Abstract
mRNA localization is an essential mechanism of gene regulation and is required for processes such as stem-cell division, embryogenesis and neuronal plasticity. It is not known which features in the cis-acting mRNA localization elements (LEs) are specifically recognized by motor-containing transport complexes. To the best of our knowledge, no high-resolution structure is available for any LE in complex with its cognate protein complex. Using X-ray crystallography and complementary techniques, we carried out a detailed assessment of an LE of the ASH1 mRNA from yeast, its complex with its shuttling RNA-binding protein She2p, and its highly specific, cytoplasmic complex with She3p. Although the RNA alone formed a flexible stem loop, She2p binding induced marked conformational changes. However, only joining by the unstructured She3p resulted in specific RNA recognition. The notable RNA rearrangements and joint action of a globular and an unfolded RNA-binding protein offer unprecedented insights into the step-wise maturation of an mRNA-transport complex.
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Acknowledgements
We thank V. Roman for her support, and M. Seiler and M. Feldbrügge for helpful discussions. We acknowledge the use of the X-ray crystallography platform of the Helmholtz Zentrum München, NMR measurements at the Bavarian NMR center and SAXS measurements at the facility of the SFB1035, Technische Universität München. This work was supported by the Deutsche Forschungsgemeinschaft (SFB1035 to M.S.; SPP1935 to M.S. and D.N.; FOR2333 to R.-P.J. and D.N.; SFB646 to D.N.) and by the Bayerisch-Französisches Hochschulzentrum (BFHZ) to D.N.
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F.T.E., A.S., R.G.H., A.J., A.N.-B., M.I.S. and R.S. conducted the experiments. F.T.E., A.S., R.G.H., R.-P.J., J.-C.P. and D.N. designed the experiments. F.T.E., A.S., R.G.H., A.N.-B., R.S., R.J., M.S., R.-P.J. and D.N. analyzed the data. F.T.E. and D.N. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Assessment of synergistic RNA recognition by the She2p-She3p complex in electrophoretic mobility shift assays (EMSA).
(a) E3-(51 nt) LE was previously shown to mediate full synergistic She2p-She3p binding and is incorporated into motile particles (Heym, R.G. et al., J Cell Biol. 203, 971-84 (2013); Müller, M. et al., PLoS Biol. 9, e1000611 (2011)). The single-stranded regions at the 5’ and 3’ ends of E3 (51 nt) relate to a large bulge in the context of full-length ASH1 mRNA (Fig. 1a). Deletion of either the 5’end or part of te 3’ regions in the E3-(51 nt) mutants Δ1 and Δ2, respectively, resulted in wild-type binding. In contrast, a complete deletion of the single-stranded bases at the 3’ end abolished binding (Δ3). The results indicate that bases 1812-1814 are indispensable. Final She2p concentrations were 0.02 μM, 0.06 μM, 0.18 μM, 0.54 μM, 1.61 μM and 4.86 μM. (b) A deletion of the upper part of the stem and of the nona-loop (nt 1786-1802) combined with an insertion of a more compact tetra-loop (38 nt-loop) still allowed for high-affinity binding by She2p and She3p. She2p concentrations were 0.01 μM, 0.03 μM, 0.10 μM, 0.30 μM, 0.90 μM and 2.70 μM. (c) Comparative EMSAs of She2p wild type and a cystein-mutated, N-terminally truncated She2p version (She2p(6-246, C-S)). The latter shows wild type-like ternary complex formation with She3p and the E3-(51 nt) RNA. Each EMSA is representative for three independent experiments.
Supplementary Figure 2 NMR analysis of the free RNA in solution.
