Our representation of an RNA molecule is centered on secondary structure elements . By fitting a cylinder to the helix axis, these elements are carried over into a coarse-grained 3D structure representation. Integration with Biopython allows for handling of all-atom 3D information.forgi can deal with a variety of file formats including dotbracket strings, PDB and MMCIF files. We can handle modified residues, missing residues, cofold and multifold structures as well as nucleotide numbers starting at arbitrary positions. We apply this library to the study of stacking helices in junctions and pseudoknots and investigate how far stacking helices in solved experimental structures can divert from coaxial geometries.We present The creation of such potentials requires knowledge extraction from solved RNA structures, usually taken from the Protein Data Bank (PDB)2. PDB files and their newer replacement, MMCIF files, contain atomic coordinates and additional information in the header fields, but do not contain any base-pairing annotations. To extract information about base pairs and their types3, dedicated software like MC-Annotate4, RNAView5, FR3D6, DSSR7 or the RNApdbee web server8 is required. Due to the hierarchical organization of the RNA energy landscape9, it is often most convenient to treat secondary and tertiary structure of RNA molecules separately and predict tertiary structures given a secondary structure10.In RNA 3D structure prediction, knowledge-based potentials are commonly used, especially for coarse-grained approaches that are suitable for larger RNA moleculesFor knowledge extraction from RNA-containing PDB files, it is highly desirable to have a software library at hand, which understands the semantics of RNA secondary structures and makes tasks like iterating over loops of a certain type straightforward. Ideally, such a library should be written in an easily accessible scripting language, be well documented and tested and should be available under an open-source license.forgi library presented here only partly fill these needs. The Vienna RNA package11 can be used to predict secondary structures from sequence, including advanced features like G-Quadruplex prediction and incorporation of SHAPE data. It provides Python and Perl bindings, but deals exclusively with secondary structure. Biopython13 is useful for dealing with RNA sequence data and can be used to load RNA 3D structures, but has no dedicated support for RNA secondary structure. PyCogent14, a library for genomic biology, has extensive support for nucleic acid sequences, but contains only a lightweight class for RNA secondary structures and code for pseudoknot removal15. A stand-alone version of the latter was included into theforgi library under the terms of the GNU General Public License 3.0. More specialized libraries includemodeRNA16  and the FR3D suite6 . Here, we present theforgi library17, which is centered on RNA secondary structure elements  and makes them usable for 3D structure analysis.forgi aims at providing a high level API for many common operations, but can be easily extended with new functionality. The flexibility of providing an open source library in a scripting language is a clear benefit over other programs that are only distributed as binaries. While not restricted to work with PDB or MMCIF files,forgi shines especially where 3D information is analyzed in the context of its secondary structure environment.Libraries other than theforgi library17 is strongly object-oriented, but takes advantage of module-level Python functions where appropriate. The core object representing the secondary structure is theBulgeGraph object, which holds aSequence instance for the primary sequence. To include secondary structure based 3D coordinates, theBulgeGraph’s subclass,CoarseGrainRNA is available. For all-atom 3D analysis, theforgi library has a built-in integration with Biopython.TheWe will briefly describe the three main data structures that hold the sequence, secondary structure and the tertiary structure representation of an RNA.Sequence class.Primary structure: The EachBulgeGraph holds aSequence object. Sinceforgi supports loading of data from PDB files (see below), thisSequence object has to account for many special cases arising from experimental considerations, which will be detailed in the following paragraphs. There are two numbering schemes commonly used for sequences: 1-based indexing and indexing based on an external reference. In particular, many sequences in structural experiments use “1” to dedicate the first residue of a biological macro-molecule, whereas the first residue actually used in the experiment can be upstream of the functional RNA (leading to negative indices) or after the start of the biological unit (leading to an index above 1). The latter is especially common if fragments of larger RNA molecules like the ribosome are studied. Finally, experimenters might decide to insert nucleotides in the middle of a molecule. In order not to affect the numbering of subsequent residues, these inserted residues get the same number as the previous one, followed by a letter .Sequence class distinguishes indices by type. Integer indices always refer to 1-based indexing, while tuples compatible to the indices used in Biopython’s PDB module are interpreted as the second kind of indices.To handle both kinds of indexing, the2 groups and A to I editing, have been implicated with a variety of biological functions18. During parsing of PDB files, we automatically convert such modified residues to the unmodified parent, but in addition store the modification as an annotation in theSequence object. The corresponding unmodified parents for 3-letter codes of common modified residues were obtained from PDBeChem19 (http://www.ebi.ac.uk/pdbe-srv/pdbechem/) and theforgi library has the ability to query this database on the fly if it encounters a new 3-letter code.For many applications, it is necessary to restrict the RNA alphabet to 4 letters (i.e. 4 residue types), “G”, “C”, “A” and “U”. However, in the cell many RNA molecules are post-transcriptionally modified at certain positions. Many modifications, including the methylation of OH or NHforgiSequence class can store two version of the sequence, with and without missing residues, and the file parser can extract this information from PDB and MMCIF files.Finally, many experimental 3D structures do not contain coordinates for all residues present in the experiment. The20 and is related to the independently developed RAG (RNA as Graph) approach21.The secondary structure of an RNA is internally represented as a graph, the Bulge Graph, where secondary structure elements  form the nodes. Whenever these elements are adjacent along the backbone, they are connected by an edge in this graph. During the Bulge Graph creation, each node gets a unique name such as “s0” for the first stem or “h0” for the first hairpin. The concept of the Bulge Graph, illustrated inforgi supports element-based transformations of the Bulge Graph, such as condensing the secondary structure to an representation similar to RNAshapes22, which we use to classify pseudoknots, for example (see below).BulgeGraph object allows for easy identification, selection and classification of structural domains, such as multi loops, helices consisting of multiple stems and bulges (termed “rods” inforgi), and pseudoknots.TheCoarseGrainRNA class holds 3D structures.The 3D structures are loaded from PDB or MMCIF files using Biopython’s PDB module13. In order to assign a secondary structure to the RNA,forgi can call either MC-Annotate4  or DSSR7 (binary available after free registration athttp://forum.x3dna.org). As an alternative,forgi has a built-in heuristic for the detection of canonical base pairs and GU wobble pairs. This heuristic is based on distances along the hydrogen bonds and the coplanarity of the bases, and is not intended to compete with the power of more specialized tools since it will fail in some edge cases, e.g. involving modified residues or residues with missing atoms. This heuristic is a useful fallback, if the above mentioned programs are not available.20. For each stem, we store start and end coordinates of the helix axis as well as two twist vectors that point towards the minor groove at the beginning and the end of the stem. Similarly, start and end coordinates for bulges, loops and single-stranded regions are stored and can be accessed using the element’s name.During loading of the RNA, a helix axis is assigned to stems as described previouslyforgi 2.0 now fully supports co- and multi-fold structures and can load multiple chains that are connected by base pairs into a singleCoarseGrainRNA object, while chains not connected by any base pair are loaded into separate objects.Bio.PDB.NeighborSearch), a list of residues within 6 angstrom from a non-RNA C or N atom is obtained. These residues are considered protein-/ligand interacting. Knowledge of interacting residues is particularly useful to avoid biases in statistics about structural features of bare RNA.Using Biopython’s KD-Tree implementation  or Anaconda.Helpful utility scripts.forgi comes with the two very useful scripts:rnaConvert.py can be used to convert between many common file formats for RNA structures, including the Vienna format and fasta variants, the bpseq format, the connectivity table (ct) format, MMCIF format and PDB files.visualize_rna.py can be used to display a coarse-grained representation of an RNA’s secondary structure inPyMol alongside the all atom structure from PDB files, producing visualizations like those inAnalysis of stacking geometries. To illustrate how theforgi library makes secondary structure elements usable for 3D structure analysis, we used it to analyze the stacking of adjacent helices in multi loops and pseudoknots in a representative set of RNA 3D structures28 .forgi, DSSR7 (Version v1.7.1-2017nov01) was called to obtain the secondary structure and nucleotide level reference stacking annotations. We count a pair of connected helices as stacking, if at least one nucleotide of the first helix’s closing/opening base pair is in a continuous stack with at least one nucleotide of the second helix’s opening/closing base pair. This allows for any number of stacking nucleotides between the stems (not necessarily connected via the backbone) and for bulged out nucleotides which do not contribute to the stack. Our definition of stacking is more relaxed than stricter criteria used elsewhere29.While loading these 3D structures intoforgi to calculate a number of properties, such as the angle between the stem vectors, the separation vector between the stems’ ends and an offset value between the stems.For each pair of adjacent stems within a junction, we usedvh as pointing away from the multi loop segment along the helix axis, wheresh andeh are the helix’s start and end coordinates.These properties are based on the axes that were fitted to the helices (see above) and the multi loop segment that connects the two stems. We define the stem vectorsi andh is then defined as the angle between their stem vectorsvi andvh:The angle between the adjacent stemshR that start at the helix’s end closer to the multi loop segment,ch, and extend the helix axis:The offset between stems is calculated as distance between two raysDepending on the helix orientation, the distance between these rays is either the distance between two lines or the distance between two points or between one point and one line, all of which can be calculated with standard formulas.forgi was used to calculate these properties for multi loops:The following code shows howfrom forgi import load_rnafrom forgi.threedee.utilities.vector import vec_angle, vec_distancefrom forgi.threedee.utilities.vector import line_segment_distancedef main:    # Load the PDB into CoarseGrainRNA instances    # rnas is a list, because a PDB can contain multiple connected components     # This also loads the DSSR json annotations.    rnas = load_rna    for rna in rnas:        for junction in rna.junctions:             print_junction_parameters          def print_junction_parameters:    """    Prints the angles and offsets of a junction.    :param rna: A BulgeGraph Object    :param junction: A list of element names (strings)    """    for ml in junction:        # rna.edges holds adjacent nodes in the BulgeGraph        stem1, stem2 = rna.edges[ml]        # rna.coords holds the 3D coordinates of structure elements        # rna.coords["s0"] returns a tuple start-, end-coordinates of stem "s0"        # Furthermore rna.coords has the get_direction function to give the        # vector pointing from the start to the end coordinate.        