The Elav-like proteins are specific mRNA binding proteins which are required for cellular differentiation. They contain three characteristic RNP2/RNP1-type RNA binding motifs. Previously we have shown that the first and second RNA binding domains bind to AU-rich elements in the 3′-UTR of mRNA. In this paper we show that the Elav-like proteins exhibit poly(A) binding activity. This activity is distinct from poly(A) binding activities that have been previously described. The Elav-like proteins specifically bind to long chain poly(A) tails. We have shown that the third RNA binding domain encompasses this poly(A) binding activity. Using poly(A)-Sepharose beads in a ‘sandwich’ assay we have shown that the Elav-like proteins can bind simultaneously to the AU-rich element and to the poly(A) tail.
The Elav-like proteins are a group of closely related RNA binding proteins that were first described in Drosophila (1). Recently, Elav-like genes have been cloned from higher organisms (2–8). There are four members of the human family, HuD, HuC, Hel-N1 and HuR (3–5,7). HuR is expressed at the RNA level in all proliferating cells, whereas HuD, HuC and Hel-N1 are normally expressed on terminal differentiation of neurons (7,9,10). The human members of this family are of particular interest since they are tumor antigens associated with a wide variety of human tumors. (5,11,12). The Elav-like proteins contain three highly conserved RNA recognition motifs (RRM). Two of these RRMs are in tandem and are separated from the third by a basic segment (3,4,7,13). A significant insight into the mechanism of action of these proteins was provided by the observation that they specifically bind to AU-rich elements in the 3′-untranslated region (UTR) of mRNA. (7,13–17). These AU-rich elements were originally characterized by Shaw and Kamen, who were the first to show that the AU-rich element in the 3′-UTR of GM-CSF mRNA regulates its expression at the post-transcriptional level (18). The current model is that the AU-rich elements are recognized by a specific endonuclease, which cleaves the transcript and renders it acessible to an exonuclease (19). Thus, mRNAs that contain these elements have a very short half-life and are usually present at a very low steady-state level. The level of these mRNAs can be dramatically increased by factors that also bind to the AU-rich elements and inhibit the degradative activity. Recent evidence has indicated that the Elav-like proteins are such factors and selectively inhibit the decay of mRNAs that contain AU-rich elements (20–23). In previous studies we have shown that the first and second RRMs of HuD and HuR bind specifically and with high affinity to AU-rich elements (7,16). In this paper we show that these Elav-like proteins also bind to the poly(A) tail of mRNA. This is mediated by the third RRM and may promote an interaction between AU-rich elements and the poly(A) tails of mRNA.
Materials and Methods
HuR, HuD and deletion mutants of HuD were purified as GST fusion proteins as previously described (7,16). Poly(A)-Sepharose 4B and Sepharose 4B were from Pharmacia. BA 85 nitrocellulose filters were from Schleicher & Schuell. RNase T1 was obtained from Calbiochem. Poly(A), poly(G), poly(U) and poly(C) were from Sigma.
Preparation of RNA transcripts
RNA transcripts were synthesized from plasmid DNA using [α-32P]ATP or [32P]GTP and were gel purified before use (16). The specific activity of these transcripts is expressed as c.p.m./pmol nucleotide. The 3′ myc and 3′ myc(A)87 transcripts were derived from AflII and HindIII digests of pMycSD3, a gift of Dr Gary Brewer (24). The 3′ myc(I) transcript was derived from a SspI digest of pMycSD3. The 3′ myc(II) transcript was derived from an AflII digest of pMycSD3/Δ5. pMycSD3/Δ5 was created by deletion of an EcoRI-ApoI fragment from pMycSD3. The (A)87 transcript was derived from a HindIII digest of pSD3, also a gift of Dr Gary Brewer. Poly(A)600av was hydrolyzed with alkali and end-labeled using T4 kinase and [γ-32P]ATP to a specific activity of 1 × 106 c.p.m./pmol ends and gel purified.
RNase T1 selection assay
Reaction mixtures (0.02 ml) contained 50 mM Tris, pH 7.0, 150 mM NaCl, 0.25 mg/ml BSA, 0.25 mg/ml tRNA, radiolabeled RNA and purified HuR as indicated. After 10 min incubation at 37°C, RNase T1 (5 U) was added and the reaction continued for a further 10 min. The mixtures were diluted 1:6 with buffer F (20 mM Tris, pH 7.0, 150 mM NaCl) and filtered through nitrocellulose (BA 85; Schleicher & Schuell). After washing the nitrocellulose twice with buffer F, the bound RNA was eluted by phenol/chloroform extraction. The resultant RNA was mixed with formamide buffer, denatured at 65°C for 3 min and analyzed by 12% polyacrylamide-urea gel electrophoresis. The gel was fixed with 1:1:8 acetic acid:methanol:water, dried on DE-81 paper with a backing of gel drying paper and exposed to the XAR5 film at −70°C overnight.
