Article Figures & Data
Figures
- Figure 1. Structure of the Mei-P26 NHL domain and its specific ssRNA recognition.
(A) Mei-P26 domain organization with NHL domain shown in blue (top left). R, RING domain; B, B-Boxes; CC, coiled coil, Q, glutamine rich region. Size-exclusion chromatography profile and SDS–PAGE gel for Mei-P26 NHL protein with mass markers indicated at the top. Blue line on the chromatogram corresponds to 280 nm wavelength, orange line to 254 nm wavelength (top right). Cartoon and surface representation of the crystal structure of the Mei-P26 NHL domain in top surface and side orientations (encompassing amino acids 931–1,203) (left bottom). (B) Microscale thermophoresis results for the fraction of bound DNA, dsRNA, or ssRNA (U7) ligands for different concentrations of Mei-P26 NHL. The dissociation constant (Kd) was calculated from at least three independent experiments (n ≥ 3). (C) EMSAs employing various concentrations of the Mei-P26 NHL domain (as indicated above each lane) on a single-stranded DNA oligonucleotide, an RNA hairpin structure (both as indicated in the microscale thermophoresis panel), or a single-stranded U9 RNA oligonucleotide. Depicted is a representative gel of three independent replicates.
Source data are available online for this figure.
Source Data for Figure 1[LSA-2022-01418_SdataF1.pdf]
- Figure S1. Mei-P26 NHL stability and structural comparison of the Mei-P26, Brat, DrLin41, and Thin/Abba NHL domains.
(A) Mei-P26 NHL stability test. Legend: Ctrl—freshly purified protein as a control, RT—sample incubated at RT. (B) Silver-stained acrylamide gel from Mei-P26 NHL crystal washing trials. Legend: P, protein sample; D, drop from the crystallization conditions; W1, wash1; W2, wash 2; Xtal, crystal. (C) Schematic representation of the domain organization of the D. melanogaster Mei-P26 (DmMei-P26), D. melanogaster Brat (DmBrat), D. rerio Lin41 (DrLin41) proteins, and D. melanogaster Thin/Abba (DmThin/Abba). BB, B-boxes; CC, coiled coil; F, Filamin domain; NHL, NHL domain; Q, glutamine-rich region; R, RING domain. Comparison of the architecture of the NHL domains derived from Mei-P26 (light blue), Brat (pink), DrLin41 (ochre) and Thin/Abba (green), N- terminus of each protein in red, C terminus in dark blue. (D) Representation of top surface charge distribution of the NHL domains of the three different proteins (red, negative charge; blue, positive charge) with depicted oligonucleotides bound by Brat and Lin41NHLs. (E) Overall amino acid conservation of Mei-P26, Brat, Lin41, and Thin/Abba NHL domains generated using ConSurf platform (dark green—low conservation, dark violet—high conservation).
Source data are available online for this figure.
Source Data for Figure S1[LSA-2022-01418_SdataFS1.pdf]
- Figure S2. Sequence alignment for Mei-P26, Brat, DrLin41, and Thin/Abba NHL domains.
Alignment of the C-terminal sequences of the Mei-P26 (aa 798–1,206), Brat (aa 713–1,037), Lin41 (aa 548–824), and Thin/Abba (aa 992–1,353) proteins. Arrows indicate the beginning of the individual NHL domains. Conservation of individual residues is shown in blue-scale color. Amino acid residues demonstrated to be involved in RNA recognition in the DmBrat (red boxes) or DrLin41 (yellow boxes) are highlighted. Asterisks mark positions in Mei-P26 that were analyzed by mutagenesis in this study.
- Figure S3. The RNA binding of Brat NHL.
(A) Complex formation of the wt Mei-P26 NHL domain with two different RNA substrates at elevated salt concentration. 32P-radiolabelled RNA fragments derived from Hrb27c (left) and Col4a1 mRNA (right, compare Fig 4F) were incubated with the recombinant NHL domain under conditions comparable for the mutant NHL derivatives (150 mM salt) and complexes were analyzed by native PAGE. (B) Incubation with the Mei-P26 NHL domain does not affect RNA integrity. Radiolabeled U8 and SEQ3 RNAs were incubated with the recombinant Mei-P26 NHL domain (as indicated at the top), buffer only served as a control. Subsequently, RNA integrity was assessed by denaturing PAGE. (C) Computational analysis of RNAcompete experiment conducted for Brat NHL. Consensus motif recognized by Brat NHL was calculated from set A and B. Scatter plot with Z-scores for set A and B. A list of six highest scored RNA 7-mers are depicted. (D) Normalized microscale thermophoresis data of Brat NHL–binding curves for BRAT1 and Mei-P26 SEQ3 and SDS–PAGE gel from Brat NHL purification. Binding curve for Brat NHL with estimated Kd was marked in solid line, whereas binding with Kd > 5 μM was presented as experimental points. Kd was calculated from three independent experiments (n = 3).
