichment of GAPDH mRNA in UPF1 immunoprecipitates.