direction1 = rna.coords.get_direction(stem1)        direction2 = rna.coords.get_direction(stem2)        # Get the indices into rna.coords for the stem sides closer to        # and further away from the multiloop segment ml        c1, f1 = rna.get_sides        c2, f2 = rna.get_sides        # Make sure the stem vector points away from the multiloop        if c1 == 1:            direction1 = - direction1        if c2 == 1:            direction2 = - direction2        angle = vec_angle # imported above        is_stacking_dssr = (ml in rna.dssr.stacking_loops)        closer1 = rna.coords[stem1][c1]        closer2 = rna.coords[stem2][c2]        # Calculate the offset as distance between rays.        offset = vec_distance)        print)27, Matplotlib30 and a custom library (https://github.com/Bernhard10/filterAndView) to analyze and visualize the collected data. The results were collected for different classes of RNA independently. This was necessary, because the representative sets of RNA structures contain, by design, homologs of the same molecule in multiple species.We then used pandas31 based on the definition of the mathematical genus is used. Classes of pseudoknots are defined based on their shadow representations, which contain only crossing base pairs and only one base pair each. On the level of these shadows, only four distinct classes exhibit genus 1, two of which are well known: the H-type pseudoknot and the kissing hairpin. pseudoknots with higher genus contain, among others, the case where a genus 1 pseudoknot is nested within another pseudoknot.For the classification of pseudoknots, Reidys’ conceptforgi library, we wrote a tool that is able to convert structures to their shadow representation, identify and classify the pseudoknots within the structure and describe their helix arrangement in the 3D structure. This tool, calledpseudoknot_analyzer.py, is distributed with theforgi library in the folder “examples”. We used it to gather statistics about simple H-type and kissing hairpin pseudoknots.α andβ restrict the possible values of the angleγ. We also include the representative structure of an intermolecular kissing hairpin interaction  and two angles for the relative orientation in 3D space. InThe distribution of angles between adjacent stems in tRNAs multi loops see shows thThe second family of RNA molecules with lots of data available is ribosomal RNA. Here we find that the angle of adjacent stems in multi loops is almost uniformly distributed above 20° until a peak from 135° to 170°, where DSSR annotates roughly half of the geometries as stacking see. 7% of t34).Additionally we analyzed the angle distribution between the stems forming simple H-type pseudoknots or kissing hairpins. Most angles measured within an H-type pseudoknot are between 120° and 180° see. In thisβ and especiallyα. One additional angle measurement between the two regular stems (seeγ) shows one peak at about 20° and one at about 65°. Within the class of kissing hairpins we often find coaxial stacking, but most of the time only one of the two regular stems stacks with the kissing stem in the middle. With the help of the coarse grained representation we were able to divide the class of kissing hairpins into three different structural families  with the second one (counting from the 5’ end) stacking onto the kissing stem. The anglesα andβ are above 130°. One example is the structure with PDB id 5KPY35 shown inα > β).The first family is especially common among (A-)riboswitches. Here the two regular stems are oriented almost parallel (36) shown inThe second structural family is shown inforgi is also able to model multiple RNA chains that interact with each other forming an intermolecular complex. One example is the dimerization initiation site of the HIV type 1 (PDB id 1ZCI37), illustrated inβ is close to 0°, because stacking takes place at the other side of the kissing stem and thus the complementary angle is close to 180°.The two families as well as some conformations in between are observable within intramolecular pseudoknots. As mentioned,forgi library17 we have analyzed the relative orientation of helices in junctions and pseudoknots. While stacking between adjacent helices is common, we found that their orientation often deviates significantly from co-linearity, suggesting that junctions introduce flexibility into the RNA structure while still maintaining the benefit of stacking interactions. Previous studies that assign coaxial geometries based on manual inspection38 miss this deviation from coaxiality that becomes apparent once you actually calculate the helix axis. Our approach allowed us to perform this analysis on the scale of stems, as opposed to the smaller all-atom scale used by the program DSSR and in previous surveys39 or the level of networks of  base pairs38.Using the40.Conversely, we found that out of 1257 pairs of adjacent stems in ribosomal RNA structures with an angle above 140°, only roughly half (608) are annotated as stacking by DSSR. This becomes clear if we consider that the angle between stems is only a proxy for a 5-dimensional orientation parameter. In particular, a large offset means that stem axes with an angle close to 180° can be parallel without being coaxially stacked. Indeed, it is not uncommon that all three stems in a 3-way junction are roughly parallel, with two stems forming a coaxial stack and the third having a higher offset41. Theforgi library allows us to easily identify pseudoknots in RNA 3D structures and gather statistics on the frequency of pseudoknot types, sizes, and composition. Kissing hairpins formed between two RNA molecules are known to often form continuous stacks between all three helices, such as the pseudoknot inThere is growing interest in predicting pseudoknots in RNA structures as they are involved in a variety of biological functionsSimilar to stacking conformations in multi loops, many stacking helices in pseudoknots form angles below 180°. A deviation of the coaxiality between stems can have multiple reasons such as a higher flexibility of short helices within a pseudoknot as well as strain from short stem connections. But there are also structures that show more classical coaxial stacking like PDB id 2XD0, seeforgi to the analysis of pseudoknots are described in more detail in the thesis by Beckmann42.Results and challenges of the application of20 based on our findings about stacking and pseudoknots.Varying additional parameters like the minimal number of base pairs required to count an interaction as a helix allow for a better understanding about the principles of the formation of three-dimensional RNA structures. The insight in the world of pseudoknots and multi loops presented here can support the improvement of the prediction of RNA structures and help identify unrealistic multi-loop conformations predicted by current RNA structures prediction tools. We are now in the process of implementing additional features for the RNA 3D structure prediction program Ernwinforgi17 is a multi-purpose Python library for dealing with RNA on the levels of sequence, secondary structure and tertiary structure. By providing our code as a Python library, we give users of our tool more flexibility than a single executable program could provide. Furthermore, our code is fully open source, giving researchers the possibility to comprehend and where needed extend the inner workings of the code.forgi is well documented, with a tutorial available athttps://ViennaRNA.github.io/forgi/ and a complete API documentation following the Python standard of docstrings parsable by Sphinx. It contains an extensive automatic test suite (unit and integration tests) and has been optimized for speed, maintainability and usefulness.28 at a cutoff of 4 Å,http://rna.bgsu.edu/rna3dhub/nrlist/release/3.36/4.0A.The PDB ids analyzed were taken from version 3.36 of the representative sets of RNA 3D structuresforgi 2.0 Extended Data.https://doi.org/10.17605/OSF.IO/HDJRU43. This project contains the following extended data files:assignment_of_classes_to_pdbids.csv: Assignment of PDB ids to RNA type. These annotations were generated in a semi-manual way.multiloop_angles.csv: Angles and offsets measured in PDB structures for multi loops, generated with the scriptdescribe_rna.py, which is available in the examples folder offorgi.pseudoknot_angles.tsv: Angles measured in pseudoknots, generated withpseudoknot_analyzer.py, which is available in the examples folder offorgi.Open Science Framework: 3D based on 2D:Extended data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Software available from:https://pypi.org/project/forgi/  andBioconda.Archived source code at time of publication:https://doi.org/10.5281/zenodo.258287017.License:GNU General Public License 3.0. I'm satisfied with all modifications the authors made.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors present a new version of the forgi software, a python library to analyze the tertiary structure of secondary structure elements in RNA. It takes as input almost any format of 2D or 3D information. This manuscript describes a new method for easily obtaining the angles and stacking patterns in a multi-branched loop and a pseudoknot/kissing hairpin. The authors validate their results by testing the software on known pseudo-knots and junctions, and obtain results that are consistent with the state-of-the-art knowledge of RNA local structure organization. The authors highlight significant improvements compared to previously published packages and specifically all-atom based methods. For instance, a deviation from coaxiality is very common but can be easily missed if a manual inspection is performed without doing computations. The software is clearly presented, well-validated, freely available and open source. The results presented are significant and the tool will be very useful to the RNA bioinformatics community. Although, this manuscript does not discuss extensively the analysis of the results, the latter is included in the first author’s thesis, and freely accessible as further reading. `Minor remarksPage 4: ‘’with and without missing residues’’, it is easy to imagine how the graph without the missing residue is, but how are the ‘’missing residues’’ added? Page 5: The authors mention a ‘’cleaned version’’ of the PDB. Is the only ‘’cleaning’’ performed is the conversion of the residue names?Page 5: It would be helpful to clarify what is a ‘’simplified example’’ of the code? What sort of simplification was applied to it? Is the real code significantly more complicated to use than the example? If it is not, the authors might want to consider rewording this as it appears to undermine the real simplicity of use of forgi (which is very user friendly).We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for the constructive comments.Our PDB and CIF parsers extract the information about missing residues from the files. For this purpose we have patched the biopython PDB parsing routine; this has already been merged into the biopython source. Following your input, we have revised the text to reflect this.Yes, the cleaned version only has modified residues renamed and non-rna molecules removed. We hope this is now clear from the revised text.We have expanded the sample code to compute the proper direction of helices, thus it is now fully functional and no longer "simplified".We have also added the code used to calculate the offset. For unknown reasons, the authors introduced a new spelling of the name pseudoknot. Throughout the whole article, they use spelling with space: "pseudo knots". I haven't met such a form before. The authors of the article also used the term "pseudoknot" and not "pseudo knot" in their previous works. So, the spelling should be corrected.On page 3, the authors write about FR3D suite, but they do not cite the paper about the tool. Please, include the appropriate citation here.As the paper subject is calculating helix angles and stacking parameters, I would expect to see the formulas used to calculate these data . The lack of such details is serious negligence.The article is written in a clear and concise way. Illustrations perfectly complement the content. I have only 3 comments to the authors and I would like them to be taken into account in the revision:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for the constructive comments.We have now included the exact formulas for the helix angles as requested. In particular this makes clear what sign we use for the helix vectors.The FR3D reference was present in our LaTeX source and the resulting HTML version, but got lost in the pdf conversion. This has been fixed in the revised version.


