Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: Crystal structure of the C-terminal part of D. rerio LIN41. a The crystal structure of the DrLIN41 filamin-NHL domains is displayed in a cartoon mode, with a transparent grey surface in two orientations rotated by 90°. The molecule is colored from blue (N terminus) to red (C terminus) to indicate the topology. Protein domains, termini, and β-propeller blades are labeled for better clarity. b Top view of the RNA-binding site of the DrLIN41 NHL domain with the electrostatic surface potential mapped onto the molecular surface. Surface potential is computed by using the APBS plugin implemented in PyMOL ( www.pymol.org ) and is displayed from – 5.0 kT/e (red, acidic) to + 5.0 kT/e (blue, basic). c DrLIN41 filamin-NHL domains in complex with the lin-29A stem-loop RNA. The filamin and NHL domains are shown in cartoon mode in blue, with a white transparent surface. The lin-29A RNA fragment, forming a hairpin, is displayed as a cartoon with nucleotides in different colors (guanine: green, adenine: blue, cytosine: orange, uracil: cyan). d Magnified view of the lin-29A RNA stem loop bound to the DrLIN41 NHL surface (colors as in c ). A diagram detailing the nucleotide composition of the stem loop is shown above

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: Labeling, RNA Binding Assay

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: Molecular interactions underlying LIN41 binding to RNA SLs. a , b Detailed views of interactions between the lin-29A RNA and the DrLIN41 NHL propeller. Nucleotides and protein side chains are highlighted and their directly interacting residues are shown as sticks; the remaining parts are shown as lines (RNA) or ribbons (protein). Hydrogen bonds are presented as dotted lines and hydrophobic interactions as solid lines. Nucleotides are colored as in Fig. , whereas protein side chains are colored according to the mutational analysis. c Schematic representation of the lin-29A RNA hairpin and of its interactions with DrLIN41 NHL residues (type of interaction and color code as in a , b ). d Expression of mutant HsLIN41 proteins did not severely down-regulate Renilla luciferase (RL) reporter expression unlike the wild-type HsLIN41, when a fragment corresponding to the mab-10 condensed 3′-UTR was transplanted into an unregulated 3′-UTR of the reporter construct. Bars in the graph represent the mean between three biological replicates

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: Binding Assay, Expressing, Mutagenesis, Luciferase, Construct

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: The LIN41 response element. a Schematics depicting RNA features used to build the LIN41 Response Element (LRE) model. Considered were all possible bases in the three loop positions (I, II, and III) and all possible base pairs at the stem position 1 (– 1/ + 1). The pairing probability of stem position 1 was determined by the relative occurrence of all possible structures that a particular RNA sequence can acquire. The pairing probabilities were grouped into seven bins on a log2 scale. Combining the sequence and structure features resulted in 2688 (6 × 64 × 7) RNA motif variants. b A heat map showing the average CeLIN41-binding scores from the RNAcompete experiment for all RNAs containing any particular motif variant as described in a . Pairing probability and base pairs at stem position 1 are shown on the left and the right of the heat map respectively. The loop (I, II, and III) sequences are shown in two rows, for clarity, at the bottom of the heat map. The data were clustered based on the CeLIN41-binding score. The overall distribution of pairing probabilities is shown on top of the pairing probability scale. Bottom right: the drawing represents a stem-loop motif, based on the model, referred to as the LIN41 Response Element (LRE). Yellow: data not available (RNA motif variants supported by < 20 oligo sequences)

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: Sequencing, Binding Assay, Variant Assay

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: LIN41 binds to LREs both in vitro and in vivo. a Fluorescence polarization (FP) assays determining binding constants of CeLIN41 to LRE variants in the position III of the loop. Raw FP data of CeLIN41, interacting with a wild-type LRE (SL I in Supplementary Fig. ), a control stem-loop RNA with five nucleotides in the loop, and LREs mutated at loop position III, are shown in units of millipolarization (mP). The equilibrium dissociation constant ( K D ) is shown for the WT LRE. Each data point is a mean of three experiments and the error bars represent the standard deviation. b The FP assays determining binding constants of CeLIN41 to LRE variants in the position 1 of the stem. Raw FP data of CeLIN41, interacting with WT LRE and LREs mutated at stem position 1, are shown in units of millipolarization (mP). Each data point is a mean of three experiments and the error bars represent the standard deviation. The WT LRE data is the same as in a . It is replotted for easy comparison with the mutants. c Contribution of LREs of varying strengths, predicted by the model in Fig. , present in 5′-UTRs, coding sequences (CDS) and 3′-UTRs, to CeLIN41 binding as determined by linear regression. RNA binding was assayed by co-precipitation with CeLIN41, followed by RNA sequencing (RIP-seq). The error bars represent SEs for the coefficients obtained from the linear regression

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: In Vitro, In Vivo, Fluorescence, Binding Assay, Control, Standard Deviation, Comparison, RNA Binding Assay, RNA Sequencing

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: RNA binding preferences of LIN41 and Brat. a Left: crystal structure of the DmBrat NHL domain in a complex with a single-stranded linear RNA (PDB 4ZLR ). The protein surface is colored by the electrostatic surface potential from – 8 kT/e (red, acidic) to + 8 kT/e (blue, basic) and the RNA is shown as a cartoon. The approximate footprint of the RNA interaction on the protein surface is shown as a dotted red line. Right: magnified view of the RNA-binding site. A fragment of the interacting protein surface is shown and colored as on the left. The RNA is shown in surface mode, with carbon atoms in green and other atoms in standard colors. Nucleotide positions are labeled as in the RNA sequence displayed below. b Left: crystal structure of DrLIN41 in complex with the lin-29A RNA stem loop. The protein surface is colored as in a . Right: magnified view of the RNA-binding site in an orientation rotated by 90°. The RNA stem loop is shown in surface mode, with carbon atoms in gold and other atoms in standard colors. The corresponding RNA sequence is on the right

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: RNA Binding Assay, Labeling, Sequencing