We quantified the abundance of four picornaviruses and the reovirus monthly from August 2014 to May 2015. Patterns of viral abundance in the >5 μm size fraction and representation in microbial-sized community RNA libraries over time suggest that one picornavirus genotype (TS24835) and the reovirus (TS148892) may infect small (<5 μm) eukaryotic microorganisms, while two other picornaviruses (TS24641 and TS4340) may infect larger (>5 μm) eukaryotic microorganisms or metazoa. Our data also suggest that picornavirus TS152062 may originate from an allochthonous host. All five viral genotypes were present in at least one size fraction across all 3 lakes during the year, suggesting that RNA viruses may easily disperse between adjacent aquatic habitats. Our data therefore demonstrate that RNA viruses are widespread in temperate lacustrine ecosystems, and may provide evidence of viral infection in larger eukaryotes (including metazoa) inhabiting the lakes.Decades of research have demonstrated the crucial importance of viruses in freshwater ecosystems. However, few studies have focused on the seasonal dynamics and potential hosts of RNA viruses. We surveyed microbial-sized (i.e. 5–0.2 μm) mixed community plankton transcriptomes for RNA viral genomes and investigated their distribution between microbial and macrobial plankton over a seasonal cycle across three temperate lakes by quantitative reverse transcriptase PCR (qRT-PCR). A total of 30 contigs bearing similarity to RNA viral genomes were recovered from a global assembly of 30 plankton RNA libraries. Of these, only 13 were found in >2 libraries and recruited >100 reads (of 9.13 x 10 Nearly 30 years of research has highlighted the crucial role of viruses in aquatic ecosystems, where they cause significant mortality of microbial and metazoan hosts, facilitate gene exchange, and influence elemental cycling –5. ViralTemperate lacustrine ecosystems represent interesting habitats in which to study the diversity and temporal dynamics of aquatic viruses for several reasons. Lakes receive constant and episodic allochthonous inputs from terrestrial environments (i.e. runoff). Lakes at high latitudes experience distinct seasonal shifts in biological phenomena, including seasonal (primarily spring) blooms, post-bloom clear-water phases, and ice cover in winter. They are also subject to greater variability in natural disturbance events  than lakes at lower latitudes. Lakes also experience various mixing regimes, from meromictic to polymictic which influence their productivity and microbial ecology. These distinct mixing regimes and productivity cycles provide ideal conditions to examine dynamics of viral groups by providing stark temporal changes in host abundance.-1 [-1) [The composition of viral communities in aquatic ecosystems is a function of production of new virus particles through autochthonous infection and import from allochthonous sources and their decay –38. Runo-1 , while i-1 [-1) , 44, and-1 [-1) , 46. ThuNumerous studies in the last two decades have focused on surveying viral composition in aquatic ecosystems to understand patterns of diversity as they relate to environmental parameters and hosts –27. MostThe purpose of this study was to understand RNA viral composition in three temperate lakes with variable hydrology and allochthonous inputs. We first surveyed RNA viral genomes using a community transcriptomics approach targeting microbial-sized (0.2–5 μm) material. We identified RNA viral genomes within transcriptomes, then evaluated their seasonal distribution in the lakes on particles >5 μm . Our results revealed that several RNA viral genotypes are temporally transient and lake-specific, may relate to precipitation and runoff, and may follow seasonal patterns of productivity.2) relative to its size, and is surrounded by agricultural land [The Finger Lakes  are a series of 11 freshwater lakes oriented approximately north-south, ranging in length from 4.8 km to 64 km, and in maximum depth from 29 m to 188 m. Cayuga Lake, which is the longest of the Finger Lakes, is mesotrophic , having ral land . As a coral land .oN, 76.470780oW; OL); Seneca Lake  and Cayuga Lake  , chlorophyll a concentration (~ 5 L), and plankton RNA (~20 L). No specific permissions were required for these locations/activities, and this work did not involve endangered or protected species.Samples were collected monthly between September 2014 and August 2015 from three locations: Owasco Lake  . In Febra analysis (0.1–1 L) were diafiltered through 47 mm diameter GF/F filters, which were subsequently encased in alumninium foil and frozen at -20°C prior to analysis. Samples for chlorophyll a were analysed by the acetone extraction-fluorometry approach [a concentration determined without correction for phaeophytin in a hand-held Turner Designs Fluorometer.Duplicate samples for chlorophyll approach . Filters-1) for 2 min before being filtered through black 0.2 μm polycarbonate filters. The filters were then mounted on glass slides using PBS:Glycerol (1:1) as mountant. Slides were kept frozen prior to microscopy. Bacteria