Kinetoplastid RNA (kRNA) editing takes place in the mitochondria of kinetoplastid protists and creates translatable mRNAs by uridine insertion/deletion. Extensively edited (pan-edited) transcripts contain quadruplex forming guanine stretches, which must be remodeled to promote uridine insertion/deletion. Here we show that the RRM domain of the essential kRNA-editing factor TbRGG2 binds poly(G) and poly(U) RNA and can unfold both. A region C-terminal to the RRM mediates TbRGG2 dimerization, enhancing RNA binding. A RRM-U4 RNA structure reveals a unique RNA-binding mechanism in which the two RRMs of the dimer employ aromatic residues outside the canonical RRM RNA-binding motifs to encase and wrench open the RNA, while backbone atoms specify the uridine bases. Notably, poly(G) RNA is bound via a different binding surface. Thus, these data indicate that TbRGG2 RRM can bind and remodel several RNA substrates suggesting how it might play multiple roles in the kRNA editing process. Trypanosoma brucei, T. cruzi and Leishmania spp., which are the causative agents of African sleeping sickness, Chagas disease and leishmaniasis  transcript (T. bruceiRGG2 (TbRGG2) protein is required for editing all nine pan-edited mRNAs  procyclic form (PF) or the stage that exists in mammalian hosts, called the bloodstream form (BF). TbRGG2 was shown to be essential for growth in both PF and BF forms of the parasite -rich region composed of GWG and RG repeats and a C-terminal RNA recognition motif (RRM) . Gly-ricEscherichia coli C41(DE3) cells. Protein expression of each was induced by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for 4 hours (h) at 37°C when the cells had reached an A600 of 0.4–0.6. The expressed proteins were found in the soluble fraction and were purified using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography. The His6 tags were cleaved using thrombin cleavage capture kits (Qiagen). The His-tag free proteins were further purified by Superdex 75 size exclusion column chromatography. RRM and RRM-Cterm mutants were made using the Quikchange mutagenesis protocol and expressed and purified as per the WT proteins.The DNA region encoding the TbRGG2 RRM domain (residues 203–269) and the TbRGG2 RRM-Cterm (residues 203–320) were PCR amplified and cloned into pET15b between the BamHI and NdeI restriction sites. Each expression plasmid was then transformed into P41212  by mixing protein at 10 mg/ml 1:1 with a crystallization reagent consisting of 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5 and 1.4 M trisodium citrate. Crystals took several weeks to grow to final size and were cryo-protected straight from the drop. Data were collected to 1.8 Å at the Advanced Light Source (ALS) beamline 8.3.1, processed with MOSFLM and scaled with SCALA. The crystals take the tetragonal space group, 12 Table , the modCrystals of the WT TbRGG2 RRM domain complexed with a 4-mer poly(U) RNA (herein termed U4) were obtained by mixing protein at 10 mg/ml) and RNA (in a 1:1 molar ratio) with a crystallization solution 1:1 consisting of 1.2–1.3 M sodium citrate and 0.1 M HEPES pH 7.0. X-ray intensity data was collected at the Advanced Photon Source beamline 22-ID and processed with HKL-2000 . The str0 mg/ml aN-1-methylguanine oligonucleotides were used for FP experiments to analyze RNA binding by WT RRM, WT RRM plus C-terminal domain (RRM-Cterm) and mutant RRM and RRM-Cterm proteins. For the experiments, purified proteins were titrated into 0.990 mL buffer composed of 20 mM Tris–HCl pH 7.5, 5% (v/v) glycerol and 50 mM NaCl, 20 mM and containing 1 nM  fluorescently labelled RNA. Data were fit using Logger Pro. The binding curves were normalized  using the equation: (mP – mP0)/(mPmax – mP0) × 100, where mP0 is the minimal mP for the binding assay and mPmax is the maximum mP value. Each binding curve is a representative curve from at least three technical replicates.FP experiments were performed using a PanVera Beacon 2000 FP system at 25°C. 5′-Fluoresceinated 20-mer poly(A) (A20), 20-mer poly(C) (C20), 20-mer poly(G) (G20), 20-mer poly(U) (U20), 7-deazaguanine and c oxidase (12.4 kDa), carbonic anhydrase (29 kDa) and albumin (66 kDa). For the SEC experiment with protein and RNA, purified TbRGG2 RRM-Cterm at 16 mg/ml was mixed with equimolar U20 RNA. The protein/RNA mixture was loaded onto a Superdex 75 size exclusion column and eluted in the same buffer as used for the protein alone. Two peaks were obtained from the size exclusion column, one corresponding to TbRGG2 RRM-Cterm with U20 RNA and one corresponding to excess U20.For the SEC analyses of the RRM and RRM-Cterm, the proteins were concentrated to 10 mg/ml and each were loaded onto a Superdex 75 size exclusion column and eluted with a buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, and 1 mM DTT. The elution volumes were compared to a series of protein standards to determine the molecular weights. The standards used were cytochrome http://www.glenresearch.com). Samples were ethanol precipitated, air dried, and dissolved in water. All other RNA oligonucleotides used for crystallization and RNA-binding assays were purchased from Dharmacon. To assay binding by the 7-deazaguanine modified RNA all guanines in the quadruplex-forming sequence, 5′-UAGGGUUAGGGU-3′, were replaced with 7-deazaguanine while for the m1G modified sequence, only the first three guanines were replaced with m1G. The purities of the modified and unmodified oligonucleotides were verified by NMR as described (The quadruplex-forming 5′-UAGGGUUAGGGU-3′ oligonucleotide and modified derivatives of this RNA were synthesized using a MerMade 6 Oligo Synthesizer employing 2′-tBDSilyl protected phosphoramidite (ChemGenes) on 1 μM standard synthesis columns (1000 Å) (BioAutomation). Fluorescein and 7-deazaguanine phosphoramdites were purchased from Glen Research and ChemeGenes, respectively. The synthesized oligonucleotides were cleaved from the 1 μM column using 1 ml ammonia methylamine followed by 2 h incubation at rt for base deprotection. The solutions were then air-dried and dissolved in 115 μl DMSO, 60 μl TEA and 75 μl TEA-3HF for 2′O deprotection followed by 2.5 h incubation at 65°C. Samples were purified using Glen-Pak RNA cartridges (Glen Research Corporation)  and showed no measurable binding to C20 RNA  and the RRM(L209M) structure contains one. The RRM molecules in the two structures are essentially identical; the Cα atoms of the RRM in both structures can be superimposed with root mean square deviations (rmsds) of 0.4–0.6 Å. The structure shows that the TbRGG2 RRM indeed harbors the characteristic RRM fold composed of a five-stranded anti-parallel β-sheet surrounded by two α-helices  Figure . StructuAlthough the TbRGG2 RRM adopts an RRM fold, as noted it does not contain the conserved aromatic residues located on the RNP motifs, characteristic of canonical RRM proteins. Instead it harbors valine, arginine and glutamine residues in these positions Figure , D. The 5)RY(V/I/L)F, respectively   RNA. The structure was solved by molecular replacement and refined to final s) Table . Comparis) Table  and 3A. s) Table . In thisAs noted, Trp215 is analogous to the tryptophan in the pseudoRRM motif that was shown to bind RNA in the SRSF1-RNA structure . HoweverIn addition to the RNA contacts from the Cys231 backbone and the side chains of Phe230 and Trp215, several basic residues either make contacts to the RNA phosphate backbone (Lys219) or are positioned to provide electrostatic contacts to the RNA, such as Lys230. In fact, examination of the electrostatic surface representation of the two RRM subunits interacting with the RNA reveals that the face of the dimer that contacts the RNA is notably electropositive Figure .The TbRGG2-RNA structure revealed that the TbRGG2 RRM binds U4 RNA as a dimer. However, the TbRGG2 RRM domain itself is not dimeric and there is currently no information as to the oligomeric state of full length TbRGG2. Gly-rich regions such as the N-terminal domain of TbRGG2 are primarily disordered in the absence of substrates and not known to oligomerize. Moreover, the Gly-rich domain of TbRGG2 orthologs are not conserved in sequence or length as might be expected for an oligomerization region Figure . SequencKds of 240 ± 37 nM compared to 1.4 μM, 1.9 ± 0.7 nM compared to 27 nM, 1.0 ± 0.1 μM compared to 6.3 μM and 2.6 ± 0.4 μM compared to no binding, respectively  sequences than poly(U) RNA . These dThough the TbRGG2 RRM does not employ RNP residues for poly(U) binding, the possibility existed that it may use these residues to bind poly(G) even though they do not contain the conserved aromatic residues found in canonical RRM domains. To address this possibility we generated Q205A and R238A mutations in the TbRGG2 RRM and examined G20 binding. These experiments showed that these substitutions had no significant effect on G20 binding Table . GuanineKd of 28 ± 6 nM (Poly(G) RNA repeat elements like the ones we utilized in binding studies with the TbRGG2 RRM are predicted to form higher order structures, in particular G-quadruplex arrangements . This fi8 ± 6 nM . We next8 ± 6 nM . The dis8 ± 6 nM  of G-C8 8 ± 6 nM  revealed8 ± 6 nM . These dN1-methylguanosine (m1G). These RNA substrates do not form quadruplexes as they cannot make stable Hoogsteen base pairs. This was confirmed by 1D NMR analyses of each substrate  and poly(G) RNA. A previous study using full length TbRGG2 protein as a GST fusion construct (GST-TbRGG2) also showed poly(U) binding . Howeverd to 25% . Our datRecent data have shown that OB-fold proteins, which dock on the surface of the editosome, harbor RNA chaperone activity and hence appear to play crucial roles in the editing process including, importantly, G-quadruplex unfolding . Our datIn conclusion, our combined data have revealed that the RRM from the essential kRNA editing factor TbRGG2 utilizes a non-canonical mode of RNA binding by an RRM protein and furthermore, harbors multimodal binding capability allowing it to bind multiple RNA substrates on different surfaces. It shows a preference for binding RNA poly(U) and poly(G), which are repeats elements characteristic of the kRNA editing process. How the unique RNA binding and remodeling features of the TbRGG2 RRM are involved in kRNA editing remains to be determined.Coordinates and structure factor amplitudes for RRM(L209M), WT RRM and the WT RRM-U4 complex have been deposited with the Protein Data Bank under the Accession codes 6E4N, 6E4O and 6E4P.Supplementary DataClick here for additional data file.