Nitrocellulose filter binding assay
Reaction mixtures (0.02 ml) contained 50 mM Tris, pH 7.0, 150 mM NaCl, 0.25 mg/ml BSA, 2.5 µg/ml tRNA, labeled RNA and purified protein as indicated. After 10 min incubation at 37°C the mixtures were diluted 1:6 with buffer F and filtered through nitrocellulose (BA85; Schleicher & Schuell). After washing the filter twice with buffer F, bound radioactivity was determined by Cerenkov counting. Each point is corrected for the amount of RNA bound in the absence of protein, which is usually <1% of the input.
The Elav-like proteins bind to AU-rich elements and to poly(A)
We have previously shown that the human Elav-like proteins bind specifically to the 3′-UTR of c-myc mRNA (7,16). Using RNase T1 selection analysis we discovered that the 3′ myc mRNA contains two independent HuR binding sites that we have labeled AU1 and AU2 (see Fig. 1). The two sites were confirmed by RNase T1 selection analysis of transcripts 3′ myc(I) and 3′ myc(II) (Fig. 1). In view of the relationship betweeen mRNA decay and polyadenylation (25), we decided to examine interaction of the Elav-like proteins with a polyadenylated transcript. Using pMycSD3 and [32P]GTP we synthesized a c-myc transcript that contained a poly(A) tail of 87 nt. The HuR binding sites on this transcript were assayed by the RNase T1 selection assay (7). As expected, HuR bound specifically to the AU1 and AU2 sites. (Fig. 2A, lanes 1–4). We were surprised, however, to observe that a larger band was also selected (Fig. 2A, lanes 3 and 4). This band was the same size as the (A)87 fragment present in the total RNase T1 digest of the transcript [the (A)87 fragment is labeled by virtue of a G residue in the restriction site at the end of the template DNA]. Thus we concluded that HuR exhibits a poly(A) binding activity. This activity was intrinsic to the HuR protein, as no fragments were selected by high concentrations of GST. We next determined whether HuR could directly bind to poly(A). Using the pSD3 plasmid (see Fig. 1) we synthesized the (A)87 tail itself. To increase the sensitivity of the assay we labeled the transcripts with [32P]ATP. Figure 2B shows that HuR bound to (A)87, even in the absence of the AU1 and AU2 sites. Thus HuR has an independent poly(A) binding activity. It is important to point out that HuR exhibits a significantly lower affinity with (A)87 than with the AU1 and AU2 elements.
Characterization of the poly(A) binding activity
Next we investigated the properties of the poly(A) binding activity in more detail. We were surprised to observe that HuR did not bind to poly(A)30. Thus we investigated whether HuR had a requirement for longer poly(A) tails. End-labeled poly(A) of uniform size distribution was prepared and incubated with HuR or GST. The bound poly(A) was selected by nitrocellulose filtration and analyzed by gel electrophoresis. Figure 3 shows that binding was first detectable with poly(A)80, was half-maximal with poly(A)180 and saturated at poly(A)300. This result was confirmed by purification of poly(A) of defined size and determination of their binding affinity. As in previous studies, the interactions between the Elav-like proteins and RNA were quantitated using a nitrocellulose filter binding assay (7,26). A low concentration of labeled RNA was incubated with increasing concentrations of HuR. The reaction mixtures were filtered through nitrocellulose and the bound radioactivity determined. As predicted from the selection analysis, virtually no complex was formed with poly(A)30 (Fig. 4A). Increasing reactivity was observed with poly(A)100, poly(A)150 and poly(A)200, reaching saturation at poly(A)300. A plot of log[complex/free poly(A)] versus log(HuR concentration) revealed a straight line in each case (Fig. 4A). This suggested a simple interaction with little cooperativity. The affinity of HuR for poly(A) is significantly less than that for the AU-rich elements. This is quantitatively shown in Figure 4B. The apparent Kd for 3′ myc is 4 nM, whereas the apparent Kd for poly(A)300 is 146 nM. As before, the intrinsic nature of these activities is indicated by the lack of reactivity with GST (Fig. 4B). There is also little difference in binding between 3′ myc and 3′myc(A)87 (Fig. 4B). Thus the AU-rich element is the primary determinant of HuR binding. Next we examined the specificity of the poly(A) binding activity. We investigated whether the HuR-poly(A) complex could be displaced by other homopolymers. Figure 4C shows that the HuR-poly(A)87 complex was displaced by poly(A)600 (50% displacement at 0.2 molar excess) and to a lesser extent by poly(G)600 (50% displacement at 0.8 molar excess). Neither poly(U)600 (50% displacement at 32 molar excess) nor poly(C)600 (no displacement at 1000-fold molar excess) significantly displaced the HuR-poly(A)87 complex.