were counted on an Olympus BX-51 epifluorescence microscope under blue light excitation and at 1000X magnification. Ten random fields on each slide containing >200 cells were counted for each slide and abundance calculated from the average bacterial abundance per field.Samples for bacterioplankton abundance (10 mL) were collected and fixed in 2% formalin and stored at 4°C prior to analysis. Preparation of slides for bacterial enumeration occurred 7–14 d after sample collection. Bacterial abundance was determined in fixed samples by DAPI epifluorescence microscopy . DuplicaDuplicate samples for plankton RNA . Contigs matching the RNA viral database at an e-value < 10−10 were then subject to BLASTn against the non-redundant (nr) database at NCBI to confirm viral identity. RNA viral contigs (i.e. those matching RNA genomes via tBLASTx) were then subject to fragment recruitment using a minimum overlap of 0.1 and minimum similarity of 0.95 for all environmental RNA libraries. Only contigs recruiting ≥ 100 reads across ≥ 2 libraries were considered further. Contigs fulfilling these criteria were then subject to tBLASTx against the nr database to identify their phylogenetically closest relative. Sequence data is available at NCBI under BioProject accession PRJNA417956.Sequence libraries were first vetted for poor quality sequence (Ambiguous nucleotides ≤1), trimmed for adapters and length using the CLC Genomics Workbech 4.0. Following this, libraries were assembled using the program’s http://www.primer3.com) [-1 based on linearity of standards and absence of amplification in no template controls; this equates to <0.001 genotype copies mL-1 based on the volumes of water filtered.Five candidate RNA viral phylotypes were selected for focus that were represented in ≥ 5 libraries (er3.com) . qRT-PCRAll statistical analyses were performed in Microsoft Excel using the XLStat plugin (Addinsoft SARL). Pairwise correlation significance (Pearsons ρ) α values were corrected for Type II error by dividing 0.05 by the number of comparisons made.RNA viral communities inhabiting lacustrine ecosystems are a milieu of temporally and spatially variable agents which may arise from both aquatic infections and from infection of hosts in non-aquatic habitats. Our data provide evidence that RNA viruses observed in microbial-sized (0.2–5 μm) plankton RNA libraries may, in fact, represent viruses of larger (>5 μm) microbial eukaryotes, aggregates of eukaryotes, or metazoa. Some RNA viral genotypes had a seasonal trend which may be related to overall patterns of lake productivity  and precipitation/runoff. Our data provide evidence that several RNA viral genotypes were dispersed between all three lakes, but their presence in different size fractions varied between lakes.All three lakes demonstrated a seasonal pattern of temperature consistent with mean air temperature . Lakes ea concentration between August and December 2014 were concurrent with decreased water temperature across all three lakes. Elevated chlorophyll a from March to June 2015 suggested a spring bloom occurred  library at NCBI ; S2 Fig,ustralis  and one crophyte . These dPicornavirales. TS24835 and TS24641 do not contain sufficient genome architecture or phylogenetic information to assign them to a specific family. TS4340 has genome architecture and amino acid sequence consistent with Iflaviridae. TS152062 is phylogenetically most similar to Nora viruses, which are within the Picornaviridae, but remains unclassified to family. Finally, TS148892 is most similar to orbiviruses (Reoviridae), which typically have segmented double stranded RNA genomes and represent arboviruses infecting vertebrates and invertebrates.We chose to focus on 5 viral genotypes (4 Picornaviruses and 1 Reovirus) that matched most closely metazoan RNA viruses at NCBI and were represented in ≥ 5 libraries. These were: 1) Contig TS152062, which was most similar to Wenling Crustacean Virus  ; 2) ContRead recruitment of all 5 viral genotypes revealed that reovirus TS148892 had the greatest representation in plankton RNA libraries (i.e. 5–0.2 μm) . All fivQuantitative PCR targeting the 5 genotypes was applied to the >5 μm size fraction cDNA from the time series samples . The fiv2 = 0.37) in Owasco Lake, with bacterial abundance (R2 = 0.81) in Seneca Lake, and weakly with chlorophyll a concentration (R2 = 0.34) in Cayuga Lake . TS24641 abundance correlated with water temperature  size fraction in the fall of 2014. Reoviruses infect a wide range of hosts, including fungi, plants, invertebrates and vertebrates. Since TS148892 occurred in both microbial and >5 μm size fractions and was especially enriched in fall samples, we speculate that it may infect phytoplankton or a eukaryote that consumes decaying primary production.a in Seneca Lake in fall and spring suggests that it may infect a host influenced by enhanced resource availability. Zooplankton in the lake ecosystem exhibit seasonal abundance shifts according to resource availability, with peak abundances during spring and fall blooms, followed by declines in summer