(a) Agarose gel with the denatured RNA sample before crystallization and after extraction from crystals indicates no degradation, suggesting that the lack of electron density in this region is due to structural flexibility. (b) 2D-imino NOESY spectra of ASH1-E3 (28 nt-loop) and E3-(42 nt TL-TLR) RNAs showing the sequentially assigned observable imino protons. Resonance labels are color-coded as indicated in Fig. 1f. Solid lines in the scheme represent unambiguous hydrogen-bonds as obtained from the sequential assignments. The broken line for G1781-C1805 in the 42-mer indicates an assignment of the G1781 imino proton inferred by exclusion, although no cross peak to adjacent nucleotides was observed. Overall the spectra are consistent with the existence of the base pairing observed in the crystal structure (Fig. 1d,e) also in solution. However, significant line broadening of imino resonances and undetectable imino signals in the bulged region between H1 and H2 indicate dynamics and flexibility in this part of the RNA. Bases marked with apostrophe indicate heterologous GAAA tetra-loop containing sequences, not belonging to the E3 LE. Bases highlighted with red boxes show peak broadening in the 1D imino spectrum indicating dynamics.
Supplementary Figure 3 Assessment of She2p-RNA structure.
(a-e) She2p binding of She3p(382-405) is not physiologic. Native crystals of She2p with RNA diffracted best when soaked with a short She3p peptide (residues 382-405). (a) In the She2p-RNA co-structure (Fig. 2a) additional electron density was observed that could not be assigned to She2p or RNA. The location of this not well-ordered electron density, however, overlaps with a previously reported interaction site of She3p (364-368) (Singh, N. et al., Proc Natl Acad Sci U S A. 112, 142-7 (2015)). (b) The corresponding residues from fragment 382-405 were modeled into electron density (398-405, green) and compared to the previously published peptide 363-368 (red). Interactions were similar but not identical. (c) A sequence comparison of the previously described interaction motifs of She3p(364-368) (grey box) and of She3p(382-405) revealed similarities. (d) Crystal structure of She2p(6-246, C-S) with the ASH1-E3 (28 nt-loop) RNA at 2.41 Å resolution in front view and rotated 90° around the vertical axis. Non-physiologically bound She3p fragments are depicted in green. (e) Isothermal titration calorimetry (ITC) experiments with She2p and She3p(382-405) (top) showed no binding even at final She3p concentrations of 157 μM in three independent experiments. Since the positive control (She3p residues 364-368) (Singh, N. et al., Proc Natl Acad Sci U S A. 112, 142-7 (2015)) showed binding to She2p (bottom) in two independent experiments, we conclude that the electron density of She3p(382-405) in the co-structure with She2p and RNA is non-physiologic. (f) Close-up of interactions in the kinked region of E3 RNA in the She2p-bound state. Watson-Crick base pairings, hydrogen bonding, base stacking, and water interactions are shown. For better visualization, She2p is hidden. (g) Superposition of the two RNA chains E3 (pink) and E3’ (grey) observed in the She2p-bound form. The sole difference is visible at the very 3’ base (RMSD of all atoms = 0.5 Å).
Supplementary Figure 4 Comparison of free RNA with She2p-bound RNA in solution by small angle X-ray scattering.
(a) Overlay of the RNA models from the E3-(42 nt-TL-TLR) RNA crystal structure (green), where the heterologous GAAA-donor acceptor has been replaced in silico by a shorter GAAA tetra-loop (grey), and of the E3 (28 nt-loop) RNA from the She2p-She3p bound complex (magenta). Strong rearrangements in the RNA secondary structure pinpoint to a large conformational change upon complex formation (see also Fig. 2c). (b-d) Different RNA models (red) are fitted against the scattering curve of E3-(28 nt-loop) RNA recorded at 1.5 mg/ml concentration in single experiments. (b) The calculated scattering curves for both the bound (in (c): pink, kinked) and unbound (in c: green, elongated) RNAs, assuming that both conformations could be present (with fitted fractions kinked RNA: elongated RNA = 0.72: 0.27) does not fit well to the scattering curve especially at low (0.03 Å-1) and high (0.3 Å-1) q values, which is also reflected by the high χ2 value of 7.6. (c) When testing a kinked and a melted, single-stranded RNA model (ssRNA) (depicted in blue; ratio kinked RNA: ssRNA = 0.74: 0.26) the fit becomes better and χ2 decrases to 3.9. (d) Assuming that an elongated and a single-stranded species (ratio elongated RNA: ssRNA = 0.65: 0.34) exists in solution, the χ2 value improves to 1.7. (e) The most accurate fit could be achieved by fitting a combination of the kinked, single-stranded and elongated RNA (ratio kinked: ssRNA: elongated = 0.12: 0.33: 0.55). Here χ2 was 1.6. SAXS data support the observation that the E3-(28 nt-loop) RNA has an elongated conformation in solution. The fact that a single-stranded species is needed to fit the data suggests an ensemble of RNA conformations. Notably, the elongated form represents a major population consistent with the SAXS and NMR data.