Source data are available online for this figure.
Source Data for Figure S3[LSA-2022-01418_SdataFS3.pdf]
- Figure 2. The Mei-P26 NHL domain specifically recognizes single-stranded, U-rich RNA motifs.
(A) Analysis of the Mei-P26 NHL RNAcompete data. Scatter plot representing Z-scores for two halves of the RNA pool (set A and set B) shown at the bottom; six highest scoring 7-mers were highlighted in color in right corner of the plot. The consensus motif derived from the indicated sequence motifs is indicated at the top. (B) Comparison between the Mei-P26 NHL in complex with UUUUACA (left) and UUUUUUU (right) (color-coded RNA sequences depicted at the top). The complexes were computationally modeled using molecular dynamics simulations by introducing oligonucleotides onto the surface of the Mei-P26 NHL domain. (C, D) Microscale thermophoresis–binding studies of the NHL domain to the six highest scoring 7-mer sequences (C), variants thereof, or the motif recognized by the closely related protein Brat (D). Kd values at least three independent experiments (n ≥ 3). Microscale thermophoresis–binding curves for protein-oligonucleotide complexes with calculated Kd were marked in solid lines, curves with Kd > 3 μM were presented as experimental points.
- Figure S4. Molecular dynamics simulations of Mei-P26 NHL–RNA complexes.
(A) Molecular dynamics simulations of Mei-P26 NHL domain in complex with different RNAs. The MeiP26 NHL domain was simulated with SEQ3: UUUUACA, SEQ1: UUUUUUU, inverted SEQ3: ACAUUUU and BRAT1: UUGUUAA. For each of these simulations, the left panel shows the superposition of representative models from the top five clusters during a 1 µs molecular dynamics (MD) simulation. The heat map of pairwise root mean square deviation (r.m.s.d.) of different frames, with points are colored based on the r.m.s.d. (in Å2) values during the MD simulations are shown in the middle panel. The right panels show the r.m.s.d. maps of different frames with respect to the starting frame of the simulation. (B) A maximum likelihood superposition of the top five clusters from MD simulations of Mei-P26 models with SEQ3 (left panel) and SEQ1 (right panel) obtained from Theseus (blue color indicates little variance, whereas yellow the maximum variance). Individual RNAs of the top five clusters were marked with different colors.
- Figure S5. Molecular dynamics simulations of Brat NHL–RNA complexes.
(A) Molecular dynamics simulations of Brat NHL domain in complex with different RNAs. The Brat NHL domain was simulated with BRAT1: UUGUUAA, co-crystallized sequence UUGUUGU (PDB ID: 5EX7) and SEQ3: UUUUACA. For each of these simulations, the left panel shows the superposition of representative models from the top five clusters during a 1 µs molecular dynamics (MD) simulation. The heat map of pairwise root mean square deviation (r.m.s.d.) of different frames, with points are colored based on the r.m.s.d. (in Å2) values during the MD simulations are shown in the middle panel. The right panels show the r.m.s.d. maps of different frames with respect to the starting frame of the simulation. (B) A maximum likelihood superposition of Brat NHL crystal structure (PDB ID: 5EX7) with Brat model (left panel) and the top five clusters from MD simulations of Brat model for crystal structure with UUGUUGU (PDB ID: 5EX7) (right panel) obtained from Theseus (blue color indicates little variance, whereas yellow the maximum variance). Individual RNAs of the top five clusters were marked with different colors.
- Figure S6. Microscale thermophoresis measurements for Mei-P26 with SEQ1-6.
Results of single microscale thermophoresis experiments for Mei-P26–binding to oligonucleotides SEQ1-6 with depicted calculated averaged Kd and number of replicates (n) for each measurement.
- Figure 3. Identification of amino acids in the NHL domain important for RNA binding.
