Uridylation by TUT4 and TUT7 marks mRNA for degradation

Abstract

Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than ∼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.

Figure 1. TUT4 and TUT7 Are Required for mRNA Uridylation in Human Cells

(A) Uridylation frequency measured by small-scale TAIL-seq (with Illumina MiSeq) following RNAi of the indicated genes. Frequency (y axis) is the fraction of uridylated reads among the total number of mRNA reads with short poly(A) tail (5–25 nt). Light blue refers to mono-uridylation (U), blue indicates di-uridylation (UU), and dark blue represents ≥ 3 uridines (U ≥ 3). Uridylation frequency significantly decreased in siTUT2/4/7 (p = 0.0378 for U; 0.0388 for UU; 0.0201 for U ≥ 3 by one-tailed t test). Error bar represents SEM from two biologically independent replicates (n = 2). (B) Uridylation frequency of mRNAs with short poly(A) tails (5–25 nt) measured by small-scale TAIL-seq in knockout HeLa cell lines. Uridylation frequency was reduced modestly in TUT4 and TUT7 knockout cells (p = 0.109 for U, 0.0273 for UU, 0.142 for U ≥ 3 of TUT4 KO; p = 0.150 for U, 0.00685 for UU, 0.0713 for U ≥ 3 of TUT7 KO by one-tailed t test). Error bar represents SEM from two replicates (n = 2). (C) Uridylation frequency of mRNAs with short poly(A) tails (5–25 nt) measured by TAIL-seq following simultaneous TUT4 and TUT7 knockdown (siTUT4/7). Uridylation was reduced when both TUT4 and TUT7 were depleted (p = 0.0941 for U, 0.00922 for UU, 0.0105 for U ≥ 3; one-tailed t test). Error bar represents SEM from three biological replicates (n = 3). (D) Changes in uridylation of individual mRNA species upon TUT4/7 knockdown. “Average U length per tail” (y axis) is the sum of the number of all uridines on short A-tails (5–25 nt) divided by the total number of reads with short A-tails. Note that unlike “uridylation frequency,” average U length per tail weighs every uridine in oligo-U-tails. Each dot represents a transcript with ≥ 15 reads in both samples. Uridylation was significantly decreased following TUT4/7 knockdown (p = 7.69 × 10⁻¹⁰⁰, one-tailed Mann-Whitney U test). The full list is shown in Table S1. (E) Examples of gene-level uridylation changes. Twenty-one most abundant mRNAs (not including ribosomal protein mRNAs and histone mRNAs) are shown in the order of mRNA abundance. See also Figure S1.

Figure 2. Short A-Tails Are Selectively Uridylated by TUT4 and TUT7

(A) Distribution of mono-uridylation (top) and oligo-uridylation (bottom) according to the length of poly(A) tails. Poly(A) tail lengths from 5 nt to 231 nt are pooled into equal-width bins in the logarithmic scale (base 2) (x axis). The left edges (inclusive) of bins are 5, 7, 9, 12, 15, 21, 28, 38, 50, 67, 89, 119, 159, and 212 nt. Uridylation frequency (y axis) indicates the percentage of uridylated reads within each poly(A) tail size range. Error bar represents SEM (n = 3). (B) Top: illustration of chemically synthesized RNA substrates. Grey bars represent the last 20 nt of SHOC2 3′ UTR and “A” indicates an adenosine. Bottom: in vitro uridylation assay using immunopurified FLAG-TUT4. RNA (0.45 nM) was used in each reaction. The products were resolved on 6% polyacrylamide sequencing gel containing 7 M urea. The average length of uridylation is shown below each band. See Extended Experimental Procedures for quantification method. (C) Top: illustration of chemically synthesized RNA substrates. Green bars represent the last 20 nt of CALM1 3′ UTR and “A” indicates an adenosine. Bottom: in vitro uridylation assay using recombinant TUT7 C-terminal fragment (951–1,495 aa) purified from E. coli. RNA (0.45 nM) and 14 nM of recombinant TUT7 were used in each reaction. Extension products were resolved on 6% polyacrylamide sequencing gel containing 7 M urea. The average length of uridylation was quantified as in (B). See also Figure S2.

Figure 3. PABP Inhibits Uridylation of Polyadenylated mRNA

In vitro uridylation assay by using recombinant TUT7 (951–1,495 aa) with a varying concentration of recombinant PABPC1 (0, 10, or 40 nM). 0.45 nM of RNA and 160 nM of recombinant TUT7 (rTUT7) were used in the reaction. Extension products were resolved on 6% polyacrylamide sequencing gel containing 7 M urea. The average length of uridylation was quantified as described in Extended Experimental Procedures and shown below each band. See also Figure S3.