Supplementary Figure 5 Control EMSA and surface representation of the ternary complex.
(a) Control EMSA related to Fig. 3b showing that none of the used She3p truncation mutants (331-405 or 331-425) is able to bind E3-(28 nt-loop) RNA in the absence of She2p at the given experimental concentrations. (b) Schematic drawing of She2p(6-246, C-S) and the N-terminally fused flexible linkers consisting of five (GGSGG) or ten amino acids (GGSGG)2. (c) EMSA with She2p-She3p fusion constructs. Since She2p and She3p interact with a 1:1 ratio, they also form synergistic complexes when fused to each other with a glycine-serine linker with different length. The last lane on the right side contains She2p(6-246, C-S) fused to the (GGSGG)2 linker as a control, not showing any band shift. (d) She2p alone is depicted in surface representation with its contact sites for She3p (green) and RNA (magenta) in front view and 90 ° rotated around the vertical axis. (e) The electrostatic surface potential of She2p shows that E3-(28 nt-loop) RNA (magenta) binds at the positively charged area (blue) in the middle of the She2p tetramer, whereas negatively charged residues (red) surround the binding region. She3p is depicted in green. (f) Stereo view of the She2p-She3p complex with ASH1 E3 (28 nt-loop) RNA. (g) Ternary complex as depicted in Fig. 3b with She2p in surface representation. Red area marks She2p amino acids 164-179 that were previously shown to UV crosslink with E3 (51 nt) (Müller, M. et al., PLoS Biol. 9, e1000611 (2011)). Dashed blue line shows the anticipated projection of the RNA over the crosslinking site. This depicted single-stranded loop region followed by a double-stranded stem (right side) is consistent with the experimentally validated secondary structure shown in Fig. 1a. EMSAs in a and c are representatives for three independent experiments each.
Supplementary Figure 6 Representative EMSAs for apparent KD determination.
(a-d) Binding affinities of E3 variants that showed ternary complex formation in Fig. 4 were quantified in EMSA experiments. Respective control EMSAs show unspecific RNA binding of She3p at high protein concentrations. Nevertheless, when She2p is additionally present, band intensities of the shifted complex increase, thus reflecting the specific ternary complex of She2p-She3p and RNA. In (a) radioactively labeled E3-(51 nt) RNA was used. In (b) E3 (28 nt-loop) was assessed. (c) and (d) show EMSAs where RNA mutants “M1” and “M2” were tested. (e) Table summarizing apparent mean KD values ± s.d. for ASH1 E3-RNA mutants in complex with She2p and She3p. KDs were calculated from three independent experiments using the one-site binding equation. While E3 (51 nt) forms the ternary complex with a KD of 0.20 ± 0.03 μM, the minimal E3-(28 nt-loop) RNA bound She2p and She3p just slightly weaker. Replacing U1780 by the pyrimidine C in mutant “M2” decreased the affinity to a KD of 0.44 ± 0.09 μM, whereas mutating U1780 to purine A in mutant “M1” had a more severe effect with a KD of 1.05 ± 0.33 μM. (f) Representative EMSA of the crystallized She2p(6-246, C-S)-(GGSGG)2-She3p(331-405) fusion protein with ASH1 E3-(28 nt-loop) RNA shows high affinity binding. The apparent KD was 112 ± 29 nM. Square brackets marked by an asterisk delimit the area of the gel, which was used for quantification in a-d and f. All EMSA were performed in three independent experiments.