(A) Model of the Mei-P26 NHL domain in complex with a UUUUACA RNA sequence. Select amino acids predicted to be in close proximity to the RNA are highlighted. (B) Size exclusion chromatography profile and SDS–PAGE gel for selected Mei-P26 mutated variants. Abbreviations for K1172A/R1175A (KR) and R1150A/K1172A/R1175A (RKR) are used in the inset. (C, D) Microscale thermophoresis (panels C and D [left]) or EMSA-based (panel D [right]) in vitro binding analyses of the recombinant NHL domain or variants thereof (as indicated in each graph) to RNA oligonucleotides with the sequence UUUUACA (C), U9 (D [left]), or U16 (D [right]). Dissociation constants (Kd) were calculated from three independent experiments (n = 3).
Source data are available online for this figure.
Source Data for Figure 3[LSA-2022-01418_SdataF3.pdf]
- Figure S7. Purification and biophysical characterization of Mei-P26 NHL protein variants with amino acid substitutions.
(A) Elution profiles of the wild-type Mei-P26 NHL domain (wt) and variants thereof (color coded, legend on the top left) after separation via a Superdex 200 Increase column and SDS–PAGE analyses of the individual recombinant proteins after purification. (B) Analyses of thermal stability of the individual recombinant proteins (plotted are the first derivatives of the data obtained from thermal shift assays). Color code as in panel (A). Melting temperatures (Tm) were calculated from three technical replicates (n = 3) and are given for each protein; vertical dashed line indicates the Tm for wt Mei-P26 NHL (58.3°C).
Source data are available online for this figure.
Source Data for Figure S7[LSA-2022-01418_SdataFS7.pdf]
- Figure S8. Differences in ssRNA sequence specificity between Mei-P26 and Brat NHL domains.
(A) Modeled Mei-P26-NHL–RNA complex with Ala in position 1046 (yellow). (B) Brat-NHL–RNA structure with Arg in position 875 (orange). (C) RNA binding analyses of the A1046R variant using as a substrate either the BRAT1, SEQ3, or a U9 RNA by microscale thermophoresis assay.
- Figure S9. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) of Mei-P26.
(A) Analysis of cultured Drosophila Schneider 2 cells transfected with plasmids encoding full-length Mei-P26 (lanes 1 and 2) or the NHL domain only (lanes 3 and 4). Lanes marked RKR represent samples with expression of protein constructs that carry the R1150A, K1172A, and R1175A substitutions. Molecular weight markers (in kD) are indicated on the right. Western blotting was performed with an antibody directed against the FLAG tag of the transfected constructs (top panel). Bottom panel: re-probing of the same membrane with an antibody directed against tubulin as a control (bottom panel). Asterisks indicate leftover signals from the anti-FLAG blot after stripping of the membrane and re-probing. (B) Analysis of immunopurified, UV-crosslinked Mei-P26 ribonucleoprotein complexes. Drosophila Schneider 2 cells were transfected with constructs encoding FLAG-tagged Mei-P26 full-length protein (FL), its NHL domain only (NHL), or the respective RKR mutant derivatives thereof (FL in lanes 3 and 4, NHL domain in lanes 7 and 8). Immunopurified RNP complexes were subjected to a limited ribonucleolytic digestion with different amounts of RNase I (as indicated above each lane), and radioactively labeled. Nucleic acids covalently crosslinked to the respective proteins reduce their gel mobility, resulting in a smear visible in the autoradiography of the membrane after transfer (as indicated on the left). Molecular weight markers (in kD) are indicated on the right. (C) Summary of the iCLIP results obtained with full-length Mei-P26 protein or its NHL domain as baits. Numbers are provided for identified crosslink clusters and genes that contain the clusters, along with their respective locations within transcription units. 5′ UTR, 5′ untranslated region; CDS, coding sequence; 3′ UTR, 3′ untranslated region.
Source data are available online for this figure.
Source Data for Figure S9[LSA-2022-01418_SdataFS9.pdf]
- Figure 4. Identification of Mei-P26 target RNAs in cultured cells.
