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1 Supplementary Table 1 Mean inclination angles a of wild-type and mutant K10 RNAs Base pair K10-wt K10-au-up K10-2gc-up K10-2gc-low K10-A-low ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.7 a Inclination angles were determined with the program CURVES using the optimization procedure for the best curvilinear axis and are displayed as mean values ( ) ± the standard error of the mean (s.e.m.). 1

2 Supplementary Table 2 Mean major groove width of wild-type and mutant K10 RNAs. Groove width at base position a K10-wt K10-au-up K10-2gc-up K10-2gc-low K10-A-low ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 a Major groove widths were calculated using the program MOLMOL as closest phosphate-phosphate distance across the major groove Å. Displayed are the mean values (Å) ± the standard error of the mean (s.e.m.). 2

3 Supplementary Figure 1 NMR spectra of wild-type K10 RNA. (a) 1 H, 13 C-ct-HSQC (constant-time heteronuclear single quantum coherence) spectrum of aromatic pyrimidine C6-H6 and purine C8-H8 correlations in 13 C, 15 N-labelled K10-WT RNA. Most aromatic resonances (labelled according to Fig. 1a) display excellent chemical shift dispersion, except for several uracil C6-H6 resonances, which show severe chemical shift overlap in both the carbon and proton dimensions (indicated by an oval). (b) 1 H, 13 C-ct-HSQC spectrum of adenine C2-H2 correlations in 13 C, 15 N-labelled K10-WT RNA. Several C2-H2 correlations from adenines in helical regions display strongly upfield-shifted proton resonances (below 7.0 ppm) indicative of unusual base-base stacking 1. (c) Overlay of a homonuclear TOCSY (green) and NOESY (purple) spectrum of unlabelled K10-WT RNA presenting a part of the aromatic-anomeric walk. Strong NOESY cross-peaks from pyrimidine H5-H6 protons (also indicated as green TOCSY peaks) obscure part of the aromatic-anomeric walk indicated with orange lines (e.g. U13 or U14). Intraresidue correlations are labelled with residue numbers according to Fig. 1a. (d) Overlay of a homonuclear TOCSY (green) and NOESY (purple) spectrum of site-specifically pyrimidine H-5 deuterated K10-WT RNA showing the same part of the aromatic-anomeric walk as in Supplementary Fig. 1c. Strong NOESY cross peaks (purple) from pyrimidine H5-H6 protons are now absent due to the site-specific pyrimidine H-5 deuteration and only two residual TOCSY peaks (green) are visible as indicated by an asterix. The pyrimidine H5-H6 crosspeak-free aromaticanomeric walk is indicated with orange lines. 3

4 Supplementary Figure 2 Refinement with angular restraints derived from RDCs is crucial to define the overall structure of wild-type and mutant K10 RNAs. (a-f) Each plot displays the fit of measured and back-calculated RDCs of the best-fitted low energy structure calculated without RDCs (pink) and the lowest energy structure calculated with RDCs (blue) using the program PALES 2. Corresponding RMS values (Hz) are displayed in the right-bottom corner and the K10 RNAs are indicated in the top-left corner. The corresponding alignment tensors for WT and mutant K10 RNAs are listed in Table 1. (e) The lowest energy structure of the A-form hairpin is displayed with bases in blue and ribose-phosphate backbone in grey. (f) Same coloring scheme as in a-e. In addition, the fit of measured and back-calculated RDCs from the A-form hairpin which were used in the refinement of K10-A-low, is displayed (yellow). 4

