CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding

Abstract

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.

Figure 1. Overview of the SELEX procedure

(A) The 36 nucleotide invariant sequences of the random RNA library were those flanking the c-mos EDEN in the GbORF/mosEDEN RNA. The forward and reverse primers, used for the PCR amplification steps, hybridize to approximately 20 nucleotides of these invariant sequences. The forward primer has a T7 promoter overhang (underlined). (B) Coomassie blue staining of a sample of the recombinant CUG-BP1 used in the experiments (lane 2). Lane 1, molecular mass markers, sizes in kDa are given on the left.

Figure 2. SPR and EMSA evaluations of SELEX enrichment

The negative control [S0(1) RNA] (A), the positive control (c-mos EDEN RNA) (B), the initial random RNA population (S0 pool) (C) and the enriched RNA population (S8 pool) (D) were tested for their capacity to interact with recombinant CUG-BP1 by SPR (left-hand panels) and EMSA (right-hand panels). For SPR, RNAs were injected at 100 nM over a sensor chip containing approx. 11000 RU of immobilized CUG-BP1 (corresponding to 11 ng of CUG-BP1/mm²). The grey and black curves represent the first and the second injection of each RNA sample respectively. The end-point values (in RU) correspond to the response signal reached at the end of the RNA injections (t=120 s). For EMSA, uniformly labelled RNA probe was incubated with the following concentrations of CUG-BP1 (nM): lane 1, 0; lane 2, 0.5; lane 3, 2; lane 4, 8; lane 5, 31; lane 6, 125; lane 7, 500; lane 8, 2000. The free probe (F) and two complexes (C1 and C2) were resolved by native electrophoresis. The gel was dried and analysed using a phosphoimager and ImageQuant software. The concentrations (nM) of CUG-BP1 required to achieve 50% of shifted probe are indicated on the top of each gel (means±S.D. of three independent experiments).

Figure 3. EMSA analysis of individual aptamers

The indicated aptamers were analysed by EMSA for their capacity to bind CUG-BP1 as described in Figure 2. For the EMSA with the s3′Eg5 and s3′Eg5C6 RNAs the protein concentrations were (nM): lane 1, 0; lane 2, 1; lane 3, 2; lane 4, 4; lane 5, 8; lane 6, 17; lane 7, 33; lane 8, 67; lane 9, 133 and lane 10, 266. The concentrations (nM) of CUG-BP1 required to achieve 50% of shifted probe are indicated at the top of each gel (means±S.D. of three independent experiments). The positions of the free probe (F) and the complexes C1 and C2 are indicated on the right of each panel.

Figure 4. In vitro deadenylation assay of selected aptamers

(A) SELEX sequences [S8(2), S8(7), and S8(12)] and c-mos EDEN (mosEDEN) were cloned upstream of a synthetic (A)₆₅ tail in the 3′UTR of a β-globin reporter gene. Capped, in vitro synthesized radiolabelled transcripts were micro-injected into Xenopus embryos. At different time points, total RNA was extracted from batches of embryos and analysed by electrophoresis on a denaturing polyacrylamide gel. The positions of the fully adenylated (A)₆₅ and the fully deadenylated (A)₀ forms of the transcripts are indicated on the left of each panel. (B) Quantification of the experiments shown in (A). The percentage of (A)₀ RNA at each time point was calculated as described in the Experimental section.

Figure 5. CUG-BP1 binding sites are enriched in UGU trinucleotides

The number of UGU trinucleotides in the individual sequenced aptamers of the S0 pool and families 1 and 2 are shown as a histogram. Family 1, grey bars; family 2, black bars; S0 pool, white bars.

Figure 6. XCl100α maternal mRNA is a target for EDEN-BP and is deadenylated after fertilization

(A) Maternal RNA isolated from unfertilized eggs (0 h) or 2 h and 4 h after fertilization was separated into poly(A⁺) and poly(A⁻) populations and analysed by Northern blotting. XCl100α was revealed using a ³²P-labelled probe as described in the Experimental section. RNA loading was 1 and 11 embryo equivalents for total RNA and poly(A⁺) or poly(A⁻) RNAs respectively. (B) ³²P-labelled RNAs containing the globin ORF with or without the c-mos EDEN, respectively gbORF and gbORFmos, and the UGU rich region of XCl100α 3′UTR were incubated in Xenopus egg extracts and processed for UV-induced cross-linking. After separation by SDS/PAGE the proteins that had become radiolabelled were revealed by phosphoimager analysis. The positions of molecular-mass markers (kDa) and of EDEN-BP are indicated on the right. (C) Xenopus 2-cell embryos were injected with a reporter mRNA containing the XCl100α 3′UTR. At the indicated times, samples were taken for extraction and analysis of the RNA. After separation by electrophoresis on denaturing gels the radiolabelled RNAs were revealed by phosphoimager analysis. The position of the fully adenylated (A⁺) and deadenylated (A⁻) RNAs are indicated on the left. Lane M, RNA markers of 888 and 715 nucleotides; lane A⁻, the reporter RNA containing the XCl100α 3′UTR synthesized devoid of a poly(A) tail.