(A) Comparison of iCLIP data derived from either full-length Mei-P26 protein (depicted in blue) or its NHL domain (depicted in salmon). Number of genes containing a statistically significant local enrichment of cross-link positions (cross-link peaks) and percent values are given for each fraction. (B) Chromosomal origin of the 249 shared target genes identified in both iCLIP datasets. (C) Location of the full-length Mei-P26 (left) or its NHL domain-derived (right) iCLIP clusters within transcripts. (D) Presence of potential Mei-P26 RNA motifs in vicinity of the cross-link peaks in the shared target genes. No overlap: cross-linking of the full-length protein and the NHL domain occurred in different regions of the gene locus. (E) Gene Ontology term analysis of biological processes enriched in the shared target genes. (F) Validation of select Mei-P26 binding sites identified by iCLIP. Top left: schematic depiction of the Hrb27c and Col4a1 gene loci. Introns are depicted as lines, exons as boxes; the grey shading indicates the protein coding region. Below: iCLIP read depth analyses of 3′ UTR regions (as indicated by the solid black lines) from experiments performed with either the full-length Mei-P26 protein (light blue), its NHL domain (dark blue), or from control experiments (grey). Potential RNA motifs recognized by Mei-P26 are highlighted in yellow. Right: EMSA analyses using RNA fragments derived from the iCLIP clusters (as indicated by the red arrows, sequences provided at the bottom of the gels), using different concentrations of the recombinant Mei-P26 NHL domain or its mutant derivative (Mei-P26RKR: R1150A, K1172A, and R1175A, as indicated above the gels). Free RNA probe and NHL:RNA complexes are indicated on the left
Source data are available online for this figure.
Source Data for Figure 4[LSA-2022-01418_SdataF4.pdf]
- Figure S10. Examples of iCLIP reads in select genomic loci.
(A, B, C) Left: Genome browser tracks of iCLIP reads aligning to the lost (panel A), LanA (panel B), and Mlc-c (panel C) loci. Schematic gene models are provided at the top of each panel (color coding as in Fig 4F). Read depths aligning to the genomic regions indicated by the lines are shown below (summed-up from three independent, biological experiments, tags from control experiments shown in grey, color coding as in Fig 4F). Right: EMSA analyses using RNA fragments derived from the iCLIP clusters (as indicated by the red arrows, sequences provided at the bottom of the gels), using different concentrations of the recombinant Mei-P26 NHL domain or its variant (Mei-P26-NHLRKR: R1150A, K1172A, and R1175A, as indicated above the gels). Free RNA probe and NHL:RNA complexes are indicated on the right.
Source data are available online for this figure.
Source Data for Figure S10[LSA-2022-01418_SdataFS10.pdf]
- Figure 5. Mei-P26 regulates gene expression via 3′ UTR binding sites.
(A) Tethered function assay using Brat, Gw182 (positive control) and full-length Mei-P26. Activities are calculated relative to proteins that lack the tag for tethering. (B) Mei-P26–mediated repression of a reporter that either bears the nanos mRNA 3′ UTR (nos wt) or a version thereof in which a U-rich sequence element previously implicated in regulation was mutated (nos mut). Reporter activity was determined in the presence of co-expressed wild-type Mei-P26 protein (grey bars), or a Mei-P26 protein carrying substitutions that affect RNA interaction. (Mei-P26RKR:R1150A, K1172A, R1175A; black bars). All activities are expressed relative to control reactions without overexpression of Mei-P26 (using an empty plasmid). (C) Reporter assays using 3′ UTR sequences derived from various Mei-P26–bound genes (as indicated at the bottom) and uisng wt Mei-P26-FL protein (grey bars) or its mutant derivative (black bars) as described for panel (B). All activities are expressed relative to control reactions without overexpression of Mei-P26 (using an empty plasmid). (D) Reporter assays using 3′ UTR sequences of nos (grey), Hrb27c (blue) and spz (dark blue) derived from Mei-P26-FL protein or its mutant derivatives (as indicated at the bottom) as described for panels (B and C). (A, B, C, D) For panels (A, B, C, D) mean values ± SD are depicted of at least three independent biological experiments performed in three technical replicates each. P-values were calculated with a two-sided t test relative to the control reactions described for each panel; n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Schematic depiction of Mei-P26–mediated post-transcriptional regulation of gene expression. Mei-P26 (NTD in grey, NHL domain in blue, mutated residues that are crucial for RNA binding are highlighted) associates with U-rich RNA motifs present in the 3′ UTRs of its RNA targets (depicted schematically) to regulate their expression. Regulation involves additional factors such as Ago1, Sxl, Bam, Bgcn, and Wuho that have been implicated in Mei-P26–dependent repression, whereas potential co-factors involved in Mei-P26–dependent gene activation remain to be identified.