5 Supplementary Figure 3 Structural details of wild-type K10 RNA. (a) Structure of the K10 octaloop (5 -A18UUAAUUC25-3 ). The hairpin loop displays a compact fold with continuous stacking of the first three loop bases (A18, U19, U20) above the loop-closing A17-U26 base pair on the 5 side. The basestacking is followed by a so-called U-turn in the backbone 3 (indicated by an arrow), which exposes the bases of A21 and A22 into the minor groove. The following residue U23 is dynamic and its base is looped out while the last two loop bases (U24 and C25) stack inside the loop as seen on the 5 side. Residues are numbered according to Fig. 1a. All nitrogen atoms are blue, oxygen atoms of the bases and ribose O4 atoms are red; all carbon atoms of the bases and ribose-phosphate backbone atoms are light pink, except phosphorus atoms (yellow spheres). The lowest energy structure of the final K10-WT RNA ensemble is shown. (b) The single nucleotide bulges flanking the middle helix adopt different conformations and modulate the relative orientation of the lower and upper helix. The base moiety of adenine A37 stacks between the purines of the adjacent U7-A38 and G8-C36 Watson-Crick base pairs and thereby increases the helical twist between them to about 50 as compared to a mean value of 34.1 in regular A-form RNA 4. The bulged residue C33, on the other hand, resides in the major groove flanked by a U11-A32 Watson-Crick base pair and a dynamic A10-U34 base pair below the bulged nucleotide, which appears in an intermediate conformation between open (towards the minor groove) and closed form in the final ensemble. NMR evidence for formation of a A10-U34 base pair could only be obtained from 2D NOESY spectra in water recorded at low temperature (5 C) and therefore no hydrogen-bonding restraints were applied (data not shown). The major groove positioning of residue C33 maintains A-form helical twist of about 36 in the final ensemble of K10-WT RNA. The helical twist was measured by drawing a line through the carbon C6 atom of pyrimidine bases and the carbon C8 of purine bases of the corresponding base pairs (indicated by circles) in the final ensemble of K10-WT RNA. The lowest energy structure of the final K10-WT RNA ensemble is shown. Numbering of residues and coloring scheme as in a. The minor and major grooves are indicated. (c) The G4-U41 base pair in the lower helix does not adopt a wobble conformation. In the context of a Watson-Crick base paired helix, G-U pairs usually adopt a wobble conformation with two imino-carbonyl hydrogen bonds indicated by a strong imino-imino crosspeak in 2D NOESY spectra recorded in water. For the G4-U41 base pair of the K10 RNA no imino-imino crosspeak is observed in NOESY spectra at 25 C and only a very weak imino-imino crosspeak is observed at low temperature (15 C) (data not shown). In the final ensemble of structures the G4-U41 base pair is opened towards the major groove and only a single hydrogen bond is formed (G4-N2 U41-O2 = 2.9 ± 0.05). Numbering of residues and coloring scheme as in a, except that phosphate oxygens are displayed as small red spheres. Hydrogen bonds are indicated by dotted lines. 5

6 Supplementary Figure 4 CD analyses of wild-type and mutant K10 RNAs support A -form conformation of helices. (a) CD spectra of K10-WT RNA in the absence and presence of trifluoroethanol (TFE). K10-WT RNA displays a positive band at 282 nm characteristic for B-form DNA and indicative of lower base inclinations than usually found in A-form RNA 5. Increasing amounts of TFE, which causes dehydration and leads model B-form DNA to adopt an A-form conformation 5, shift this band towards 260 nm, characteristic for the A-form conformation. (b) Comparison CD spectra of the corresponding K10-WT DNA in the absence and presence of TFE. K10-WT DNA displays a positive band at 282 nm characteristic for B-form DNA. Increasing amounts of TFE up to 75% shift this band towards 260 nm characteristic for A-form RNA and DNA 5. The observed shift is smaller than usually observed for a B-A transition in DNA, but might be due to the high A-T base pair content (e.g. poly d(a).d(t) cannot adopt A-form conformation 6 ). (c) CD spectra of upper wild-type and mutant K10 RNA helices. The wild-type and mutant K10 upper RNA helices comprise nucleotides with 2 additional G-C base pairs serving as a clamp. Naming of the mutants is according to Fig. 2a. The WT-up helix displays a positive band at 278 nm characteristic for B-form DNA and indicating low base inclinations 5. The au-up mutation, which preserves A -form inclination angles in the upper helix (Supplementary Table 2), also displays a positive band at 279 nm, while the 2gc-up mutation, which displays higher inclination angles in the upper helix (Supplementary Table 2) shows a positive band shifted towards 260 nm (broad maximum at 272 nm). (d) CD spectra of lower wild-type and mutant K10 RNA helices. The wild-type and mutant K10 lower RNA helices comprise nucleotides 1-7 and with 2 additional G-C base pairs serving as clamp and capped by a 5 -CUUCG-3 tetraloop. Naming of the mutants according to Fig. 2a. The WT-low helix also displays a positive band at 277 nm characteristic for B-form DNA and indicating low base inclinations 5. The A-form mutant (A-low) shows a positive band shifted towards 268 nm indicative of A-form RNA or DNA, and the 2gc-low mutation, despite lowered inclination angles (Supplementary Table 2) is also shifted to 269 nm as compared to WT-up, which we do not currently understand, but may be associated with the influence of base pair identity on CD spectra 6. (e) CD spectra of K10-WT and K10- C33 RNAs. Both RNAs display a positive band at 282 nm characteristic for B-form DNA and indicating low base inclinations 5. This strongly suggests that the upper and lower helices adopts A -form conformation independent of the presence of the single nucleotide bulge C33. Overall the results of our CD analyses are consistent with the presence of A -form or B-form-like helices in the K10 TLS. 6

7 Supplementary Figure 5 Localization activity of additional mutant K10 RNAs. Representative confocal images of blastoderm embryos injected with transcripts not shown in Fig. 2b or Fig. 4c,d, as indicated. Transcripts were visualized by direct incorporation of fluorochrome-coupled UTP. Arrow indicates the approximate site of injection in all experiments. Apical is to the top and basal is to the bottom in all images. Scale bar, 50µm. 7