and fall [2 = 0.67). The correspondence between these viral genotypes may further indicate the presence of a potential pathogen of phytoplankton (TS24641) and a potential pathogen of its grazer (TS4340).In contrast to reovirus TS148892, iflavirus TS4340 was only detected in two lakes (Owasco and Seneca), only during fall 2014 and spring 2015, and did not contribute substantially to microbial-sized libraries of plankton RNA . Our obsand fall , 69. Ifla concentration in Owasco lake (R2 = 0.59) but did not correlate with phytoplankton biomass in Seneca Lake. The absence of TS152062 from the >5 μm size fraction in early Fall 2014 and sporadic presence in Spring 2015 suggests that it may infect a rare host that was seldom captured in our survey. The larger contribution of this virus to microbial-size fraction plankton RNA libraries during months that it was absent in the >5 um size fraction by qPCR suggests that it either infects a smaller eukaryotic microorganism that form aggregates during enhanced phytoplankton production, or that it may be present in cell debris during peak phytoplankton abundance. TS152062 also correlated with picornavirus TS24641 in Cayuga Lake (R2 = 0.44), but did not significantly correlate with any genotype in Seneca Lake. A key question in our study was whether there is evidence for allocthonous viruses in lake plankton. Such viruses may be present on soil particles, animal carcasses or cell debris, or fecal matter that is washed into the lake by freshwater inputs or runoff [Biomphalaria sp.) and several terrestrial arthropods  is consistent with either a terrestrial or shallow-water invertebrate host.TS152062 was sporadically present in two lakes (Seneca and Owasco) and correlated with chlorophyll r runoff , 70. Of Our data also provide evidence that some viruses were widespread between adjacent lakes, while others were specific to one or two lakes. For example, picornavirus TS24641 and reovirus TS148892 were present in all three lakes in >5 μm plankton, while iflavirus TS4340 and picornavirus TS152062 were present in larger plankton only two lakes, and picornavirus TS24835 detected in only one lake. All five viral genotypes were, however, observed in microbial size fraction (5–0.2 μm) libraries in all three lakes at least for some part of the sampling period. These data suggest viruses of larger unicellular eukaryotes or metazoans may persist in cell debris for some time after host lysis. Alternatively, our data could indicate that small eukaryotic hosts aggregate at certain times during the year and therefore are detectable in the larger size fraction. However, our data indicate that all viruses detected in our study are widely distributed across all three lakes.RNA viruses are constituents of all aquatic viral communities. However, their predicted hosts, seasonal dynamics and biogeography have not been extensively investigated in temperate lakes , 71. OurS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigPhylogenetic annotation occurred via the MG-RAST server ( as at Ma(TIF)Click here for additional data file.S2 FigTime Series library dates and locations can be found with reference to (TIF)Click here for additional data file.


Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and  The design of chemical tools for gene regulation is one of the most promising approaches to the development of new drugs. Among the variety of molecules proposed for this scope, Peptide Nucleic Acids (PNAs) are one of the most established and efficient artificial systems for targeting DNA or RNA so far described; these compounds are nucleic acid analogues in which the deoxyribose phosphate backbone is substituted by a polyamidic chain of N-(2-aminoethyl)-glycine units 12. Due t12346791011An emerging challenging application is the use of PNA targeting micro-RNA (miR). Up- or down-regulation of miRs has been correlated with many different diseases, including cancer15161821γ-position  can assist this process allowing for an atomistic understanding of the physics behind the PNA structural organization and enabling a computer-based strategy for γ-radiation induced cross-linking in PNA:DNA heteroduplexes by means of a 300 ps long MD, allowing to explain experimental data on cross linking in terms of the effect of terminal capping groupset al.et al.et al. studied PNA:DNA and PNA:RNA duplexes focusing on the definition of the most relevant structural parameters51Orozco and coworkers pioneered the exploration of PNA properties employing classical MD techniques. They compared triplex PNA:DNA:PNA structures as reported in literature recording 1.05 ns long trajectories and they found structural parameters in good agreement with experimental ones45In this work, we define a computational strategy based on Well-Tempered Metadynamics (WT-MDMT)5355565758In particular, starting from an experimental PNA:RNA NMR structure (PDB ID: 176D)We further investigate the stability of the PNA:RNA duplex by simulating dissociation and re-annealing processes. This study revealed the critical importance of the central base pairing of the sequence. This