In this study, we have identified a conserved three-dimensional RNA motif, present in the four ASH1 localization elements and in two other bud-localized transcripts, that is essential for She2p recognition and for the localization function of these elements. An in vivo selection approach, using partially randomized libraries of the localization element RNAs coupled with a modified yeast three-hybrid assay, led to the identification of the nucleotides essential for She2p binding in each element. While the interactions detected by the modified three-hybrid assay are indirect (since we used the C-terminal domain of She3p as the bait), we have also shown that more direct assays (like using She2p as the bait or using RNA band-shift assays) did not provide comparable readout and sensibility to measure the effect of mutations in the ASH1 localization elements on their interaction with She2p.
This approach has been vindicated by the consistent results obtained from the screens, where a motif consisting of a CGA triplet and a single conserved cytosine in two loops separated by a short stem was found to be the only conserved feature among the localization elements. Mutation of this motif resulted in a decreased interaction with She2p and loss of the localization function of the elements. Interestingly, a distance of ≈28 Å between the two cytosines must be maintained in order to preserve the interaction with She2p, suggesting that three-dimensional features in these RNAs are essential for She2p recognition. However, our experimental results cannot determine if it is the distance between the cytosines or the spatial orientation of these residues or both that are important, since any insertion or deletion within the helix that separates the cytosines will affect both.
Our results suggest that the RNA motif identified contains the main specificity determinants of the She2p binding domain. These determinants are highly conserved in three of the four ASH1 localization elements (E1, E2A, and E2B). For the element E3, several features of the She2p binding RNA motif are conserved: the two essential cytosines on opposite strands, separated by a stem and at a distance of 28 Å from each other. However, variations from the classic motif are present in this element: one of the loops is replaced by a single bulged cytosine and the GA of the CGA triplet is not essential. In this case, a particularity of this localization element, a single cytosine bulging from a stem instead of being incorporated in a loop, may result in a conformation where She2p binding becomes less dependent on the GA of the CGA triplet. Interestingly, among the variants isolated for the element E1, we found three variants with mutations in the GA of the CGA triplet (mutants 8, 26, and 43). These variants can be folded like the element E3 (with a stem of 5 or 6 base pairs between the two conserved cytosines; data not shown), suggesting that the GA of the CGA triplet may be less important for She2p binding in some structures.
While these determinants are essential for She2p recognition, other elements around this RNA motif have been found to be important. For instance, the stems around the internal loops of the elements E1, E2A, and E2B-D1 were also shown to be important for She2p binding, possibly by participating in the proper folding of these RNAs. From the randomized libraries, we also identified highly conserved nucleotides in each individual element that were not present in the other three elements (see Fig. ). The nucleotides surrounding the highly conserved CGA triplet, present in all four localization elements, provide such example (see the elements E1 versus E2B-D1). These nucleotides may be involved in noncanonical base pairs and may improve the accessibility of the single cytosine or of the CGA triplet for She2p binding in the particular context of a given RNA structure. Overall, our results suggest that the She2p-binding motif requires a set of conserved specificity determinants around which several nucleotide sequence combinations are tolerated only if they maintain the proper folding of this motif.
Recently, more than twenty new mRNAs have been found to be specifically localized at the bud tip of yeast cells during mitosis (38
). The localization of these mRNAs depends on Myo4p, She3p, and She2p, suggesting that the localization pathway of the ASH1
mRNA is not restricted to this transcript. No specific zipcodes have yet been characterized in any of these transcripts, but since they were identified by coimmunoprecipitation with She2p, it is highly probable that they all contain She2p-binding domains similar to the one found in the ASH1
zipcodes. Indeed, using the She2p binding RNA motif in a computer search, we have been able to identify functional localization elements in two of these transcripts: IST2
. Ist2p is a membrane protein targeted to the plasma membrane via a new trafficking pathway that requires the localization of its mRNA to the daughter cell cortex (21
). The function of YMR171c
is still unknown. The identification of the same RNA motif in the localization elements of the bud-localized ASH1
, and YMR171c
mRNAs strongly supports our conclusion that this motif contains the main determinants required for their recognition by She2p.
These results raise questions about how She2p may recognize these determinants. The recent publication of the three-dimensional structure of She2p revealed that this protein acts as a homodimer and that a series of basic residues essential for the interaction between this protein and the ASH1
mRNA localization elements are clustered in a specific region that folds as a basic helical hairpin (30
). Interestingly, this region of the protein, from R44 to K57, covers a distance of around 27 Å in length (data not shown), which is very close to the conserved 28 Å distance between the CGA triplet and the single cytosine of the She2p-binding motif. Therefore, we can speculate that this RNA motif may dock into the basic helical hairpin, where amino acid residues at the extremities of this RNA binding motif (R43, R44, K57, and K60) may interact with the conserved CGA triplet and the single cytosine of the RNA motif, while basic residues in the middle (R52 and R63) may interact with the phosphate backbone of the helix that separates the two loops.
While the presence of two RNA-binding domain per homodimer would suggest that two RNA molecules could bind the homodimer (2:1 ratio), Niessing et al. have shown instead that a She2p homodimer binds only one RNA molecule (1:1 ratio) (30
). They proposed that the RNA molecule arches over the upper, uncharged surface of the She2p homodimer and binds simultaneously to the RNA-binding domain of both monomers. However, our electrophoretic mobility shift assays suggest the presence of both 1:1 and 2:1 ratios of RNA zipcode versus She2p homodimer. The ratios observed vary with the localization element (E2B-D1 has only one shifted species while E2A has two shifted species of equivalent intensities), and may depend on the size of the RNA zipcode used in the electrophoretic mobility shift assay (the E2B-D1 RNA has 75 nucleotides while the E2A RNA has 103 nucleotides).
The low level of sequence conservation and the dependence on secondary and tertiary structure of their target RNA is a common feature found in RNA-binding proteins involved in the transport of several different mRNAs within the same cell. For instance, in Drosophila
, the K10
, and hairy
mRNAs are localized during oogenesis and in blastoderm embryos by the Egalitarian (Egl) and Bicaudal-D (BicD) localization machinery (5
). The localization of these transcripts depends on zipcodes containing short stem-loop structures without sequence similarities (6
), suggesting that structural features in these RNAs are recognized by the Egl-BicD localization pathway (6
). Another example is Staufen, an RNA-binding protein containing double-stranded RNA-binding domains, which interacts with the localized mRNAs bicoid
, and prospero
mRNAs in neurons (28
). Although Staufen interacts with specific mRNAs in vivo, it binds nonspecifically to double-stranded RNA in vitro (18
). Our study show that mRNAs with zipcodes lacking primary sequence similarity can rely on a few conserved nucleotides properly oriented in their three-dimensional structure in order to be recognized by the same localization machinery.