Figure 4. Uridylation Promotes mRNA Degradation

(A) Transcriptome-wide change of mRNA half-life determined by RNA-seq. Left: experimental scheme. HeLa cells were transfected twice and harvested at 0, 1, 2, and 4 hr following actinomycin D treatment. Center: changes of average mRNA half-life upon TUT4/7 knockdown from two biological replicates. The range of display is limited to between 0 and 30 hr for the better visual recognition (232 out of 1,829 mRNAs are outside of the view). The full list is available in Table S2. Right: distribution of mRNA half-lives in control or TUT4/7 knockdown cells. A box represents the first and third quartiles and an internal bar indicates median. Whiskers span between the ninth and the 91^st percentiles. Half-lives of mRNAs are significantly extended by TUT4/7 knockdown (***p = 4.06 × 10⁻¹⁵⁵, one-tailed paired Mann-Whitney U test). See Extended Experimental Procedures for the detailed description of procedure. (B) Measurement of mRNA half-life by qRT-PCR. Left: the experimental scheme. Right: following 0, 2, and 4 hr of actinomycin D treatment, relative abundance (y axis) of five selected genes were measured. For normalization, GAPDH mRNA was used because it was highly stable (half-life > 24 hr, data not shown) and did not change noticeably by TUT4/7 depletion. Error bar represents SEM (n = 3). Half-lives are calculated by linear fitting of the log-transformed exponential decay function. (C) Left: schematic representation of reporter assay system with the λN tethering. Center: reporter (firefly) luciferase activity was measured and normalized to Renilla luciferase activity (n=3). Right: reporter mRNA levels were determined by qRT-PCR (n = 4). Error bars represent SEM. Luciferase activity or RNA level were significantly reduced when AGO2 or TUT4 were tethered (*p < 0.01, **p < 0.001; two-tailed t test). See also Figure S4.

Figure 5. Uridylation Facilitates miRNA-Mediated mRNA Decay

(A) Changes in uridylation after miR-1 transfection. Left: experimental scheme. miR-1 was transfected into HeLa cells and the cells were harvested after the indicated time for TAIL-seq. Targets are the transcripts with ≥ 1 miR-1 3′ UTR site and down-regulated by ≥30% on 12 hr posttransfection of miR-1 (Guo et al., 2010). Right top: average Ulength change relative to 0 hr is shown in each time point. Average U length per tail is the number of uridines on short A-tails (5–25 nt) divided by the total number of reads with short A-tails. Box represents the interval between the first and third quartiles, and the internal bar indicates the median. Whiskers span between the ninth and 91^st percentiles. Average U length of miR-1 target is significantly extended after miR-1 transfection (*p = 0.0152, **p = 0.00318, ***p = 5.79 × 10⁻⁴; one-tailed Mann-Whitney U test). Right middle: poly(A) tail length change relative to 0 hr. The length change is represented by log₂ odds ratio between long tails (>25 nt) and short tails (≤25 nt) in one among 3, 6, or 9 hr and 0 hr. A negative value (<0) indicates increase of the fraction of short tails compared to 0 hr. Error bars indicate SD among mRNAs. The portion of short poly(A) tails expanded more for miR-1 targets than the others (p = 1.80 × 10⁻⁶ for 3 hr, p = 8.47 × 10⁻¹³ for 6 hr, p = 1.48 × 10⁻¹¹ for 9 hr; one-tailed Mann-Whitney U test). Right bottom: mRNA abundance (poly(A)⁺ tag counts) change relative to 0 hr. Error bars indicate SD among mRNAs. Expression levels of miR-1 targets were decreased more than the rest transcripts (p = 2.09 × 10⁻⁴ for 3 hr, p = 2.65 × 10⁻¹⁴ for 6 hr, p = 5.46 × 10⁻¹⁸ for 9 hr; one-tailed Mann-Whitney U test). (B) Measurement of half-life of miR-1 targets by qRT-PCR. Left: the experimental scheme. Following siRNA transfection for 62 hr, HeLa cells were transfected with miR-1 or mock transfected. After 4 hr, actinomycin D was treated and cells were harvested at 0, 1, 2, and 4 hr. Right: relative abundance (y axis) of miR-1 target mRNAs were measured. For the normalization, highly stable GAPDH mRNA was used because it did not change significantly by siTUT4/7 or miR-1 transfection. Error bar represents SEM (n = 3). Half-lives are determined by linear fitting of the log-transformed exponential decay function.

Figure 6. The 5′ and 3′ mRNA Decay Factors Degrade Uridylated mRNAs

(A–C) Changes of poly(A) tail and uridylation upon knockdown of decay factor(s) detected by small-scale TAIL-seq (with Illumina MiSeq). Fraction of mRNA reads out of the total poly(A)⁺ mRNA reads is shown in each poly(A) tail size range. Narrow bars represent reads without U-tails (U₀) and wider bars indicate uridylated reads (U₁–U₃₊). The “DCP1/2 mut” sample derived from cells coexpressed of dominant-negative mutants of DCP1 and DCP2 (DCP1a-GSSG and DCP2-E148Q, respectively). See also Figure S5.

Figure 7. Model for Uridylation-Dependent mRNA Decay in Humans

mRNA decay is generally initiated by deadenylation. PABP proteins are dissociated from mRNA as poly(A) tail becomes shorter (less than ∼25 nt). TUT4 and TUT7 act redundantly to uridylate mRNAs with a short A-tail. The U-tail is in turn recognized by the downstream decay factors (uridylation-dependent mRNA decay pathway). The LSM1–7 complex binds to the U-tail and facilitates decapping by the DCP1/2 complex. Decapped mRNAs are degraded by the 5′–3′ exonuclease XRN1. Alternatively, the U-tail is recognized by exosome or DIS3L2 that degrade mRNA exonucleolytically from the 3′ end. It is currently unclear if and what fraction of deadenylated mRNAs are degraded through uridylation-independent alternative pathways (indicated with gray dashed lines).