Halimeda has been elucidated.We assess 90 years of change on a Low Wooded Island , employing drones and topographic profiling to accurately survey ramparts, mangroves, the reef flat and the sand cay. A comparison with maps from the 1928–1929 Great Barrier Reef Expedition revealed the redistribution of an outer rampart and inward movement of shingle ridges. Remarkable lateral expansion of the mangrove woodland some 400 m has occurred as carbonate sand deposition has increased reef flat elevation, obscuring coral microatolls. The sand cay has stayed relatively constant in size, moving approximately 44 m in a northeasterly direction and rotating slightly. We conclude that the existing configuration of landforms probably represents an equilibrium with local biophysical conditions, including sea level, wave dynamics, vegetation growth, storms and cyclones. The variable nature of ramparts and the presence of a trough that prevents the continuous spread of mangroves across a uniformly flat colonization surface precludes the interpretation of landform changes with respect to a geomorphic evolutionary sequence. Moreover, longer-term implications of environmental change for these landforms can only be evaluated once the specific nature of the local carbonate budget, including the relative contribution of corals, foraminifera and  ChaThe past 90 years have seen remarkable developments in mapping technology, such that the activities that constituted ‘mapping’ on coral reef environments in the early twentieth century are now completely unrecognizable from those employed today ,12. The 1.1.Low wooded islands comprise a collection of landforms that have formed on planar reef platforms of relatively high elevation including windward shingle ridges and islands, elevated conglomerate platforms, mangrove swamps and leeward sand cays . While sThe present gross geomorphic form of the Great Barrier Reef islands reflects both their history of growth and their localized environmental setting. Microatoll evidence of a smoothly falling Holocene sea level from five low wooded island sites visited during the 1973 expedition to the northern Great Barrier demonstrated that this region of northern Queensland experienced a higher sea level in the mid-Holocene  and thatOver shorter timescales, observed changes to reef top landforms, including expansion of lagoonal reef flat mangroves and movements of sand cays and shingle ridges, have given rise to questions about the manner in which the reef top form is controlled by contemporary environmental conditions. Two distinct perspectives regarding this topic arose from the Royal Society expedition led by Sir Maurice Yonge in 1928–1929.It is well known that the sea reached its present level on the northern GBR by about 6000 years BP . Spenderet al. [An alternative viewpoint held by Steers & Kemp  suggesteet al.  noted laet al. [The 45-year assessment of change at Low Isles did not favour either of these viewpoints on the interplay between environmental conditions and reef flat geomorphic landforms. Rather, it highlighted the complexity of the recent history of low wooded islands, the difficulty of proposing simple models of their evolution and the need for continued monitoring to elucidate processes and rates of change . Of partet al.  suggesteSince the last major update of geomorphic changes to Low Isles, an increased awareness of the impacts of global climate change has raised further questions about how reef islands respond to phenomena such as sea-level rise, coral bleaching and ocean acidification, both historically and in the future. Globally, there are widely perceived notions that reef islands are some of the most vulnerable coastal landforms that will probably erode or even disappear by the end of the century . This isHow the effects of coral bleaching and ocean acidification will be felt on reef-associated landforms ultimately depends on the extent to which reef top morphology relies on carbonate sediments produced by the organisms that inhabit the reef environment . While the accretion of reef platforms and the geomorphic integrity of associated landforms are fundamentally reliant on the reef carbonate budget , the impThus, the uncertainty of linking current observations and anticipated future projections of environmental change to associated behaviour of reef landforms emphasizes the value of retrospective studies that can provide some evidence-based insights into what can be reasonably expected in the future . This st1.2.Low Isles is situated at 16°23′ S, 145°34′ E on the inner shelf of the Great Barrier Reef (GBR), some 65 km north-northeast of Cairns and 15 km northeast of Port Douglas on the Queensland coast. The outer barrier of the GBR is 40 km to the east of Low Isles. A unique characteristic of the underlying reef platform shape of Low Isles is a southward-pointing indentation along the northern reef margin known as the ‘Anchorage’. This gives the reef its distinctive horseshoe-shaped formation, and supports a reef flat composed of ‘an uninhabitable mangrove swamp and an oval sand cay’ [The prevailing winds come from a southeasterly direction for the majority (85%) of the year, except during summer months (December to February) when the northwest monsoon dominates, characterized by heavy rainfall and occasional cyclones from the Coral Sea. A southeasterly swell transports sediments length-wise (i.e. in a northwesterly direction) across the lagoon .low wooded island with limited reef top mangroves and a separate sand cay’ [Tournefortia, Scaevola, Casuarina and Ipomoea as well as a lighthouse and associated buildings. Approximately a quarter of the 1.78 km2 reef flat (0.46 km2) is covered by a mangrove forest, dominated by Rhizophora sp., and known as Woody Island, which lies some 600 m to the southeast of the sand cay. These are separated by a shallow trough or depression that lies across the northern central reef flat.Low Isles is the southernmost of a distinct type of GBR reef classified as a ‘low-wooded island’  or ‘islaand cay’ . On the Halimeda and large benthic foraminifera that inhabit the algae and seagrass beds of subtidal ponds and around the reef crest [The majority of the carbonate production takes place around the periphery of the shallow outer reef slope, particularly on the windward side. Up on the reef flat, the main carbonate producers are the calcified macroalgae ef crest .et al., provide a review of previous studies of the biophysical character of Low Isles [Since 1928, several high energy events including storms and cyclones have been observed to generate and remobilize sediment around the reef flat, forming a series of inner and outer ramparts and associated shingle ridges comprising coarse coral fragments notably around the windward reef margin ,8,30. In2.2.1.Fieldwork was undertaken at Low Isles from 8 to 12 August 2017. This combined aerial drone surveys with differential positioning accuracy to continue a 45-yearly update of observational changes to the ramparts, mangroves, central reef flat and sand cay using the best contemporary technology available. While several mapping exercises have recorded the distribution of reef flat features at Low Isles  in September 1928’. A mosaic of some of these photographs is reproduced as Plate XXVII in , p. 19. c).A topographic survey was undertaken using a Leica Sprinter automatic optical level across the ramparts in the southeastern section of the reef. This followed the same line as that surveyed in 1973 with a Kern automatic dumpy level during the Royal Society and universities of Queensland expedition to the northern Great Barrier Reef . The twofigure 22.3.Changes to the mangrove woodland boundaries were mapped across the reef flat following a procedure applied for the same purpose at Diego Garcia atoll in the central Indian Ocean to derive a continuous estimate of shoreline change . Briefly2.4.A total of 6762 images were collected via drone at an altitude of 120 m across the entire Low Isles reef system over a period of the 3 days in August 2017 during which all fieldwork was undertaken. Approximately 4000 of these images were taken from the reef flat area between the sand cay and Woody Island at low tide, such that the reef flat features including live and dead coral, invertebrates and areas of sand, seagrass and algae were exposed. Images were processed using the photogrammetric software AgiSoft Photoscan [Five topographic profiles were surveyed along ground transects, including the aforementioned rampart profile, the longest of which was a greater than 600 m traverse across the reef flat south-southwest of the cay . For further details on the drone survey and image processing, see . As a ba3.3.1.a,b). These changes include further mangrove colonization to the south in the area of P10, and across the inner rampart at the southern tip of Woody Island, where the main body of the mangrove forest appears to have coalesced with the initial colonizing patches of Rhizophora evident in the 1928 photo (a).A detailed picture of the changes that have taken place along the eastern windward reef flat over the past 89 years can be gained through comparison of the 1928 aerial photomosaic with the 2017 drone image mosaic a,b. Thes28 photo a.et al. [bare surface of reef rock  and occ50 m wide veneer of loose coral shingle over reef rock; andc, photos 6 and 5) and bassett edges .a 30 m zone of shingle conglomerate , similar to their antecedents in 1928–1929. A 1973 survey of one transect above LWN shows such a wall-like ridge 15 m wide and 2 m high separated from the mangroves by a 20 m wide coral rubble terrace 30–40 cm above the floor of the mangrove swamp, the terrace being the surface of a relic inner rampart. Our 2017 survey of this same transect indicates further northwesterly movement of the overlying shingle ridge some 40 m across the former inner rampart terrace and into the adjacent mangroves . This mobile shingle ridge is part of the former outer rampart, as mapped in 1973, which has migrated inland to form dual coral shingle ridges over the inner rampart terrace as shown in the 1973 profile, which are now spilling into the mangrove swamp. The basal portion of this deposit (the old inner rampart) has not moved as shown on the two transects (figure 2c).Some degraded remnants of the old indurated inner rampart were also present in 2017, located in the same position as it had been mapped in both 1973 and 1928, carpeted with Discontinuous outcrops of inner rampart were backed by a ‘breastwork’ or bank of coral shingle well above HWN whose movement was first described by Moorhouse . Furtherc, photos 1 and 2). Eastward migration of the mangrove swamp is constrained by the inner rampart, which rises directly from muddy substrate at a steep slope of some 60° to a crest around 2 m above the reef flat. While this distinct ridge and similar ridges along the inner margin of the eastern reef flat form a barrier to seaward migration of the Rhizophora woodland, further westward movement of the ridge is limited by the prop roots, stems and branches of the mangroves .Transects 4 and 5 comprise two or three minor ridges that suggest episodic development of the inner white coral rubble deposits indicating recent wash-over into the mangrove swamp (figure 23.2.2 (a 270% increase in area of reef flat covered). Moreover, internal expansion of the open glades in the mangrove swamp, described as ‘sinister places’ by Stephenson et al. [b). The peripheral boundary of the mangrove swamp (Woody Island) showed variability in the distance and rate of mangrove movements . The northern half of the ‘Sand Flat’ is now occupied by a mixed coral shingle and muddy sand, while the ‘Boulder Tract’ in the extreme west of the reef flat is similar in both position and form, although additional reef blocks have been deposited by tropical storms.The locations and surface characteristics of several parts of the reef flat mapped and named in the 1928–1929 expedition reports have changed over the 90-year period. These include the ‘Thalamita Flat’ in the west, the ‘Middle Moat’ around the southern and western reef rim moats, the ‘d coral’ , p. 54. 