evidence confirms and characterizes the experimental results by Igloi and co workers conducted on mismatch positioningγ-modified single stranded PNA  as described in the methodology section. It is worth mentioning that either for the ssPNA and the et al.In In particular, the first (i.e. magnification γ-modified PNA (γ-ssPNA). The starting helical conformation  turned to be significantly more persistent than the non modified ssPNA above discussed. The most relevant structural modifications occur only in the latest stage of the unbiased MD run suggesting a remarkable pre-organized behavior. . Structures sampled in F and E show several combination of possible stacking between bases. In particular, those sharing a folded configuration (R < 1 nm) are stabilized by two consecutive base stacking  would drive to misleading conclusions being the ssPNA helical structure relatively stable for shorter timeframes. On the contrary, MD and WT-MDMT runs conducted on the γ-ssPNA structure demonstrated a remarkable stability of the pre-organized helical conformation imputable to the –CH2OH functionalization placed in γ. This finding is in agreement with earlier experimental observation proposed by Ly and co-workersγ-ssPNA  allowing the –CH2OH occupying non hindered regions and favoring several polar interactions with the surrounding solvent. Unlike these, structures A and B of unmodified ssPNA showed, for H–N–C–H with the C-H bond of equivalent stereochemical position (pro-R hydrogen), a different value and wider range (−90.7° ± 76.3°). Moreover, the hydrogen-hydrogen average distance between sequential amino acids  always higher for the gamma modified PNA and enforces the view that entropic contribution are decisive for this enhanced affinity. Most importantly, the present results allow to enlarge the view of this type of “preorganization” and assess more precisely what can be expected from this type of modified backbones; in fact, the presence of the γ-side chains determines a local helical arrangement, and not a entirely helical single-stranded structure29These results are compatible with Circular Dichroism (CD) experiments conducted on similar PNAThe dissociation study, on the other hand, allowed to understand the role of the single bases and of their positioning in the stabilization of the PNA:RNA duplex. It was reported that structural variations affect the stability of PNA:DNA duplexes mainly by slower dissociation processes, while that of association is much less affected681–5 in 3 reaches an optimal and complete base pairing within the nanoseconds time scale. In this case the re-annealing is particularly favored having the complementary bases of the two strands properly oriented each other. The re-annealing is limited by diffusion of the two strands originally placed at ~6 Å and the driving force of this process are long-range Van der Waals interactions. Structures 1 and 2  were generated by insertion of serine groups in gamma position of the PNA moiety of 176D. Duplex was solvated with TIP3P water molecules in a 58.9 × 55.1 × 57.5 Å3 cubic box, counting in total about 18300 atoms (see γ-ssPNA) .All the MD simulations reported in this work were carried out with GROMACS 4.5toms see . As no ctoms see  is availtoms see , while sγ-ssPNA)  was solvet al.γ-ssPNA we carried out 400 ns long WT-MDMTs, and the simulations were stopped after verifying that the local minima converged. For discriminating among all possible conformations explored by WT-MDMT we post processed (i.e. reweighting scheme)Å and the PME algorithm was employed for the electrostatic contribution. The simulations were performed with a 2.0 fs time step, constraining all the bonds with the LINCS algorithmFor simulations where RNA moieties were present, we employed ff99SB7678How to cite this article: Verona, M. D. et al. Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics. Sci. Rep.7, 42799; doi: 10.1038/srep42799 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Flavivirus RdRps.Viral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5′-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A–G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by  Fidelity of RNA synthesis is an estimation of the frequency of non-Watson-Crick base pairs formed during RNA chain growth driven by RNA polymerases. The same phenomenon is described for DNA polymerases ,2,3,4,5.In this review, we will extend the meaning of fidelity to the process of selecting a correct 5′-triphosphate ribonucleoside (see below). Although incorporation of a modified ribose in a growing RNA strand does not generally produce a mutation in fine, the nucleotide selection process is of utmost importance in the design of nucleoside drug analogues. Fidelity of nucleotide selection is an important part of fidelity of RNA synthesis and, thus, of fidelity of viral RNA genome replication.