8 Supplementary Figure 6 Mutations in the upper or lower helix of K10 monitored by NMR spectroscopy. (a) Overlay of 1 H, 13 C-ct-HSQC spectrum of adenine C2-H2 correlations in 13 C, 15 N-labelled K10-au-up (pink) and K10-WT (purple) RNA. Only the crosspeaks in the K10-WT spectrum are labelled and the numbering corresponds to Fig. 1a. Resonances affected by the au-up mutation in the upper helix are labelled in black and unchanged resonances from the hairpin loop and the lower helix are labelled in purple. (b) Overlay of 1 H, 13 C-ct-HSQC spectrum of adenine C2-H2 correlations in 13 C, 15 N-labelled K10-2gc-up (pink) and K10-WT (purple) RNA. Crosspeak labelling, color schemes and numbering as in a. (c) Overlay of 1 H, 13 C-ct-HSQC spectrum of adenine C2-H2 correlations in 13 C, 15 N-labelled K10-2gc-low (pink) and K10- WT (purple) RNA. Only the crosspeaks in the K10-WT spectrum are labelled and numbering as in a. Resonances affected by the 2gc-low-mutation in the lower helix are labelled in black and unchanged resonances from the hairpin loop and the upper helix are labelled in purple. (d) Overlay of 1 H, 13 C-ct-HSQC spectrum of adenine C2-H2 correlations in 13 C, 15 N-labelled K10-A-low (pink) and K10-WT (purple) RNA. Crosspeak labelling, color schemes and numbering as in c. (e) 1 H, 13 C-ct-HSQC spectrum of adenine C2-H2 correlations in 13 C, 15 N-labelled orb-wt (pink) and K10-WT (purple) RNA. Several C2-H2 correlations from adenines in helical regions of orb RNA display similarly strong upfield-shifted proton resonances (below 7.0 ppm) as seen for K10 RNA in a-d suggesting that orb RNA preserves the unusual A -form helices of K10 RNA. The secondary structure of orb RNA is shown beside the spectrum. Only the crosspeaks in the K10-WT spectrum are labelled and numbering is as in a. (f) Overlay of 1 H, 13 C-ct-HSQC spectrum of purine C8-H8 and pyrimidine C6-H6 correlations in 13 C, 15 N-labelled model A- form hairpin (pink) and K10-A-low (purple) RNA. Only the crosspeaks in the K10-A-low spectrum are labelled and numbering as in a. Resonances, which exhibit the same or very similar chemical shifts in the model A-form hairpin and the lower helix of K10-A-low mutant RNAs are labelled in purple. The aromatic resonances of first seven base pairs in both RNAs exhibit almost identical chemical shifts indicating that the same local structure is formed. The following G6-U39 base pair displayed slightly altered chemical shifts due to the different chemical environment as indicated by arrows (G6-U39 is followed by U7-A38 base pair in K10-A-low and by a G-C base pair in the model A-form hairpin), but identical cross peak patterns in NOESY spectra in water (data not shown). Other resonances from the K10-A-low mutant are labelled in black. 8

9 Supplementary Figure 7 Comparison of purine base-base stacking in the upper and lower helix of wild-type K10 and mutant RNAs. (a) View down from the top of the upper helix of K10-WT, K10-au-up, and K10-2gc-up RNAs displays the degree of stacking of purine bases. In K10-WT RNA, seven adenine bases form a continuous base-base stacking (A17-A32) giving rise to B-form-like inclination angles and a widened major groove (see Supplementary Tables 1 and 2). Inverting the U13-A30 base pair to a A13-U30 base pair in the K10-au-up mutant RNA disrupts the continuous base-base stacking at the site of mutation, but results in two separate stacks of bases (A17-A29 and A13-A32) which maintain K10-WT helical features. Replacing the U12-A31 and U14-A29 base pairs by G-C base pairs disrupts the purine base-base stacking in the lower part of the helix (K10-2gc-up). Only the top 3 adenines form a stack of three bases (A17-A28), giving rise to lowered inclination angles and a narrower major groove as compared to K10-WT (see Supplementary Tables 1 and 2). Pyrimidine bases are shown in blue and the purine bases in pink; ribose-phosphate backbone and chemical groups on the bases are omitted for clarity. Numbering according to Fig. 1a. The lowest energy structure of each ensemble is displayed. (b) View down from A5-U40 base pair in the lower helix of K10-WT, K10-2gc-low, and K10-A-low RNAs displays the degree of stacking of purine bases. In K10-WT RNA, five purine bases display continuous base-base stacking (A5-G44) giving rise to B-form-like inclination angles and a widened major groove (see Supplementary Tables 1 and 2). The K10-2gc-low mutant RNA (U3-A42 and G4-U41 base pairs mutated to C3-G42 and G4-C41 base pairs) maintains purine bases in equivalent positions as K10-WT RNA and thereby the continuous base-base stacking (A5-G44) and helical features. Replacement of the lower helix by a sequence (K10-A-low), which disrupts the continuous purine base-base stacking, results in A-form helical features in this mutant RNA (see Supplementary Tables 1 and 2). Color scheme and numbering as in a. The lowest energy structure of each ensemble is displayed. 9

