Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation

Abstract

RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity.

Graphical abstract

Figure 1

Structural and Sequence Context of the GGAG Motif Determine Lin28a Binding and Functionality (A) Schematic of the secondary structure of wild-type and conserved terminal loop (CTL) mutants of pri-let-7a-1. The mutated nucleotides are in green, and the GGAG motif is in red. (B) Western blot (WB) analysis of Lin28a and DHX9 proteins in RNA pull-downs from day 0 (d0) P19 teratocarcinoma cell extract using wild-type pre-let-7 or its CTL mutants. (C) In vitro processing uridylation assays performed with internally radiolabeled pre-let-7a transcripts (50 × 10³ cpm, approximately 20 pmol) in the presence of d0 P19 cell extract. (−) represents an untreated control. Reactions were supplemented with 0.25 mM UTP. The products were analyzed on an 8% denaturing polyacrylamide gel. (D) Schematic of the secondary structure of wild-type pri-miRNA-363 and its CTL mutant. (E) Western blot analysis of Lin28a protein in RNA pull-downs from day 0 P19 cell extract using wild-type pre-miRNA-363 or mutant pre-miRNA-363@1. (F) In vitro processing uridylation assays performed with internally radiolabeled pre-miRNA-363 transcripts (50 × 10³ cpm, approximately 20 pmol) in the presence of d0 P19 cell extracts. (−) represents an untreated control. Reactions were supplemented with 0.25 mM UTP. The products were analyzed on an 8% denaturing polyacrylamide gel.

Figure 2

Lin28a Inhibits Pri- and Pre-let-7 Processing, but the Efficiency Depends on Fine Features of the Let-7 CTL (A) Northern blot analysis with a probe for let-7a of total RNA from HeLa cells transfected with only a pCG plasmid coding for pri-let-7a-1 or one of its CTL mutants or cotransfected with a pri-let-7 plasmid and pCG T7-Lin28a. (B) As a loading control, ethidium bromide (EB) staining of the polyacrylamide gel before blotting is shown.

Figure 3

SILAC Combined with RNA Pull-Down and Mass Spectrometry Reveals Putative Cofactors for Lin28a-Mediated Uridylation (A) Schematic of the method. P19 cells or HeLa cells overexpressing Lin28a were grown in “light” medium containing 12C6-arginine and 12C6-lysine or in “heavy” medium containing 13C6-arginine and 13C6-lysine. Next, RNA pull-down was performed with either agarose beads incubated with extract from light HeLa cells or beads with wild-type pre-let-7a-1 or its CTL mutants covalently linked incubated with extract from heavy HeLa cells. After thorough washing, the resulting supernatants were mixed and subjected to quantitative mass spectrometry. (B) Venn diagrams representing numbers of overlapping proteins identified in the pull-downs with wild-type pre-let-7a-1, pre-let-7a-1@2, and pre-let-7a-1@3. (C) Proteins identified exclusively in the pre-let-7a-1 and pre-let-7a-1@2 pull-downs. (D) Western blot (WB) analysis of Trim25, TuT4, and Lin28a proteins in RNA pull-downs from extract of HeLa cells overexpressing Lin28a using wild-type pre-let-7 and its CTL mutants @2 and @3. (E) Quantification of the results presented in (D). The band intensities were calculated with ImageJ software and were normalized to 100% based on the wild-type pre-let-7a-1 pull-down. (F) Immunofluorescence staining of Hoechst (blue), Lin28a (green), and Trim25 (red) in P19 cells showing colocalization of Lin28a and Trim25 predominantly in the cytoplasm. Scale bar, 10 μm. (G) Coimmunoprecipitation between Trim25 and Lin28a was performed with cell extracts prepared from HeLa cells overexpressing T7-Lin28a and V5-Trim25. The extracts were incubated with anti-T7 or control immunoglobulin G (IgG) antibody bound to protein-A beads. The bound proteins were analyzed by western blotting with anti-Lin28a or anti-V5. To control for directionality of the interaction, the assay was also performed in the presence of RNase.

Figure 4

Trim25 Is a Positive Cofactor for the Lin28a-Mediated Uridylation of Pre-let-7 (A) Western blot analysis of Trim25, TuT4, Lin28a, and α-tubulin proteins in protein extracts from P19 cells depleted of Trim25 or FAM114A1 using RNAi. Serial dilutions of the total protein extracts provide estimates of the linearity and limit of detection of the western blot assay. (B) In vitro processing assays with internally radiolabeled pre-let-7a-1 transcripts (50 × 10³ cpm, approximately 20 pmol) in the presence of mock-, Trim25-, or FAM114A1-depleted P19 cell extract. Trim25 RNAi_1 and Trim25 RNAi_2 represent results from treatment with two different siRNA sets. The reactions were supplemented with 0.25 mM UTP. (C) Real-time quantitative RT-PCR of miRNAs (let-7a, miRNA-302a, and miRNA-9) in P19 cells depleted of Trim25 or FAM114A1. The values were normalized to miRNA-16 levels. The fold change in the abundance miRNAs mediated by RNAi was plotted relative to values from a mock-transfected control, which were set to one. Mean values and SDs of three independent experiments are shown. (D) Real-time quantitative RT-PCR of pre-miRNAs (pre-let-7a, pre-miRNA-302a, and pre-miRNA-9) in P19 cells depleted of Trim25 or FAM114A1. The values were normalized to pre-miRNA-16 levels. The fold change in the abundance of the corresponding miRNA mediated by RNAi was plotted relative to values from a mock-transfected control, which were set to one. Mean values and SDs of three independent experiments are shown. (E) Western blot analysis of ubiquitinated proteins in protein extracts from P19 cells treated with DMSO or 50 μM PYR-41 for 4 hr. As a loading control, Coomassie blue stain is shown. (F) Western blot analysis of TuT4 in total extracts from P19 cells treated with DMSO or 50 μM PYR-41 for 4 hr. (G) In vitro processing assays with internally radiolabeled pre-let-7a-1 transcripts (50 × 10³ cpm, approximately 20 pmol) in the presence of P19 cell extract treated with DMSO or 50 μM PYR-41 for 4 hr.