The poly(A) binding activity is resident in the third RNA binding domain
The most striking and unique structural feature of the Elav-like family of proteins is the presence and organization of the three putative RNA recognition motifs (Fig. 5). In each case the two tandemly arranged RNA recognition motifs are connected to the third RNA recognition motif by a highly basic segment that we have termed the ‘basic segment’. We have previously shown that the first and second RRMs of HuD are necessary and sufficient for binding to AU-rich elements (16). We now sought to establish which domains are involved in poly(A) binding activity. We used the mutant constructs derived from HuD (16; Fig. 5). HuD bound to the ARE and poly(A) tail of 3′ c-myc(A)87 (Fig. 5, lane 2). The first and second RNA binding domains (HuD I,II) did not bind to the poly(A) tail. Although HuD I,II does bind to AU-rich elements, it does so with significantly lower affinity (the apparent Kd for HuD is 16 nM, whereas the apparent Kd for HuD I,II is 125 nM) (16). Thus, at the concentration used here, little or no binding to the AU1 and AU2 elements was anticipated. In contrast, the third RNA binding domain (HuD III) bound avidly to the poly(A) tail (Fig. 5, lane 4). Thus we concluded that the first and second RNA binding domains interact with AU-rich elements whereas the third RNA binding domain interacts with the poly(A) tail.
The Elav-like proteins bind simultaneously to the AU-rich element and the poly(A) tail of mRNA
Next we investigated whether the Elav-like proteins can contact both sites simultaneously. To examine this, we bound HuR to poly(A)-Sepharose beads (HuR/pA-S4B beads), removed unbound HuR by washing and then examined the ability of the beads to bind the AU-rich element. Figure 6 shows that the labeled 3′ myc transcript bound to poly(A)-Sepharose beads preincubated with HuR (HuR/pA-S4B beads) but not to poly(A)-Sepharose beads preincubated with GST (GST/pA-S4B beads). The amount of transcript bound increased with increasing concentration of HuR. The labeled 3′ myc transcript did not bind to Sepharose beads that were preincubated with HuR (HuR/S4B beads). This is a difficult experiment to perform since the off-rate of the HuR-poly(A) complex is fast. Thus the demonstration that 10% of the myc transcript can be simultaneously bound at saturating HuR concentration is significant. We concluded that the Elav-like proteins can form a bridge between the poly(A) tail and the AU-rich element.
The Elav-like proteins, HuD, HuC, Hel-N1 and HuR, stabilize specific mRNAs via an interaction with AU-rich elements in their 3′-UTR (7,14–17,20–22). In this paper we have shown that HuD and HuR proteins have an additional property, namely a novel poly(A) binding activity. Recently (after this paper was submitted for publication) the HuC protein was also shown to have poly(A) binding activity (27). Thus this activity is probably a feature of all Elav-like proteins. This activity may not have been detected in previous studies of Hel-N1 since poly(A) was used as a non-specific competitor (17,28). The properties of the Elav-like poly(A) binding activity are quite different from other poly(A) binding proteins. The cytoplasmic poly(A) binding protein (PABI) binds to (A)10 and the Kd does not significantly change with increased chain length (29). The nuclear poly(A) binding protein (PABII) binds to an oligo(A) tail >10–11 nt and remains associated [in a complex with poly(A) polymerase] until the tail is elongated to a length of 250 nt (30). Thus these activities have a minimal binding site of 10–15 nt, whereas the Elav-like proteins prefer polymers >70 nt. The affinity of the Elav-like proteins for poly(A) is relatively low [an apparent Kd of 146 nM for (A)300] compared with a Kd of 5 nM for PABI (29,31) and a Kd of 2 nM for PABII (32). The Elav-like poly(A) binding activity is, however, similar to both PABI and PABII in that its activity is displaced by a molar excess of poly(A) and poly(G) but not efficiently by poly(U) or poly(C). The observation that the Elav-like proteins preferentially bind to long polymers of poly(A) is surprising. Typically, proteins exhibit increased binding to polymers as a result of protein-protein interactions. This does not appear to be the case with the Elav-like proteins. There is no obvious cooperativity in their interaction with either short or long chain poly(A). Thus the possibility remains that the third RNA binding domain recognizes a secondary or tertiary structure that is only evident in long chain poly(A).
The Elav-like proteins contain three RNA binding domains. Previous studies have shown that the third RNA binding domain is not required for AU-rich element binding (16). This domain is crucial, however, since mutation of Gly426 to glutamic acid in Drosophila Elav leads to a temperature-sensitive phenotype (33). We have shown here that the third RNA binding domain contains the poly(A) binding activity. The bifunctionality of the Elav-like proteins is not surprising. The first and second RNA binding domains are much more closely related in sequence to the corresponding domains among different organisms than to the third RNA binding domain (2,5–7,13). This supports the notion that they have different functions.
The demonstration of a poly(A) binding activity resident in the third RNA binding domain of the Elav-like proteins has important implications for understanding their mechanism of action. Our current model is that binding of the Elav-like protein inhibits the action of a specific endonuclease that recognizes the AU-rich element. It is possible that effective inhibition of the endonuclease may require association of the Elav-like proteins with both the AU-rich element and the poly(A) tail. This would ensure that deadenylated message would not be stabilized. Alternatively, the Elav-like proteins may sequester the poly(A) tail of a target mRNA, with a consequent inhibitory effect on a poly(A) exonuclease activity. The poly(A) mRNA-protein complex described here and the recent development of an in vitro system that recapitulates the regulated turnover of mRNA (21) may provide a way to answer these questions.
We thank Barry Nevins for his patience in preparing the manuscript. This work was supported by the NIH (NS29682), The Byrne Fund and an NCI core grant (P30-CA08748).