−4 s/n/c = substitutions per nucleotide per cycle of infection) are reported for human norovirus G1  and Escherichia virus (bacteriophage) Q-β  [−5 s/n/c of the mutation rate of hepatitis C virus  genome replication has recently been published. Coronavirus Murine hepatitis virus  showed a mutation rate of 3.5 × 10−6 s/n/c [In infected cells, viral RdRps have evolved to perform RNA synthesis under fine-tuned conditions. They must synthesize a faithful copy of the viral RNA genome, yet, they have to be able to generate a defined amount of mutations per genome so they can adapt to the adverse response and changing environmental conditions provided by their host cell. Viral RNA genome replication is not a massive, parallel synthesis of similar and independent RNA genomes. Rather, it is best viewed as a process during which a population of interacting or inter-dependent genomes is synthesized. From this population successful mutants are selected exhibiting a selective advantage. Comparison of viral RdRps across species indicates that they are not working at a same fixed mutation rate, or at random, either. A too faithful a synthesis would fail to generate adaptable genomes; a too error-prone an RNA synthesis would lead to a viral population from which no viable genomes would be selected, a so-called “error catastrophe” predicted by Drake  and demovivirus) ,13. An avivirus)  of 3.8 ×Enterovirus genus of Picornaviridae, Coxsackie virus B3 (CVB3) RdRp and poliovirus (PV) RdRp, have different basic fidelities. It is thus reasonable to expect that each Flavivirus RdRp has its own characteristic fidelity.Measuring fidelity at the RdRp level relies on biochemical assays of recombinant proteins. Misincorporation ) may be measured using steady state or pre-steady state measurements ,15,16,17It would be of great importance for many scientific fields  to have reliable data of fidelity of RdRp-mediated RNA synthesis, and RNA genome replication. For most viruses, a clear comparative set is not available, and there are almost no data for flaviviruses yet.If the nucleotide selection process is altered, synthesis may be terminated or the outcome may have a genetic cost. Mutants may be generated, which are or are not viable in the infected cell. In cells there is a plethora of naturally-occurring nucleotide analogues, which must be selected against in order to produce bona fide viral RNA, and an infectious genome in fine. To achieve selectivity, RNA and DNA polymerases in general have evolved selective “gates” and molecular sensors in their active sites to precisely probe the correctness of the base, the absence or presence of a hydroxyl group at the 2′ position of the ribose moiety of nucleoside triphosphates, and the presence of the triphosphate ,21,22,23To understand how the fidelity of viral RNA polymerases is achieved has implications on our understanding of not only viral mutagenesis and evolution, but also on the clever design of NA drugs to control viral infections.Nucleic acid polymerases create a phosphodiester bond between the 3′-OH and the 5′-α-phosphate of two nucleotides, in a nucleic acid template-dependent or independent manner. RdRps depend on an RNA template. Generally, the 5′-α-phosphate belongs to a 5′-triphosphate nucleotide, and receives a nucleophilic attack from either the 3′-OH of a single nucleotide or that of a short oligonucleotide primer or, in some cases, an -OH group of a peptide or protein primer.Flaviviridae, Coronaviridae, Cystoviridae, Leviviridae) are able to synthesize their own oligonucleotide primer and, hence, start nucleic acid synthesis de novo. The corresponding first phase of RNA synthesis is named “initiation”. Initiation is a distributive polymerization reaction, i.e., the enzyme dissociates frequently from the template. The next phase consists in a processive RNA synthesis reaction, called “elongation”. RdRps from some viral families  occurring much more efficiently on a mismatched base than on the regular Watson-Crick base, this reverse process represents de facto a correction mechanism operating to increase the overall nucleotide insertion fidelity. In reality, this process is very likely to occur since the time of residency of a mismatch in the RdRp active site is expected to be significantly longer than that of a regular base. A mismatch produces a stalling of the RdRp, whereas a Watson-Crick nucleotide is immediately extended by the next correct nucleotide. Interestingly, the pyrophosphate level in the cell is in the micromolar range and stringently controlled by cellular pyrophosphatases, whereas the NTP level in the cell is higher, in the millimolar range ,28. AddiFlavivirus RdRps.Since the general reaction mechanism of nucleotide addition is shared by all polymerases, the active site of viral RdRps is made of catalytic motifs with a conserved secondary structure, motifs A and C, and two aspartic acid residues common to all polymerases. Another two motifs, B and D, are common to RNA-dependent polymerases . Motifs Flavivirus RdRps in particular, have evolved features that are usually accomplished by ancillary proteins in the eukaryotic cell. They synthesize their own primer at the precise 3′-end of the (+) genomic and (−) anti-genomic viral RNA, and leave the (−) anti-genomic RNA 5′-end uncapped, but cap the genomic (+) RNA 5′-end. The de novo RdRp function of flaviviruses is harbored in the C-terminal domain (RdRp domain 74 kDa) of protein NS5, which also contains an N-terminal domain with methyltransferase (MTase domain 30 kDa) functions involved in RNA cap formation [De novo viral RdRps, and ormation .Picornaviridae RdRps, notably of foot-and-mouth disease virus  [The first crystal structure of a nucleic acid polymerase  in 1985  establistovirus, ), PV [401 (EV71) . These m1 (EV71) ) versus 1 (EV71) ). They wFlavivirus RdRp structure was determined in 2007 and was of the isolated RdRp domain of West Nile virus . Since ID: 2HFZ , PDB ID:ID: 2HFZ  and seveID: 2HFZ ,48,49), ID: 2HFZ ) and mosID: 2HFZ , PDB ID:ID: 2HFZ , PDB ID:ID: 2HFZ ). The geThe structures of three full-length NS5 proteins have been determined so far: of JEV (PDB ID: 4K6M ), DENV3 Flavivirus RdRps are able to synthesize RNA without the help of a primer. The Flavivirus NS5 binds to specific viral RNA sequences (and avoids other spurious cellular RNA 3′-ends) [Flavivirus genus, under a variety of conditions of template sequence, template presence/absence, and metal ions in vitro. The structural element decisive for de novo initiation is the priming loop that has been observed in the first Flavivirus RdRp structures [Flavivirus RdRps. Interestingly, the loop can be deleted without major impeachment of RNA synthesis. The resulting enzyme is now able to initiate RNA synthesis at various internal sites of an RNA template. Accordingly, the structure of a ternary complex enzyme:primer/template:nucleotide of HCV RdRp in the catalytic state [As stated above, 3′-ends) ,60, and 3′-ends) . The enzructures ,45. Lateructures . The stric state  was obtaic state , but theFlavivirus RdRp structures are flexible, it has been difficult to set up test systems such as transient kinetic systems with a stable elongation complex as it has been possible for PV RdRp [Flavivirus NS5 with a primer/template [Since  PV RdRp ,64. It i PV RdRp . The aut PV RdRp . More sttemplate ,54,66.Flavivirus RdRp motifs A to G and their proven or potential involvement in RNA synthesis fidelity by relating them to existing fidelity data. There are only very scarce data for flaviviruses regarding RdRp variants of nucleotide selection fidelity. Few variants and associated data have been reported for WNV [Flaviviridae family, there is no large increase of the number of fidelity variants reported in the literature either. Most studies relate to nucleoside-analogue-resistant variants mapping to the HCV RdRp gene [Picornaviridae family to benefit the Flavivirus RdRp field.In this section, we discuss in detail  for WNV  and for  for WNV . When thdRp gene ,69. HereFlavivirus RdRps . Below each WebLogo a combined structure- and sequence-based alignment of the corresponding motifs A to G is placed, including a number of relevant flaviviruses (see the figure legend) in comparison to other positive single-strand RNA virus RdRps for which fidelity data exist  belonging to Flaviviridae; PV, CVB3 from Picornaviridae; Escherichia virus Q-β from Leviviridae). The motifs are sequentially presented in the accompanying movies, which also show a model of the detailed pathway of nucleotide incorporation into RNA by ZIKV RdRp  leaving space for the 2′OH group of the aligned NTP. The proposal that the conformational change of motif A represents a fidelity checkpoint is supported by studies using Picornaviridae RdRps where mutated hydrophobic residues within motif A showed changes in fidelity  and a β-strand followed by a short helix (Motif A). Two aspartate residues coming from each motif, are universally conserved in all polymerases. In plus-strand virus RdRps the motif A signature is DxxxxD. Additionally, there is a conserved aromatic/hydrophobic residue at the beginning and a hydrophobic residue followed by S/T at the end of the motif . The saMDV RdRp  versus cMDV RdRp ) (see beFlavivirus RdRps 8 of 13 residues of motif A (xADDxAGWDTxxx) are strictly conserved , it is obvious that all Flavivirus RdRp structures contain motif A in an open conformation; the three-stranded β-sheet of motifs A and C is not formed. This is illustrated in the right panel of 2+ ion in a pre-catalytic position that has also been seen in the open form of EV71 RdRp complex structure . Motif-Flavivirus RdRp motif-A mutations having an impact on nucleotide selectivity are currently unknown. Putative fidelity-changing residues that correspond to the residues of motif A from the mutational studies of Picornaviridae RdRps are Y532, D534, T541 (ZIKV RdRp numbering), the latter are strictly conserved in Flavivirus RdRps  versus D: 2XI2,  shown inD: 2XI2, . InteresFlavivirus RdRps show important sequence conservation in motif B with an almost strictly conserved QRGSGQVxTYxLNTx signature (WebLogo Flavivirus RdRp motif B loop in the existing structures varies widely with the S603 (ZIKV numbering) side chain mainly adopting the “out” position as in the open conformation of PV or HCV RdRps or a displaced “in” position (right panel Flavivirus RdRps as an Arg (R601 in