Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability

Abstract

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.

Keywords: BRIC-seq; RNA decay; RNA-seq; UPF1; nonsense-mediated mRNA decay.

Figure 1. Validation of the UPF1 depletion and BRIC-seq. (A) UPF1 levels were quantified by RT-qPCR after RNA interference (RNAi) of UPF1. Control-1, Control siRNA-1; UPF1–1, UPF1 siRNA-1; Control-2, Control siRNA-2; and, UPF1–2, UPF1 siRNA-2. GAPDH abundance was used for normalization. Values represent mean ± errors from duplicate experiments. (B) BRIC-seq determined the half-lives of 9,229 mRNAs in HeLa Tet-off (TO) cells. White circles (left panel) indicate the fraction of mRNAs in control cells and black circles (right panel) indicate the fraction of mRNAs in UPF1-depleted cells. Transcripts with a half-life over 24 h are included in the category of 24 h. (C) Expression levels of GADD45A in control cells (gray bar) and in UPF1-depleted cells (black bar) were determined by RT-qPCR. GAPDH abundance was used for normalization. Values represent mean ± errors from duplicate experiments. (D) Decay rates in control cells (open circle and gray line) and in UPF1-depleted cells (solid circle and black line) were determined by RT-qPCR. (E) Autoregulation (feedback regulation) of NMD factors. mRNA levels upon UPF1 knockdown, determined by RNA-seq. GAPDH abundance was used for normalization. Transcripts encoding UPF2, SMG1, SMG5, SMG6, SMG7, and SMG9 are significantly upregulated in UPF1-depleted cells. Grey and black bars indicate cells transfected with UPF1 siRNA-1 and UPF1 siRNA-2, respectively.

Figure 2. Typical changes in the relative abundances of alternatively splice variants of RPL12. A chromosome diagram is shown in the upper position. Three splice variants transcribed from RPL12 gene are shown in the middle. Variant 2 (middle) is a known NMD target. Mapping data of RNA-seq tags using a Genome Analyzer (Illumina) is shown at the bottom. Block-like shadows indicate piling of RNA-seq tags. Arrows indicate the exon, contained only in splice variant 2, with increased RNA-seq tag piling in response to UPF1 depletion.

Figure 3. Steady-state abundance and decay rates of 9,229 transcripts. Transcripts were identified by expression profiling of UPF1-depleted cells by RNA-seq and by RNA stability in UPF1-depleted cells by BRIC-seq. (A) (Left) Venn diagram of overlap between mRNAs that are over 2-fold upregulated in response to UPF1 depletion (left dashed circle) and mRNAs that are over 2-fold stabilized in UPF1 knockdown (right circle). The overlap between the circles was categorized as Group C (76 transcripts), indicating upregulated, stabilized mRNAs in UPF1-depleted cells. Group A (248 transcripts) represents the over 2-fold upregulated, non-stabilized mRNAs in UPF1-depleted cells. Group B (709 transcripts) represents over 2-fold stabilized, not upregulated mRNAs in UPF1 knockdown cells. (Right) Venn diagram of the overlap between mRNAs that are over 2-fold downregulated in response to UPF1 depletion (left dashed circle) and mRNAs that are over 2-fold destabilized in UPF1 knockdown (right circle). The overlap between the circles (67 transcripts) indicates downregulated and destabilized mRNAs in UPF1-depleted cells. The left circle (98 transcripts) represents the over 2-fold downregulated, non-destabilized mRNAs in UPF1-depleted cells. The right circle (3,232 transcripts) represents over 2-fold destabilized, not downregulated mRNAs in UPF1 knockdown cells. (B−D) Representative mRNAs of Group A (panel B), Group C (panel C), and Group B (panel D). The expression levels of each mRNA in UPF1-depleted cells compared with control cells are shown as bar graphs in each panel. The dashed lines indicate 2-fold upregulation in response to UPF1 depletion. The degradation curves of each transcript in control cells (open circle and gray bar) and in UPF1-depleted cells (solid circle and black bar) are shown as scatter plots.

Figure 4. Determination of direct or indirect UPF1 targets. (A and B) Alteration of pre-mRNA (A) and mature RNA (B) expression levels in UPF1 deficient cells distinguishes direct UPF1 target mRNAs from indirect target mRNAs. Fold upregulations of RNAs in the UPF1-depleted cells relative to control cells are shown. The RNA levels were determined by RT-qPCR. GAPDH abundance was used for normalization. Values represent mean ± errors from duplicate experiments. The dashed line indicates 2-fold upregulation of transcripts in response to UPF1 depletion. (C and D) Examination of RNA binding to UPF1. RNA immunoprecipitation (RIP) of Group A, B (in C), and C (in D) RNAs with Myc-tagged UPF1 proteins was performed. Fold enrichment of indicated mRNAs in the Myc-UPF1 immunoprecipitate fraction relative to the negative control (normal IgG IP fraction) is shown. RT-qPCR determined the relative mRNA levels, which were normalized to input levels of mRNA and GAPDH mRNA levels. Values represent mean ± SD from triplicate experiments. The dashed line indicates 2-fold enrichment in the Myc-UPF1 immunoprecipitate. *, p < 0.05 (Student’s t-test) compared with the enrichment of GAPDH mRNA in UPF1 immunoprecipitates.