Supplementary Figure 7 Analyses of She2p mutants and fusion constructs with She3p.
(a) EMSA testing ASH1-E3 (51-nt) RNA-binding by different She2p-She3p fusion constructs. She2p(6-246, C-S) fused to the (GGSGG)2 linker and to She3p(331-405) shows strong ternary complex formation. When in She3p K340, R341 and Y345 are mutated to alanines, band shifts occur only at higher protein concentrations, indicating a reduced RNA-binding affinity. The fusion protein only containing She3p amino acids 331 to 346 is still able to form a weak ternary complex, confirming that this region constitutes an important interaction site. The last lane on the right side contains She2p(6-246, C-S) fused to the ten amino acid glycine-serine linker (GGSGG)2 without She3p as a control, not showing any band shift. (b) Control EMSA related to Fig. 4d. Unless stated otherwise 4.86 μM protein(s) were tested for ASH1-E3 (51-nt) RNA-binding. (c) CD spectra of She2p wild type in comparison to the double mutant She2p(E172A, F176A) confirmed the α-helical composition of both protein variants and the secondary structure integrity of the mutant. EMSAs in a and b are representatives for three independent experiments each. CD spectra in c were recorded once.
Supplementary Figure 8 Representative EMSAs of She3p mutants for KD calculations.
(a) His6-SUMO-She3p(331-405) and selected single-amino acid mutants were tested with She2p and ASH1-E3 (51 nt) for their ternary complex-formation. Since distinct She2p-She3p-RNA complexes could be detected for wild-type She3p(331-405) and She3p(331-405; R342A) apparent mean KD values ± s.d. were determined. KDs were calculated from three independent experiments using the one-site binding equation. Square brackets marked by an asterisk delimit the area of the gel, which was used for quantification. His6-SUMO-tagged proteins were used because they yielded higher amounts of protein after purification. (b) Table summarizing calculated apparent KD values for His6-SUMO-She3p mutants. Binding affinities of She3p(331-405) mutated in positions K340A, R341A, F344A, and Y345A appeared to be worse than for R342A. These were not quantified (not determined, n.d.) due to the lack of distinct band shifts. Of note, only R342 makes intra-molecular contacts. Since R342A shows reduced RNA binding, also this stabilizing interaction seems important for RNA recognition. All EMSA were performed in three independent experiments.
Supplementary Figure 9 Control experiments for in vivo studies and comparison of LEs.
(a) Western blots with anti-Myc antibody showing the expression levels of wild-type and mutant versions of She3p in corresponding yeast strains. Glucose-6-phosphate dehydrogenase (G6PD) was used as loading control. Only the triple mutant She3p(K340A, R341A, Y345A) showed no expression. Western blots with anti-She2p antibody shows similar expression level of the wild-type strain and a strain expressing the mutant She2p(E172A, F176A). Phosphoglycerate kinase 1 (Pgk1) was used as loading control. Western blots were performed in two independent experiments from different cell extracts. (b) Comparison of predicted secondary structures from ASH1 LEs. Specifically recognized, essential bases in the ASH1-E3 LE are highlighted in red (C1779/ C1813), and blue (U1780). Conserved cytosines can also be found in E1, E2A and E2B. Based on secondary-structure predictions at least E2A and E2B are likely to have both cytosines in similar distance on the opposing strand of their stems. However, the base-specific position U1780 in E3 (blue base) is not conserved in the other elements (green bases), indicating that different subclasses of LEs might exist. Since the structural rearrangements observed in E3 upon binding (Fig. 2c) are impossible to predict with current bioinformatics tools, experimental approaches are required to understand the exact modes of binding for other LEs.
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Edelmann, F., Schlundt, A., Heym, R. et al. Molecular architecture and dynamics of ASH1 mRNA recognition by its mRNA-transport complex. Nat Struct Mol Biol 24, 152–161 (2017). https://doi.org/10.1038/nsmb.3351
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DOI: https://doi.org/10.1038/nsmb.3351
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