10 Supplementary Figure 8 A -form RNA helices from Thermus thermophilus 16S rrna. Structures of helical segments from 16S rrna without RNA-RNA or RNA-protein contacts in the minor and major grooves and with three or more consecutive purines on one side of the helix as seen in the crystal structure of the Thermus thermophilus 30S small ribosomal subunit (PDB ID 2J00)7. For comparison, the structures of an ideal A-form RNA (PDB ID 1RNA)8 and an ideal B-form DNA (PDB ID 1BNA)9 and the upper and lower helix of the K10-WT TLS are shown. Pyrimidine bases are blue and purine bases are pink; ribose-phosphate backbone and chemical groups on the bases are omitted for clarity. Inclination angles (in degrees) are given for each base pair and listed in the tables beside each structure. Numbering according to the corresponding PDB entries. Helix 44 was selected for functional studies as the inclination angles are most reminiscent of those in the K10 upper helix (note that helices 6 and 33 have even lower inclination angles than the K10 upper helix). 10

11 Supplementary Figure 9 Secondary structures of minus-end-directed RNA signals. Secondary structures of minus-end-directed RNA signals suggest helices with two or more stretches of contiguous purines on the same side of the stem, which are candidates to participate in stacking interactions. The corresponding helical regions are boxed in red. Secondary structures for K10 TLS 10, hairy (hsl1) 11, orb 12, wingless (WLE3) 13, gurken (GLS) and I factor (ILS) 14, bicoid (SLV) 15 and fushi tarazu (SL1/SL2) 16 were generated with MFOLD

12 Supplementary Figure 10 Structure determination of wild-type K10 RNA. (a) Global and local heavy-atom superposition of the 10 lowest-energy K10-WT RNAs refined without RDCs. The shape of the entire RNA molecule is not well defined, since NMR-derived distance (up to 5-6Å) and torsion angle (rotation around bonds) restraints only describe the local structure, but lack long-range structural information to define the overall shape of RNA hairpins 18. Locally, on the other hand, the RNA molecule is well defined with much lower rmsds of the upper and lower helices. Helical twist and the resulting major groove widths though have to be interpreted with caution, since these parameters result from loose torsion angle and distance restraints (consistent with both A-form and A -form conformation) as well as the attractive van der Waals force between neighbouring bases introduced by the Lennard-Jones potential during final refinement, but are not a direct readout of experimental data, such as angular restraints derived from RDCs. Therefore, in order to even better define the local structure and to improve the global shape of the RNA, refinement with RDC-derived angular restraints is required, which in contrast to distance and torsion restraints contain distance-independent structural information (Fig. 1b and Supplementary Fig. 10d) 19. Bases are red and the ribose-phosphate backbone is pink. The bases of the single nucleotide bulges are green. Both the upper and lower helix are shown from the major groove side to visualize the inclination angles and groove widths. The helical axis is shown in blue and the inclination angle of base pairs is indicated by a dashed line. (b) Plot of the total energy, D a and R from iterative refinement of the K10-WT RNA with RDC restraints. Initial D a and R values were obtained from SVD-fitting of the structures determined without RDCs 20. Each round of calculation was followed by SVD-fitting of the lowest energy structures to predict new values for D a and R and calculate new ensembles. Since R is directly related to the shape of the molecule and therefore also the helicity of the RNA, the iterative refinement was stopped when the R value and therefore the overall shape of the RNA was basically unchanged ( R < 0.005) in two consecutive rounds of calculations following previously published procedures 18,21. The structures converged after four rounds of calculations (4x200 structures) with D a = and R=0.111, which agrees well with the grid search results (Supplementary Figure 10c). (c) Plot of the total energy for the lowest energy K10-WT structure of ensembles obtained as a function of D a and R. 200 structures are calculated for each of the 70 grid points (70x200 structures). The 3D plot displays all the grid points leading to the lowest energy structure, which was obtained with D a = -27 and R=0.1. This is in perfect agreement with the alignment tensor determined during iterative refinement (see Supplementary Fig. 10b). (d) Heavy-atom superposition of the 10 lowest-energy K10-WT RNAs obtained from the grid search (blue bases) and 12 lowest-energy K10-WT RNAs obtained from iterative refinement (red bases). The ribose-phosphate backbones are shown in 12

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