Identification of RNA-binding proteins’ direct effects on gene expression via the degradation tag system

RESULTS AND DISCUSSION

Loss-of-function approaches (e.g., knockout/knockdown of the target RBP) are widely used to infer how an RBP regulates its target mRNAs. However, interpreting the results from these experiments is confounded by two caveats. First, an RBP usually binds to hundreds of target mRNAs. Thus, knocking down/knockout of an RBP will disrupt numerous RBP:mRNA interactions, making it challenging to determine whether the impact on a specific mRNA is due to the direct effect of the RBP or indirect effects from other altered RBP:mRNA interactions. Second, there is a time lag between the knockdown/knockout of the RBP and the measurement of target gene expression. For example, in the siRNA/shRNA-mediated knockdown experiments, the expression of the target gene is typically assayed at 24–48 h post-transfection/transduction. Secondary effects could occur during this lag to alter the expression of the mRNA under examination. Similarly, in the knockout approach, the knockout cells may establish compensatory mechanisms to counteract the potential harmful effects from the RBP knockout, confounding the direct effects of the RBP on its target mRNAs.

To address these issues and to determine the immediate effects of an RBP on its target mRNAs, we adapted the dTAG system for the loss-of-function studies of RBPs. In this method, a small contrived protein dTAG (FKBP12-F36V-2HA, which is ∼14 kd in size) is fused in-frame with the target RBP. Thereby, the target RBP can be induced to rapid and specific degradation by a molecular “glue” (dTAG-13), a small chemical molecule that specifically binds to both the dTAG and a ubiquitous endogenous E3 ligase (Fig. 1A; Nabet et al. 2018). We applied this system to Trim71, a highly conserved stem cell-specific RBP. Independent transcriptome-wide binding studies revealed that Trim71 binds to the 3′UTR of Ago2 mRNA in mESCs (Welte et al. 2019; Liu et al. 2021a). Although overexpression of Trim71 leads to translational repression of Ago2 mRNA and decreased Ago2 protein levels, knockdown or knockout of Trim71 does not alter Ago2 protein levels in mESCs (Chang et al. 2012; Liu et al. 2021a). These inconsistent results make it puzzling to determine how the endogenous Trim71 regulates Ago2 mRNA in mESCs.

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FIGURE 1.

An inducible protein degradation system for Trim71. (A) Schematic depiction of the dTAG system for degrading Trim71. (B) Workflow for knock-in the dTAG degrader to the Trim71 locus. (C) Genotyping of the Trim71-dTAG mESCs using the two primers in (B). (D) Western blotting in the WT and Trim71-dTAG mESCs. Gapdh protein levels were used for normalization in protein quantification. (*) Nonspecific bands.

To clarify Trim71-mediated regulation of Ago2 mRNA, we knocked in the protein dTAG to the carboxyl terminus of Trim71 using genome editing (Fig. 1B) and identified biallelic dTAG knock-in mESCs (Fig. 1C). The knock-in dTAG does not alter the 3′UTR of Trim71 mRNA or Trim71's promoter, and the Trim71-dTAG is expressed at the endogenous level in mESCs (Fig. 1D). Different from the Trim71 knockout mESCs, which have defects in proliferation (Chang et al. 2012), the Trim71-dTAG mESCs proliferated at the same rate as the WT mESCs (Supplemental Fig. S1A). Moreover, the Trim71-dTAG mESCs behaved similarly to the WT mESC in self-renewal and differentiation, as assayed by the colony formation assay and the exit pluripotency assay, respectively (Supplemental Fig. S1B,C). These observations indicated that the Trim71-dTAG mESCs are phenotypically identical to the WT mESCs, and the dTAG does not abolish Trim71's function.