ZIKV) corresponds to M296 of FMDV RdRp which has been shown to be a fidelity determinant [ WebLogo . The conerminant . An equaerminant .Flavivirus RdRps with corresponding residue V579 (ZIKV). Another is CVB3 RdRp residue Y268 [Residues located in the vicinity of motif B and probably influencing its conformational flexibility have been reported to be fidelity modulators. PV RdRp residue H273 is located in a β-sheet in the fingers domain behind motif B ,76. Thisdue Y268  at the edue Y268  have repFlaviviridae inhibitors, amino acids have been identified that are directly involved in resistance to said analogues [Flavivirus NS5 [Due to the success of 2′-modified nucleoside analogues as nalogues ,69. Mutanalogues . The mainalogues . The latnalogues . So far,irus NS5 ,80,81. Nirus NS5 .Flavivirus RdRp, as for HCV RdRp [Flavivirus RdRps. In any case, motif B appears central for overall RNA synthesis fidelity, being involved both at the nucleotide probing and accommodation step and, potentially, in the mismatch–excision step.Finally, if we surmise that (i) for HCV RdRp , NTP-medDuring catalysis where the 3′-hydroxyl is deprotonated to attack the phosphorus center, an event of proton-transfer to the leaving pyrophosphate must occur to ensure overall neutrality of the reaction. Several studies ,82 propoMotif D is the least conserved motif in positive-strand RdRps at the sequence and structure levels. It starts after a conserved α-helix and is largely made of structurally-diverse loops located at the bottom entrance of the NTP entry tunnel C. SequenFlavivirus RdRps, there are three conserved residues in motif D giving a signature KxRK  affect pyrophosphorolysis of a misinserted nucleotide, accounting for the change in fidelity. Alternatively, if reaction is slowed down, a slowed-down translocation of the primer may allow more time for occurrence of a nucleotide-excision repair such as the one described for HCV RdRp .Flavivirus RdRps show a signature xFCSxHx7DGRx4PCRx  shows tficiency .Nucleotides enter the polymerase active site through a tunnel whose ceiling is made of motif F. In most of the viral RdRp structures in a catalytic state, it starts with a conserved β-strand, then adopts diverse structures to finally end up forming another conserved β-strand. Sequence conservation within positive-strand RNA virus RdRps is limited to some positively-charged residues, which can be superimposed structurally  and JEV RdRp in complex with GTP in a non-catalytic position (PDB ID: 4HDG)), motif F is only partially resolved. The end of the motif, which is present, adopts an alternative helical conformation (in Flavivirus motif F are illustrated in Flavivirus RdRps. While residue R473 seems to be in place (with some side chain changes) to probe and align the α-phosphate, K470 is displaced in the full-length NS5, and also in the isolated RdRp domain structures of ZIKV and JEV RdRps  corresponding mutation I287A (and R285A) also show altered fidelity [Flavivirus RdRps this putative fidelity determinant is an Ile (I475 In ZIKV RdRp). In conclusion, it would be interesting to mutate homotropic residues in Flavivirus RdRps and assay fidelity changes. Clearly, motif F is important for RdRp fidelity, being implicated in guiding both the nucleotide into the tunnel in order to be paired to the template base and aligned, and also in guiding the pyrophosphate out of the tunnel.Concerning fidelity determining residues in motif F, Abdelnabi et al.  have shofidelity ,90, wherfidelity . In BVDVfidelity . In FlavMotif G is situated at the entrance of the RNA template tunnel. It is merely a structural motif without any sequence conservation in viral RdRps . IntWNV RdRp ) where mWNV RdRp ) motif GFlavivirus RdRp is a mutation in a ribavirin-resistant RdRp of SLEV [Flavivirus RdRps and located four residues after motif G on the surface of the protein. Likewise, a high-fidelity EV71 RdRp variant with mutation L123F is situated just after motif G [Enterovirus RdRps forms a binding pocket for the +2 template base opposite to motif G [Once in place motif G guides the RNA template into the tunnel  distort the active site?; how do other domains, such as the MTase domain, affect the fidelity of the RdRp [A great variety of mechanisms are involved in the selection of correct versus incorrect nucleotides at the the RdRp ?; how doDrawing from AZT-resistance by HIV reverse transcriptase and the seminal paper of Jin et al.  describi