3.4.2). The cay has also rotated because of erosion in the southwest and accretion in the east . Elevations show that the beaches are slightly higher on the western and southern coastlines than the eastern and northern shores, by 80 cm and 1 m, respectively.Plan surveys of the vegetated part of the cay and surrounding beach were undertaken during the present survey b,c. Elevet al. [Surrounding the cay on the lower part of its slopes are various outcrops of ‘beachrock’ or ‘beach sandstone’; a rock formed by cementing of intertidal beach sand through the deposition of calcium carbonate between grains . Six patet al.  noted th4.−1 in the southwest compared with 2.6 m yr−1 in the northwest). Changes to the character of the reef flat include deposition of sand and shingle in the north and boulders to the west, with the appearance of a tidal moat in the southwest. The leeward sand cay appears to have migrated approximately 44 m to the northeast and rotated slightly, while maintaining a constant area.In summary, the main changes observed over the 90-year period of assessment at Low Isles include the redistribution of outer ramparts on the eastern reef flat between transects 4 and 5 , suggest4.1.redistribution of outer ramparts along the eastern sections of the reef flat,degradation of the inner rampart,c), andinward movement of shingle ridges some 40 m across the reef flat  have probably elevated the hydrodynamic energy levels in this zone of the reef flat, preventing deposition and reformation of an outer rampart in this eastern area over the past two decades. Moreover, the formation of the ramparts observed in 1991 may have been related to Cyclone Ivor (March 1990) that passed over Port Douglas some 15 km to the southwest, which was the most recent cyclone to pass Low Isles. As this cyclone approached from the southeast, it could have produced waves of sufficient height and orientation to generate ramparts on the windward reef flat. Evidence of former ramparts in this area is widespread with one example being the extensive outcrops of bassett edges, the residual partially lithified foreset beds of previous shingle ridges [figure 2c, photo 4).Our observations of the redistribution of outer ramparts, degradation of inner ramparts and inward movements of shingle ridges largely concur with those made by Frank & Jell , who sugfigure 2e ridges , as showAs also noted in the 45-year assessment of changes at Low Isles  it would4.2.Ceriops mangroves at 3.46 m and 4.37 m respectively above datum [Our data together with the 1973 survey . Our topve datum , a rangeve datum .Rhizophora seedlings become established where conditions are conducive to settlement. Broadly, these include low to moderate levels of rainfall and associated salinity levels, tidal inundation and limited subsurface current flows over the reef flat [The remarkable lateral expansion of the Woody Island mangroves some 400 m in a southwest direction across the reef flat has been possible through increases in the vertical elevation of the reef flat, itself driven by deposition of sand and shingle in the lower energy, protected leeward areas of the mangrove woodland. As the reef flat gradually accretes upward into a vertical range suitable for mangrove colonization, the patchy distribution of mangroves suggests that eef flat . Overalleef flat .et al. [Porites Pond. It was in this area that the 1934 cyclone had its greatest impact with the movement of the shingle rampart westward by 25–30 m covering the pond surface with a flattened shingle deposit. Prior to the cyclone, the moat had been rich in the coral Montipora and clam Hippopus, but after the cyclone these had been smothered by shingle from the east [Porites Pond had been encroached upon, this time from the west, by shingle from the adjacent Tripneustes Spit. They also noted that ‘Recent Rhizophora seedlings are sparsely distributed over the northern part of Porites Pond’. Later, these seedlings were reported to have died [Avicennia and 12 Rhizophora mangroves were counted during the 1973 expedition along the eastern and western sides of the pond respectively. By 2017 there was a continuous fringe of mangroves (primarily Rhizophora) comprising approximately 211 individuals visible from the drone imagery, extending all the way around the former Porites Pond and including individual plants up to 3.0 m high. This recent and rapid expansion has been accompanied by infilling of the pond such that most of the former Porites microatolls are now dead and covered with sediment. Interpreting the broader history of mangroves across low wooded islands on the northern GBR, Stoddart [Porites Pond represents a contemporary example of this latter sort.A similar situation occurs in the extreme northeast of Low Isles, where Stephenson ave died , p. 309.Stoddart  notes th2), 1973 (0.36 km2), 2012 (0.45 km2) and 2017 (0.46 km2), thereby providing an opportunity to calculate average rates of mangrove expansion over the intervening time periods. Average annual expansion rates appear to have fallen from 4.4 m2 yr−1 in the period 1928–1973 to 2.18 m2 yr−1 in the period 1973–2017, the latter figure being corroborated by the shorter 5-year window of expansion captured between the 2012 satellite image and the 2017 drone image, during which the average yearly expansion rate was 2 m2 yr−1. Such temporal variability in the rate of mangrove expansion highlights the dynamic nature of the physical processes driving expansion, drawing attention to the absence of a simplified set of consistent forces driving the evolution of the low wooded islands towards a common endpoint, as proposed by Steers & Kemp [While Fairbridge & Teichert , p. 85 ss & Kemp .At Low Isles, expansion of the mangrove front in a northwesterly direction across the reef flat is largely precluded by the deep north to south trough that separates the Woody Island mangroves from the western reef flat. Possible future changes in environmental boundary conditions including sea-level rise and a longer term reduction in the source of calcium carbonate sediments arising from coral mortality due to bleaching may compound this situation. The utility of the relative extent of mangrove coverage on the reef top as a basis for comparing the evolutionary development of a range of low wooded islands  is there4.3.et al. [While Steers , p. 15 det al. , p. 25 net al. [et al. [Acropora), interspersed with horseshoe clams (Hippopus) and occasional coral boulders’. We now know that most of these ‘slabs’ of dead coral or coral rock are fossil microatolls first recognized during the 1973 expedition when Scoffin & Stoddart [In 1928–1929 the two largest areas of the reef flat were Thalamita Flat and Sand Flat. Of the former, Stephenson et al. , p. 25 n [et al. , p. 281 Stoddart  noted thet al. [figure 4a, photo 5). Further north, the other had a much more variable substrate including dead branching coral fragments 5–10 cm in long dimension and coarse sand both with a ‘felty’ and muddy algal cover .The second largest area of the reef flat was described by Stephenson et al. , p. 25 ab) particularly through shingle rampart erosion and wash-over deposition. For instance, the leading edge of the advancing shingle rampart and its inclined slope has almost completely covered the former Fungia Moat to within a few metres of its earlier northern boundary. As a result the 30–49 cm high leading edge has been of sufficient height to provide a barrier to maintain a linear pond that has enabled coral growth of thin Porites microatolls and stunted branching corals Montipora and Millepora . Future maintenance of this moat coral and rampart morphology is unlikely as the increasing surface elevation of the potentially receiving surface of the former Thalamita Flat is above the level of surface ponding and continued coral growth (figure 4a). The erosion of shingle ramparts and dispersive influence of wash-over along the southern portion of the central reef flat may reflect the extended period of relatively low energy ‘baseline’ conditions in which hydrodynamic regimes are largely governed by the southeast trade winds, it being 28 years since Tropical Cyclone Ivor passed through the region.Between the Thalamita Flat and the outer reef slope on the southern rim of Low Isles, three parallel zones were identified, mapped and described in 1928–1929: Fungia Moat, Outer Rampart and Honeycomb Rock. All three zones were identified in 1933 , 1945 5. They wefigure 44.4.et al. [2, or less than 1% of the original cay size). Based on the edge of the vegetation line, the cay has also migrated some 44 m to the northeast. Such changes are ephemeral as the size and position of the sandy and rocky area around the cay change seasonally and periodically in line with prevailing winds, cyclones and exposure to incident waves [Comparison of the cay as mapped by Spender and described by Stephenson et al.  during tnt waves . Indeed,nt waves  report ant waves ,43,44.Some ‘characteristic’ sand cay behaviours include alongshore drift, extension of the ends of elongate islands  and migr4.5.ca 2000–0 years BP) as having extremely low rates of carbonate production and a dominance of destructive reef processes. Recent mass coral bleaching in the northern and central Great Barrier Reef [Porites bommies among the reef rock along the outer reef flat. Furthermore, the relative contributions of other carbonate producers at Low Isles, including foraminifera [Halimeda [The longer-term outlook for Low Isles will ultimately depend on the interplay between the carbonate budget and biophysical processes including wave dynamics, sea level, vegetation growth and episodic storms and cyclones. Frank  characteier Reef , which hier Reef  may compminifera  and HaliHalimeda , which c5.Episodic movements of mobile shingle across the reef flat, including the redistribution of an outer rampart and inward movement of shingle ridges.Porites Pond.The mangrove forest occupying the windward side of the reef flat known as ‘Woody Island’ expanded laterally some 400 m as the reef flat has become elevated through carbonate sand deposition, with notable mangrove colonization around the periphery of the area known as On the central reef flat surface, changes to the surface composition included disappearance, by sediment coverage, of many coral microatolls and the introduction of muds and shingle to the northern sand flat that abuts the southern shoreline of the sand cay.The sand cay itself has stayed relatively constant in size, but moved approximately 44 m in a northeasterly direction and rotated slightly.It is clear that sufficient time has elapsed since sea level reached its present elevation for the existing configuration of these landforms to have achieved an equilibrium with the physical environmental processes that shape them (e.g. wave and sediment dynamics) [In summary, the 90-year assessment of change revealed the following notable changes to the Low Isles reef flat:ynamics) . To thisynamics)  and islaynamics) . Such beynamics) , to whic