To determine how Trim71 protein degradation influences Ago2 mRNA expression, we examined mESCs cultured with dTAG-13, the molecular “glue” triggering Trim71-dTAG degradation. dTAG-13 induced efficient Trim71 degradation (Fig. 1D). At steady-state, consistent with previous studies of Trim71 knockdown/knockout (Chang et al. 2012; Liu et al. 2021a), the Ago2 protein levels were not altered in the Trim71-dTAG mESCs compared to those in the WT mESCs (Fig. 1D). Next, we performed the kinetic analysis by monitoring protein changes after the dTAG-13 treatment. dTAG-13 induced rapid degradation of Trim71, as at 4 h post-treatment, Trim71 protein levels already reduced substantially (Fig. 2A). Concomitantly, the Ago2 protein levels increased at 4 h and peaked at 8 h post-treatment (Fig. 2A,B). Interestingly, however, the Ago2 protein levels then gradually returned to the original levels despite the dramatically decreased Trim71 levels at later time points (e.g., 16, 24, and 48 h) (Fig. 2A,B). In the WT mESCs, where dTAG-13 did not cause Trim71 degradation, the Ago2 protein levels were not altered by the dTAG-13 treatment over the time course (Fig. 2A,B), indicating that the changes of Ago2 protein levels in the Trim71-dTAG mESCs are due to Trim71 degradation. The increased Ago2 at the early time points (e.g., 4 and 8 h) was not due to the potential inhibition of the global protein degradation system by the dTAG system, because when we applied the same dTAG approach to two other genes that are expressed at higher levels than Trim71, the dTAG-mediated rapid target protein degradation did not lead to increased Ago2 (Supplemental Fig. S2). Consistent with the previous finding that Trim71 represses Ago2 mRNA translation in mESCs (Liu et al. 2021a), Ago2 mRNA levels were not altered during the time course of dTAG-13 triggered Trim71 degradation (Fig. 2C), arguing that the increased Ago2 protein levels at the early time points (e.g., 4 and 8 h) are due to mRNA translational de-repression. Moreover, we performed the polysome profiling on the Trim71-dTAG mESCs harvested at 0, 8, and 24 h post dTAG treatment (Fig. 2D). We observed that the ribosome association of Ago2 mRNA first increased at the 8 h time point and then decreased at the 24 h time point. These changes of ribosome association correlate with the changes in Ago2 protein levels (Fig. 2A,B,D). Altogether, these results confirm that the endogenous Trim71 indeed represses Ago2 expression in mESCs.

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FIGURE 2.

Trim71-mediated repression of Ago2. (A) Western blotting in the WT and Trim71-dTAG mESCs at the indicated time points after dTAG-13 (500 nM) treatment. Gapdh protein levels were used for normalization in protein quantification. (B) Quantification of Ago2 protein levels in the WT and Trim71-dTAG mESCs at the indicated time points after dTAG-13 treatment. (C) Quantification of Ago2 mRNA levels in the WT and Trim71-dTAG mESCs at the indicated time points after dTAG-13 treatment. Gapdh mRNA was used for normalization in the quantification. The results represent the means (±SD) of three independent experiments. (D) Polysome analysis of the Trim71-dTAG mESCs with the dTAG-13 (500 nM) treatment at 0, 8, and 24 h. The target mRNA distribution in the polysome fractions was quantified by qRT-PCR. The quantifications from B to D represent the means (±SD) of three independent experiments. (*) P < 0.05 by one-way ANOVA.

In sum, we show that under a loss-of-function setting, Ago2 protein levels increase upon Trim71 degradation in mESCs. Our kinetic analysis upon rapid Trim71 degradation provides insights into the previous puzzling observations that knockdown/knockout of Trim71 under steady-state does not alter Ago2 levels, even though Trim71 binds to Ago2 mRNA and overexpressed Trim71 reduced Ago2 protein levels in mESCs. The different results from the kinetic analysis upon Trim71 degradation and the steady-state analysis in the Trim71 knockdown/knockout highlight a caveat in interpreting results from the traditional loss-of-function studies on RBPs: discriminating the primary effect versus the secondary effects. The dynamic changes of Ago2 levels (first increase and then go back to normal levels) upon Trim71 degradation (Fig. 2A) strongly argue that in this case (Trim71-mediated regulation of Ago2 mRNA in mESCs), the secondary effects mask the direct effects at the steady-state. Characterizing the specific secondary effects that counteract the direct effects is difficult because numerous RBP:mRNA interactions are disrupted in the loss-of-function approach. However, the time course analysis can discriminate the direct effects from the secondary effects, as the direct effects occur first and then come the secondary effects. Thus, the dTAG approach we applied on Trim71 can be used to determine the direct effects of other RBPs on their target mRNAs.