- Figure S11. Expression levels of transfected proteins.
Representative Western Blot against the FLAG-tag of transfected Mei-P26 protein and derivatives thereof as indicated above each lane (upper panel). Western Blotting against tubulin (lower panel) served as a control. Molecular weight markers (in kD) are indicated on the left.
Source data are available online for this figure.
Source Data for Figure S11[LSA-2022-01418_SdataFS11.pdf]
- Figure S12. In silico identification of Mei-P26 dimerization interface.
(A) Domain arrangement of Mei-P26 (top) and top-ranked model of AlphaFold-multimer of monomeric Mei-P26 FL. Color encodes confidence, from red (lowest) to blue (highest) (bottom). (B) Comparison of Mei-P26 NHL crystal structure with top-ranked AlphaFold model. (C) Predicted aligned error of AlphaFold predictions of a monomeric Mei-P26 FL (left), two molecules of Mei-P26 aa 1–610 (middle) and two molecules of Mei-P26 aa 540–1206 (right). Black arrows highlight high-confidence interfaces between the molecules. (D) Top-ranked AlphaFold model of the Mei-P26 aa 1–610 dimer (top) Color code as in and dimerization interface residues involved in leucine zipper. Blue and grey indicate different chains (bottom). (E) Predicted aligned error of AlphaFold predictions of two molecules of Brat (left), Lin41 (middle) and Thin/Abba (right). AlphaFold models of the respective dimer regions (bottom).
Tables
Mei-P26 NHL, PDB ID 7NYQ, BESSY II, MX14-1 Data collection Space group P21 Cell dimensions a, b, c (Å)a 34.71, 116.50, 64.70 α, β, γ (°) 90, 96.362, 90 Wavelength 0.9184 Resolution (Å)b 43.17–1.6 (1.64–1.6) Rmeas (%) 7.7 (160.9) I/σ(I) 10.15 (0.85) CC1/2 0.99 (0.46) Completeness (%) 98.8 (97.0) Redundancy 3.82 Refinement Resolution (Å) 43.17–1.6 No. reflections 66,477 Rwork/Rfree 0.196/0.215 No. atoms Protein 8,923 Ligand/ion No ligands Water 320 B factors Protein 36.7 Ligand/ion Not applicable Water 38.3 r.m.s deviations Bond lengths (Å) 0.007 Bond angles (°) 0.689 Ramachandran statistics (%) Outliers 0 Allowed regions 4.59 Favored regions 95.41 Rotamer outliers (%) 0.21 Clash score 3.02 MolProbity score 1.41
Supplemental Data 1.
Summary of iCLIP experiments. Tabulation of the boundaries of clusters of cross-link positions identified in the iCLIP experiments for the full-length Mei-P26 (Sheet 1) and the NHL domain (Sheet 2). Each entry represents a cluster of significant peaks identified in a replicate of the iCLIP experiment and extended 20 bp upstream and 10 bp downstream. Start coordinate in chromosome, first coordinate of the cluster; End coordinate in chromosome, last coordinate of the cluster; Gene ID, FlyBase ID of the identified gene; Strand, orientation of the strand to which the gene mapped.[LSA-2022-01418_Supplemental_Data_1.xlsx]
Supplemental Data 2.
Comparison of iCLIP data obtained with full-length Mei-P26 or its NHL domain. List of shared target mRNAs identified in the iCLIP experiments with full-length Mei-P26 and its NHL domain. Information on the Flybase ID of the gene loci, their chromosomal location, the gene symbol, and the gene name are provided in the first columns. Reasons for excluding individual loci from further analyses are given in the comments section. iCLIP cross-link clusters derived from the NHL domain and the full-length protein that are separated by <50 nucleotides (nt) are considered as overlapping (column CLIP peaks overlapping). The presence of potential Mei-P26 binding sites up to 30 nt upstream and 20 nt downstream of the crosslink positions is indicated in the column denoted as “motif present.” Bona fide Mei-P26 RNA binding sites (UUUUACN, UUUUANA, UUUUNCA, or UUUUUUU) or U-rich sequences with four consecutive U residues followed by 3 nt containing at least one additional U residue (U-rich motifs) were considered. The sequences of the potential binding sites are provided in the last column.[LSA-2022-01418_Supplemental_Data_2.xlsx]
Table S1. Used oligonucleotide sequences.
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