Self-replicating single-stranded RNA viruses such as alphaviruses, flaviviruses, measles viruses, and rhabdoviruses provide efficient delivery and high-level expression of therapeutic genes due to their high capacity of RNA replication. This has contributed to novel approaches for therapeutic applications including vaccine development and gene therapy-based immunotherapy. Numerous studies in animal tumor models have demonstrated that self-replicating RNA viral vectors can generate antibody responses against infectious agents and tumor cells. Moreover, protection against challenges with pathogenic Ebola virus was obtained in primates immunized with alphaviruses and flaviviruses. Similarly, vaccinated animals have been demonstrated to withstand challenges with lethal doses of tumor cells. Furthermore, clinical trials have been conducted for several indications with self-amplifying RNA viruses. In this context, alphaviruses have been subjected to phase I clinical trials for a cytomegalovirus vaccine generating neutralizing antibodies in healthy volunteers, and for antigen delivery to dendritic cells providing clinically relevant antibody responses in cancer patients, respectively. Likewise, rhabdovirus particles have been subjected to phase I/II clinical trials showing good safety and immunogenicity against Ebola virus. Rhabdoviruses have generated promising results in phase III trials against Ebola virus. The purpose of this review is to summarize the achievements of using self-replicating RNA viruses for RNA therapy based on preclinical animal studies and clinical trials in humans. Although drug development has strongly contributed to finding superior therapeutic efficacy, there is still space and need for further improvement. In addition to classic drug screening of small molecules, innovative modern approaches in biotechnology and genomics research have contributed to new therapeutic possibilities in the areas of vaccine development and gene and immunotherapy. In this context, RNA-based therapeutics have become an interesting alternative . RNA-basRelated to mRNA-based drug approaches, the employment of self-replicating RNA viruses has provided an interesting and attractive alternative to further enhance delivery and efficacy . The uniThe common feature of self-replicating RNA viruses relates to their single-stranded RNA (ssRNA) genome surrounded by a capsid core structure and a protein envelope. Alphaviruses  and flavAlphaviruses belong to the family of Togaviridiae . The mosIn a similar way as for alphaviruses, flaviviruses also possess a ssRNA genome of positive polarity . For insThe negative polarity of the MV ssRNA genome has required the engineering of packaging systems for the rescue of replicating MV from cloned DNA expression constructs . Rescue Both rabies virus (RABV)  Figure B and vesSelf-replicating RNA virus vectors have been subjected to numerous preclinical immunization studies targeting infectious diseases . In this+ T cell responses, which showed higher magnitude and broader specificity in comparison to immunizations with recombinant protein and DNA. A single administration with MHB-VLVs protected mice from HBV challenges. In contrast, immunization with SFV VLVs expressing the HBV core protein (HBcAg) did not produce CD8+ T cell responses and no HBV protection.Related to hepatitis B virus (HBV), MV vectors expressing the hepatitis B surface antigen (HBsAg) were demonstrated to elicit humoral responses in MV-susceptible genetically modified mice and rhesus monkeys immunized with MV-HBsAg remained healthy . In anotNot surprisingly, the human immunodeficiency virus (HIV) has been subjected to vaccine development applying self-replicating RNA vectors. In this context, SFV vectors expressing the HIV Env gene were administered as plasmid DNA in mice and compared to a recombinant Env glycoprotein vaccine . The humMuch attention has also been paid to vaccine development against influenza virus. For instance, SFV-based expression of influenza virus nucleoprotein (NP) elicited systemic immune responses after intravascular injections and mucosal immune responses after intranasal administration . MoreoveLassa viruses have also been subjected to vaccine development. For instance, VSV vectors expressing Lassa virus glycoproteins demonstrated protection of guinea pigs challenged with Lassa virus originating in Liberia, Mali, and Nigeria . MoreoveRelated to Middle East respiratory syndrome coronavirus (MERS-CoV), replication competent MV was used for the expression of the full-length spike glycoprotein (MERS-S) and a truncated soluble variant, MERS-solS . Both va6 IU VEE particles and 0.1 µg RNA-LNPs, whereas 1 µg naked RNA generated 10-fold lower titers. Electroporation of 20 µg DNA provided similar antibody responses as for 0.1 µg RNA-LNPs. Delivery of 0.1 μg DNA-LNPs was inefficient generating 1000-fold lower titers than for RNA-LNPs.VEE VLPs have been applied for the expression of human metapneumovirus (hMPV) and respiratory syncytial virus (hRSV) fusion (F) proteins . ImmunizSeveral studies have been carried out for simian immunodeficiency virus (SIV) vaccines based on self-replicating RNA viruses. In this context, four different KUN-based SIVmac 239 gag vaccines were evaluated in mice . In compIn support of vaccine development, gene silencing has been applied to some extent. In this context, micro-RNA (miRNA) sequences targeting RNA replication were introduced in a VEE helper vector used for replicon particle production . As cellSimilar to viral diseases, there are numerous preclinical studies carried out for cancer prevention and therapy . In this+ T cell and anti-neu IgG responses. A single administration of VEE-DCs resulted in regression of large established tumors in mice. Similarly, recombinant SFV particles carrying the vascular endothelial growth factor receptor-2 (VEGFR-2) gene were subjected to immunization studies in mice implanted with CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma [+ T cells. Furthermore, treatment of subcutaneous CT26 tumors also led to regression of CT26 lung metastases. In another approach, SFV-LacZ RNA was evaluated in mice implanted with CT26 colon tumors [+ T cell responses. Protection against tumor challenges was achieved in pre-immunized animals and therapeutic vaccination prolonged the survival of mice with preexisting tumors. Interestingly, replicon RNA did not elicit significantly more model antigen than conventional DNA vaccines in vitro, while the enhanced in vivo efficacy correlated with a caspase-dependent apoptotic cell death.Related to breast cancer, the Edmonston MV strain was engineered to express the carcinoembryonic antigen (CEA) . The MV-arcinoma . Both prarcinoma . In micen tumors . A singl+ T cell responses and prevented tumor development in mice. Moreover, vaccination resulted in efficient elimination of 7-day established tumors in 67% of tumor-bearing animals. In another study, a fusion protein of HPV E6 and E7 was expressed from an SFV vector containing a translation enhancer from the SFV capsid gene [+ T cells. However, combination with SFV-HPV 6,7 particle immunization resulted in a 10-fold intratumoral CD8+ T cell increase and the number of CD8+ T cells specific for the E7 epitope was enhanced by more than 20-fold. Irradiation also upregulated chemokines and the combination therapy provided a strong increase in the ratio of antitumoral to immune suppressive cells, thereby changing the intratumoral immune balance in favor of antitumor activity. Furthermore, a triple treatment combination regimen was established with 40 mg/kg sunitinib, a single low-dose (14 Gy) tumor irradiation, and SFV-HPV E6,7 immunization [Related to cervical cancer, VEE particles were applied for the expression of human papilloma virus-16 (HPV16) E7 protein . Immunizsid gene . Tumor-bsid gene . In compsid gene . Local lnization . This trRelated to lung cancer, it has been demonstrated that Dengue virus is able to infect and replicate in human primary lung epithelium and several lung cancer cell lines . The sus+ lymphocytes and induced protection against challenges with malignant melanoma cells. Moreover, YVF vaccination induced regression of established solid tumors and pulmonary metastases. Additionally, alphavirus vectors have been applied for melanoma treatment. VEE particles expressing tyrosine-related protein-2 (TRP-2). Evaluation in a B16 mouse transplantable melanoma model revealed humoral immune responses, durable antitumor effect, and prolonged survival [+ T cell immune responses in tumor bearing mice [In the context of melanoma, several preclinical studies have been conducted. For instance, yellow fever virus (YFV) expressing a cytotoxic T lymphocyte epitope derived from chicken ovalbumin (SIINFEKL) was evaluated in mice . Immunizsurvival . Furthersurvival . Adminissurvival . In compsurvival , demonstsurvival . Moreovesurvival . Oncolytsurvival . In conting mice . MoreovePancreatic cancer has also been the target of preclinical studies. In this context, 13 pancreatic ductal adenocarcinoma (PDA) cell lines showed superior oncolytic abilities of VSV compared to conditionally replicative adenovirus (CRAd), Sendai virus, and RSV . Moreove6 TCID50 of MV-CEA. In another approach, VEE particles expressing prostate-specific membrane antigen (PSMA) were applied for immunization of mice and rabbits [5 infectious units (IU) of VEE-PSMA was compared to immunization with purified PSMA protein. The VEE-based approach provided stronger immune responses than adjuvant PSMA protein. Furthermore, the immunogenic doses were well-tolerated. VEE particles have also been applied for expression of six-transmembrane epithelial antigen of the prostate (STEAP) [+ T cell responses and prolonged survival rate. Similarly, immunization with VEE particles expressing prostate stem cell antigen (PSCA) induced long-term protection against prostate cancer in prostate cancer-prone transgenic adenocarcinoma mouse prostate (TRAMP) mice [Related to prostate cancer, self-replicating RNA vectors have been applied for studies in several animal models. In this context, MV was shown to efficiently infect and kill PC-3, DU-145, and LnCaP prostate cancer cell lines, and was applied for expression of CEA . Signifi rabbits . A singl (STEAP) . ImmunizMP) mice . VaccinaMP) mice . Intratu7 IU to 4 × 108 IU of VEE-CEA particles were given every three weeks for four immunizations. Repeated immunization induced clinically relevant CEA-specific T cell and antibody responses. The antibody-dependent cellular toxicity against tumor cells from human colorectal cancer metastases were mediated by CEA-specific antibodies. Moreover, longer overall survival was observed in patients with CEA-specific T cell responses. Propagation-defective VEE particles expressing the PSMA have also been subjected to a phase I clinical trial in patients with castration resistant metastatic prostate cancer (CRPC) [7 IU or 3.6 × 107 IU of VEE-PSMA were administered to patients with CRPC metastatic to the bone. Vaccinations were well-tolerated at both doses although only weak PSMA-specific signals were detected. Although neither clinical benefit nor robust immune responses were achieved, the elicited neutralizing antibodies suggest that dosing was suboptimal. In another approach, SFV particles expressing IL-12 (LipoVIL12) were encapsulated in liposomes, which provided passive tumor targeting and protection against host immune recognition [9 particles per m2.Self-replicating RNA viruses have been subjected to a limited number of clinical trials . In thisr (CRPC) . Five doognition . LipoVIL6, 2 × 107 or 1 × 108 pfu) of VSV-ZEBOV to assess safety and immunogenicity of the vaccination. Some adverse event such as injection-site pain, fatigue, myalgia, and headache occurred. Lower titers were observed at day 28 for the dose of 3 × 106 pfu in comparison to the other two doses.Several VSV-based clinical trials have been conducted . Two pla7 pfu [5 pfu compared to previous doses of 1–5 × 107 pfu [Furthermore, a second dose at day 28 significantly increased the antibody titers at day 56, but the effect disappeared after 6 months. In another randomized, dose-ranging, observer-blind, placebo-controlled phase I trial, 40 participants received the attenuated VSVΔG-ZEBOV-GP vaccine . No seri7 pfu . In the  107 pfu . The low7 pfu of rVSV-ZEBOV [7 pfu and 1 × 108 pfu of rVSV∆G-ZEBOV-GP and placebo for the assessment of safety and immunogenicity. The vaccine was generally well-tolerated. Although systemic adverse events occurred in comparison to placebo, no vaccine-related severe adverse events or deaths were reported. Overall, the results confirmed the safety of vaccination of the EBV risk population with rVSV∆G-ZEBOV-GP.A randomized, placebo-controlled phase III trial was conducted in 1500 adults with the chimpanzee Ad3 (ChAd3-EBO-Z) and the recombinant VSV (rVSV∆G-ZEBOV-GP) vaccines in Liberia . AdverseSV-ZEBOV . In the SV-ZEBOV . A singlSV-ZEBOV . Vaccina3 TCID50. Complete regression of CTCL lesions was observed in one patient, while partial regression was observed in the other patients. Related to recombinant MV, a phase I trial was conducted in patients with advanced ovarian cancer by intraperitoneal injection of MV-CEA [3 to 109 TCID50 confirmed no dose-limiting toxicity. The best objective response comprised stable disease in 14 patients with a median duration of 88 days and a range of 55 to 277 days. All individuals vaccinated with higher dose levels (107–109 TCID50) accomplished stable disease, whereas only five out of 12 patients vaccinated with 103–106 TCID50) achieved it. MV-CEA has also been planned for a phase I clinical trial in patients with recurrent glioblastoma multiforme [5 TCID50 of MV-CEA escalating to the maximum dose level of 2 × 107 TCID50. One group of patients will receive direct injections into the resection cavity and in the other group MV-CEA will be administered into recurrent tumors. So far, three patients have received 1 × 105 TCID50 and three other patients 1 × 106 TCID50 in the resection cavity showing no dose-limiting toxicity. Oncolytic MV vectors expressing the human sodium iodide symporter (NIS) have been subjected to a phase I trial [6–1 × 109 TCID50) revealed that the MTD was not reached. Therefore, doses of 1 × 1010 and 1 × 1011 TCID50 were tested and the latter dose was planned to be used in a phase II trial. A complete response was observed in one patient treated with 1 × 1011 TCID50. The response persisted for 9 months after which an isolated relapse occurred in the skull without recurrent marrow involvement. Irradiation of the lesion resulted in the patient remaining disease-free for an additional 19 months. Another patient had subjective softening and shrinking of her extramedullary plasmacytomas of her back and thighs.MV-Edm vaccine strains have been tested in clinical trials against breast, ovarian, head and neck cancer, glioblastoma, and myeloma ,122. In f MV-CEA ,123. Admltiforme . The stu I trial . PatientIn summary, numerous preclinical and clinical studies have confirmed the feasibility of the approach of applying self-replicating RNA viruses for both preventive and therapeutic use for various diseases . In thisAlthough a relatively small number of clinical trials have been conducted with self-replicating RNA viruses, there has been some promising results. Especially, several phase III trials on MV-based vaccines against EBV have provided good safety profiles and protection ,115,116.One important issue related to the utilization of any delivery system is safety. In the first phase, it is essential to provide high safety standards during laboratory research and large-scale production to ensure the protection of personnel. Related to self-replicating RNA viruses, special attention has been paid to the engineering of helper virus vectors used for virus preparation both at laboratory and large-scale. Initially, introduction of point mutations in the p62 precursor of the SFV E2 and E3 envelope genes rendered generated recombinant particles conditionally infectious requiring an additional activation step with α-chymotrypsin . This seLooking into the future, continuous vector development aiming at delivery and safety improvements will certainly support the progress in therapeutic applications of self-replicating RNA viral vectors. Moreover, dose optimization studies, especially at the clinical level, needs to be conducted. As vaccine development and gene therapy approaches have taken giants leaps recently with classical approaches and more pioneering efforts using viral vectors and nucleic acids. The attractive features of self-replicating RNA viruses relate to the easy of virus production, broad host range, high safety levels due to no risk of chromosomal integration, targeting of DCs, but most importantly the extreme RNA replication in the cytoplasm, which supports high level transgene expression as the basis for generating strong immune responses. Today, RNA-based delivery provides an attractive approach, especially combined with either polymer- or liposome-based encapsulation strategies.


The IGF2 mRNA-binding proteins  are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome. ZBP1 and IMP2 belong to the IGF2BP family of RNA-binding proteins. Here the authors employed SELEX, NMR spectroscopy and mutagenesis to characterize the RNA-binding preference of ZBP1 and IMP2. RBPs participate in all aspects of an RNA’s life2, many are deposited as soon as the RNA is transcribed and they contribute to control over export, localization3, translation4, and decay5. Progress has been made in studying RBPs on the atomic level6, with single cell resolution7, as well as on a genome wide level by profiling the RNAs that interact with individual RBPs8. Many families of RBPs are involved in the regulation of normal and pathological processes, but how this regulation occurs or how differences in regulation amongst family members have evolved is not appreciated. Some prominent RBP families that have been studied on both a structural and genome wide level, include Fragile X proteins11, NOVAs14, and IGF2BPs18.Post-transcriptional regulation of RNAs is accomplished through their interactions with RNA-binding proteins (RBPs). With over 1000 RBPs expressed in humans19. The founding member of the IMP family, ZBP1, was first characterized in chicken embryonic fibroblasts and then subsequently its paralogs were discovered and named CRD-BP, IMP1, IGF2BP1, and VICKZ1. While the naming convention varied according to the lab that discovered the protein, the functions of ZBP1 as well as amino acid sequence identity are highly conserved across species. In mammals, there are three IMP family members . Each protein contains six canonical RNA-binding domains, two RNA recognition motif (RRM) domains and four hnRNP K homology (KH) domains. These RNA-binding domains are arranged into three pairs . In humans, IMP members share an overall sequence identity of 56%. The percentage increases to 70% when unstructured linker regions are excluded and the comparison is limited to the individual RNA-binding domains20. Though these proteins share similar amino acid sequences, each IMP family member regulates a different pool of cellular RNAs and knockouts (KO) of ZBP1, IMP2, and IMP3 have identifiable phenotypes22.The IGF2BP family (IMP) consists of highly conserved RBPs23, and in so doing, prevents its translation until it reaches its destination24, be it the leading edge of a fibroblast26 or a synaptic spine destined for remodeling27. ZBP1KO mice are perinatal lethal, with gross developmental abnormalities, consistent with the observation that ZBP1 expression is maximal during the period of mid to late embryonic development22. To identify targets of ZBP1, systematic evolution of ligands by exponential enrichment (SELEX) followed by biochemical and structural characterization revealed two RNA elements28. The identity of RNA bases and the spacing between them was critical for recognition29. Genome-wide searches using these stringent binding criteria identified mRNA targets that are critical for cell growth, organization, and neural development, data that is consistent with the phenotype of the ZBP1KO mouse22. These studies provided a list of targets that are critical for ZBP1’s roles in fibroblast migration30, cancer metastasis32, and synaptic plasticity34.It has been shown that ZBP1 binds to the 3′ UTR of ß-actin mRNA20. IMP2 shares less sequence homology with the other two family members and its expression persists throughout life35. Recently, IMP2KO mice have been observed to have a metabolic phenotype that extends their lifespan and renders them resistant to high fat diet induced obesity and type-II diabetes21. Further corroborating the role for IMP2 in metabolism, studies have shown that IMP2 regulates the translation of subunits critical for oxidative phosphorylation in normal cells as well as in glioblastoma36. Moreover, both IMP2 and a splice variant (p62) appear to be harbingers of a poor prognosis in gastrointestinal, hepatocellular and breast carcinomas39, possibly through its regulation of mitochondrial function36.Investigation of IMP2 has been less extensive than for ZBP1 and IMP3. ZBP1 and IMP3 share high-sequence identity  and common temporal expression patterns . Clinical studies have found their expression to be reactivated in a range of tumors, hinting at a potential role for ZBP1 and IMP3 in cancer pathogenesis18, we investigated individual targets and find, while some targets can bind to both ZBP1 and IMP2, others are specific for one or the other. The similarities and differences between the binding elements of ZBP1 and IMP2 reveal how RNAse digestion based techniques (such as CLIP) can digest away a necessary “GG” motif that is common to the consensus RNA-binding sequence of both proteins. Here, utilizing both crystallography and nuclear magnetic resonance (NMR) spectroscopy revealed how divergence in amino acid sequence can contribute to differences in RNA specificity. Targeted mutations in the amino acids of IMP2 allow the mutant to bind to ZBP1 targets. This represents how engineering of KH domains can provide insights into how this highly conserved family of RNA-binding proteins has evolved to express different genes post-transcriptionally within the cell.Though much work has gone into studying individual family members of the IMP family, it is not clear if they recognize similar or different targets. While genome wide CLIP studies show a correlation between the total RNA pools targeted by each protein29. To determine if there are differences in the overall topology of the KH34 domains, we first crystallized the KH34 domain of IMP2 and compared it to the previously determined structure of ZBP115  in their RNA-binding domains Fig. . In vivo115 Fig. .Fig. 1ZBα atoms)  and were also arranged in an anti-parallel pseudo-dimer orientation. The overall structures of IMP2KH34 and ZBP1KH34 are almost identical  Fig. . Because16). We observed that IMP2KH34 had lower affinity to the ß-actin C28 zipcode RNA (Kd = 161 nM)  . We used the minimal ß-actin zipcode element as an in vitro RNA (C28 zipcode RNAnM) Fig.  comparednM) Fig. . This re16 . The N30 pool was passed over an amylose resin bound to a recombinant MBP-IMP2KH34 fusion protein. After nine rounds of selection, we found that the RNA library was bound to IMP2KH34 with an approximately 500-fold higher affinity compared with the initial randomized RNA library Fig. . From th16 Fig. . This inTo demonstrate that the CA and GG elements found in the SELEX procedure were both necessary for binding to IMP2KH34, in vitro transcribed RNAs with either the CA or GG sequences mutated to CG or AA, respectively, were tested for binding by IMP2KH34. We observed that the mutation of either the CA or the GG element completely abolished binding of the RNA to IMP2KH34  compared with ZBP1KH34 (Kd = 8 nM)  increased across the subsequent rounds of selection   Fig. . We foun 9) Fig. . In cont15N-HSQC spectra were collected of KH34 in complex with short RNA sequences containing either the 5′ RE CCC-UCA-CC or the 3′ RE UU-UGGA-AC , therefore we determined if the two nucleotides adjacent to the CA in the ß-actin zipcode specifically inhibited IMP2 binding. We systematically mutated the individual nucleotides within and around the CA region of the ß-actin zipcode  Fig. . The GG 15. We identified 2790 human and 2138 mouse 3′ UTRs containing the bipartite IMP2KH34 RE. To enrich for bona fide IMP2 mRNA targets, we used the overlap of these two lists to identify 503 genes that had the bipartite IMP2KH34 RE evolutionarily conserved  enrichment over input or IgG controls  motif than IMP2’s (A/U)-GG-(A/U) motif16. This points towards a consistent evolutionary divergence amongst IMP family members for novel recognition sequences , sequences that have been previously shown to prevent RNAs from binding to ZBP1KH34. This specificity was mediated solely by specific variable loop amino acid mutations.It is likely that both the variable loops amino acids and target RNA sequences co-evolved when gene duplication of the IMP family occurred. Given that ZBP1 most resembles the ancestral family member and is essential for organism survival, the process of gene amplification of the IMP family members must strike a balance between loss of function and gain of function. During its evolution IMP2 has lost the ability to bind to the ß-actin zipcode and evolved away from a role in ß-actin localization to provide overall benefit to the organism by gain of function. Therefore, IMP2 has neo-functionalized with respect with ZBP1 to regulate transcripts involved in energy metabolism. Interestingly the IMP2KO mouse is resistant to diabetes through an increase in brown adipose tissue and increased thermogenesis. This phenotype is strikingly similar to the function of one of its evolutionarily conserved targets, GRB10  show extensive interactions of RNA with the variable loops. However, to date, the few didomain co-crystals do not show a single RNA spanning an interaction with both domains but rather the structures have one RNA hairpin per domain46. This is contrary to the biochemical evidence and points toward a limitation of crystallography.Like many other Type I KH domains, the overall structure of ZBP1KH34 and IMP2KH34 are very similar in the free state. As has been previously shown in the limited number structures containing of KH RNA complexes, the RNA bound state shows little change compared to the RNA free state47, invasive potential48, and patient prognosis49. Work has also shown that IMP overexpression alone is sufficient to increase invasive potential50 and that the IMP family are among the most up regulated RBPs across the TCGA51. What is still not understood is why these selective patterns of IMP expression provide benefit to the cells or tumors and the first step required to understand this is a faithful and unbiased profiling of RNA targets. The in vitro approach taken in this study is not limited by cell type and provides the framework necessary for follow up in vivo studies of the IMP family.The IMP family members are selectively expressed during different developmental time points. While ZBP1 and IMP3 are largely limited to embryonic expression, IMP2 is broadly expressed and its expression persists throughout life. Immortalized cell lines as well as cancers selectively overexpress different IMP family members and their overexpression correlates with growth rate15.For purification of maltose binding protein fusions, the coding sequences for ZBP1KH34 (404–561) and IMP2KH34 were cloned into a modified pMalc2 where a C-terminal 6×HIS tag was added by PCRFor purification of his tagged protein fusions, the coding sequences for ZBP1KH34 (404–561) and IMP2KH34 were placed in a pet22HT vector, downstream of N terminal 6xHIS tag followed by a TEV site.Mutations were introduced by synthesizing in frame DNA oligos containing the appropriate restriction sites (Invitrogen). Oligos were annealed by mixing in each oligo (1 µM) in 100 mM NaCl. The mixture was heated at 95 °C for 5 min then allowed to cool on the bench top. This reaction was then diluted 100× and 1 µL was used for ligations.13C, 2d-glucose and 1 g 15N NaCl (for NMR spectroscopy experiments) and incubated at 37 °C. The cultures were monitored by OD600 readings and induced with 1 mM IPTG when the OD600 reached between 0.6 and 0.8. Induction was performed for 4 h at 37 °C, at that time cells were pelleted and stored at −80 °C until ready for purification.Bl21 Rosetta™ 2 cells (EMD Millipore) were combined with the plasmid of interest and transformed by heat shock. Single colonies were selected for 5 mL cultures and then transferred to 1 L of either LB (for unlabeled protein expression) or M9 deuterated minimal media supplemented with 3 g MBP protein preparation was performed by a crushed complete™ EDTA-free protease inhibitor tablet (Roche) was added to the cell pellet before being resuspended in lysis buffer  and sonicated. Lysate was then centrifuged and the supernatant was passed over an amylose column (New England Biolabs) and washed for 4 h. Fractions were eluted and checked by comassie stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Protein was concentrated using centricon spin column, final protein concentration was determined by nanodrop absorbance at 280 nm and/or by Bradford assay.His protein purification was performed by lysing bacterial cell pellets in 50 mM sodium phosphate, 1.5 M NaCl, 10 mM imidazole and after centrifugation the supernatant was passed over a TALON™ column (Clontech). Fractions were eluted and checked by comassie stained SDS-PAGE gel. Protein was concentrated using centricon spin column, final protein concentration was determined by nanodrop absorbance at 280 nm and/or by Bradford assay.Totally, 100 pM RNAs were incubated at room temperature for 3 h with twofold dilutions of the purified RNA-binding protein in 10 mM Tris, 100 mM NaCl, 0.1 mM EDTA, 0.01 mg/mL tRNA, 50 μg/mL heparin and 0.01% IGEPAL CA630. Complexes were then run using 5% native PAGE in 0.5× TBE and visualized using the Typhoon 9400 variable mode laser scanner .52, analyzed using both CCPN Analysis53 and NMRFAM-Sparky54. Chemical shifts were indirectly referenced to sodium 2,2-dimethyl-2-silanepentane-5-sulfonate (DSS), using absolute frequency ratios for the 1H signal55.NMR spectroscopy used purified His-IMP2KH34. 2D 1H 15N HSQC experiments were acquired on a Bruker DRX600MHz spectrometer. 3D deuterium decoupled gradient sensitivity enhanced triple resonance experiments  experiments were acquired either on a Varian INNOVA 600 or a Bruker Avance™ 800 spectrometer with non-uniform sampling. NMR data were processed using NMRpipe/NMRDraw56.IMP2KH34 (0.5 mM) was crystallized using sitting-drop vapor diffusion at 22 °C by mixing equal volumes of the protein and reservoir solution . Crystals were cryoprotected by soaking them in the reservoir solution supplemented with 25% glycerol before flash cooling in liquid nitrogen. Data were collected to 2.05 Å resolution from a single crystal at the Advanced Photon Source SGX-CAT beam line  at a wavelength of 0.9792 Å. The diffraction data were indexed, integrated and scaled using Mosflm and the CCP4 suite of programs1 with unit cell parameters a = 76.88, b = 62.38, c = 85.74, α = γ = 90°, β = 91.32°. The structure of IMP2KH34 was determined by molecular replacement with Phaser using the structure of ZBP1KH34 (3KRM) as a search model57. The resulting model for IMP2KH34 was then used as the input for the AutoBuild routine within Phenix for automated model building58. Rounds of manual model building and refinement were then performed using Coot59 and Phenix60. Protein stereochemistry was checked using Molprobity61. The final model contains residues 425–581 of IMP2 with 100% of amino acids in the allowed region and 98.58% of amino acids in the favored region of the Ramachandran plot.The crystals of IMP2KH34 belong to the monoclinic space group P2TTTCGACGCACGCAACTATC-(N30)-GCTAAACTGCGTCGCTCTGCCC-3′ by chemical synthesis (Integrated DNA Technologies). Thirty nucleotides (N30) were randomized to 25%A, 25%T, 25%G, and 25%C. Constant sequence regions are italicized. The N30 library (200 pmol) was converted to dsDNA by reverse transcription following the manufacturer’s specifications  using the IMP2N30 top strand primer 5′-GAtaatacgactcactataggGCAGAGCGACGCAGTTTAGC-3′. T7 promoter sequence in lowercase. Then, N30 RNA pool library used for selection was generated by in vitro T7 transcription  of the dsDNA library and gel-purified. The stringency of selection was increased by progressively reducing the concentration of the purified MBP-IMP2KH34 in the binding reaction: in the first round of selection, MBP-IMP2KH34 (300 nM) was equilibrated with the library RNA pool (400 nM). In subsequent rounds of selection, the concentration of MBP-IMP2KH34 was reduced to 30 nM (rounds 2 and 3), 15 nM (round 4), 3 nM (round 5), and 1 nM (round 6–9), while RNA concentration was reduced to 300 nM (round 2–4), 90 nM (round 5), 30 nM (round 6) and 60 nM (round 7–9). After phenol/chloroform extraction, the RNA selected in every round was converted to cDNA by reverse transcription  using the IMP2N30 bottom strand primer 5′-TTTCGACGCACGCAACTATC-3′. The resulting cDNA library was then amplified by PCR using the IMP2N30 top strand and IMP2N30 bottom strand primers and then in vitro transcribed into RNA for the next round of selection. Subsequent rounds of selection were performed similarly, except that a negative selection step was included by incubating the RNA pool with amylose resin in the absence of protein to remove any RNAs with nonspecific affinity for the amylose resin. N30 Random RNA pool library and RNA from rounds 4, 7, 8, and 9 were fluorescein-labeled, and their affinity for MBP-IMP2KH34 and MBP-ZBP1KH34 (control) was quantified by EMSA.Selection of RNAs was performed by preparing an antisense degenerate library with the sequence 5′-™ TA Cloning™ Kit (Invitrogen) following manufacturer’s specifications. Colonies from the transformation were then individually picked and plated for colony sequencing (Genewiz). At least 25 colonies were sequenced per round using M13R primer 5′-CAGGAAACAGCTATGAC-3′.The N30 Random DNA pool and cDNA from rounds 7-to-9 were cloned into TOPOP-value, based on a right-tailed Fisher’s exact test, considers the number of identified focus genes and the total number of molecules known to be associated with these categories in the IPA Knowledge Base. Significance was determined when an individual pathway reached a false discovery rate of <0.05.FoldUTR3 tables were downloaded from the UCSC table browser for human (GRCh37/hg19) and mouse (NCBI36/MM8). UTRs were queried for the nucleotide sequence CUCAC-(N10-15)-(A/U)-GG-(A/U), where N represents any intervening nucleotide. Genes demonstrating conservation of this sequence motif between human and mouse genomes were identified as potential RNA ligands of IMP2. IPA Knowledge Base 9 (Ingenuity Systems) was used to determine diseases and functional category enrichment of these potential ligands. The 18 was visualized by retrieving input normalized tags and peaks for SEPT8 in the Integrated Genome Viewer.Previously published eCLIP data for ZBP1 and IMP2 H9ES cells43 and mouse brown adipose tissue21 from SRA. FASTQ files were then aligned to a transcriptome index with kallisto62 and the output TPMs were used to perform Log2(RIP/control) calculations.RIP-seq was analyzed by downloading FASTQ files from HEK293T cellsFurther information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1-2Source Data