Exosomal miRNA transfer is a mechanism for cell–cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip’s amino-terminal domain, which was previously thought to mediate protein–protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR . The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip’s RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5′ to this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences. Syncrip is a conserved RNA-binding protein that mediates selective miRNA loading into cell-secreted exosomes, which is important for cell–cell communication. Here the authors provide mechanistic insights into how Syncrip recognises its target miRNAs by combining NMR and crystallography. Exosome-mediated microRNA (miRNA) transfer is thought to be important in various processes and systems, including the immune response2 and neuron-glia communication3. In addition, it has been implicated in a number of diseases, including cardiomyopathies4, neurological diseases5 and cancers6. Exosomal miRNA delivery in cancers mediates the communication between the tumour and stromal compartments. For example, it has been shown that the exosomal miRNAs in the brain microenvironment downregulate PTEN (phosphatase and tensin homologue) in nearby tumour cells7.Exosomes are small, cell-secreted vesicles that carry specific repertoires of proteins and RNAs to recipient cells. This selective transfer of proteins and RNAs in the exosomal cargo represents an important means of inter-cellular communication1. Recent reports have shown that loading of specific miRNAs is mediated by RNA-binding proteins, four of which have been identified so far. Two of them, hnRNPA2B18  and Syncrip10, select the target miRNAs based on the presence of short G-rich RNA sequences, which are different for the two proteins. For the other two proteins, HuR11, that has been linked to miRNA function before13 and YTBX114, no target sequences have been identified. Importantly, hnRNPA2B1, YTBX1 and Syncrip14 each have multiple miRNA targets. This is consistent with a model whereby a gene regulatory signal carried by the exosome can be encoded by an ensemble of miRNA molecules that are working synergistically and that are loaded by a single regulatory RNA-binding protein. However, we have no molecular information on how these RNA-binding proteins recognise miRNA targets and mediate their exosomal localisation.The selectivity of miRNA loading encodes the inter-cellular message carried by the exosome and a key question in the field is how this selectivity is determined at the molecular level17. Mis-regulation or dysfunction of Syncrip is associated with severe cardiomyopathies and neuro-degenerative disorders20. In the fly embryo, Syncrip is important for the morphology and growth of the neuromuscular junction and regulates cytoplasmic vesicle-based messenger RNA (mRNA) transport16. In mammals, Syncrip exerts control on the length and number of neurites in mouse embryonic cortical neurons19 as well as the growth of nascent axons17, among other functions. At the molecular level, Syncrip recognises a diverse range of RNA sequences, including UACU-containing21 and polyA22 sequences, and regulates mRNA editing, transport, translation and degradation23. Importantly, we showed that Syncrip recognises an hEXO (GGCU/A) sequence in a set of miRNA targets and mediates their exosomal enrichment10. However, how Syncrip recognises its diverse ensemble of mRNA and miRNA targets is not known. Syncrip contains three conserved RRM domains -crosslinking to living cells, oligo d(T) capture of poly-adenylated RNAs, limited proteolysis and a second round of oligo (dT)-mediated isolation . Analysis of the Syncrip sequence with this algorithm revealed high-probability RNA-binding activities not only in the RRMs, but also in the ‘acidic’ N-terminal domain Fig. . This in1 with two copies in the asymmetric unit. The 2.2 Å resolution X-ray structure  . The conformation of the RRM, together with the orientation of the two domains and the inter-domain surface are essentially identical when comparing the two copies in the crystal asymmetric unit  solvent-accessible surface. This likely explains our observation that the RRM1 domain is not expressed in a soluble form in E. coli. Finally, inspection of a sequence alignment of Syncrip family members also reveals a large degree of sequence conservation in this region of the proteins further supporting the idea that the αββ extension is a defining feature of the Syncrip family  making contacts with the αββ RRM-extension, and specifically with residues of the first β-strand (eβ1). Additionally, residues of the core RRM β1-β2 loop also make contact with the N-terminal ‘acidic’ domain Fig. . The dom15N-correlation nuclear magnetic resonance (NMR) spectroscopy to assess differences in the amide resonances  of the two domains in isolation (N and eR1 constructs)  of each of the domains within the two-domain construct  that indicates that the two domains are not tightly coupled. The dynamic nature of the relationship between the domains was confirmed using a second NMR observable. We measured the 15N amide residual dipolar couplings (RDCs) for the two-domain construct and compared the experimentally measured RDC values to the RDC values back-calculated from the crystal structure using either a single tensor for the two domains or two independent tensors, one per domain; the latter representing a molecule where the domains are not tightly coupled. This comparison , with the largest, lower energy family superimposing closely with the crystal structure  Table . Next, wues Fig.  indicateuct Fig. . Surprisson Fig.  shows th33 and, also based on a predicted overall strongly negative charge, it was proposed to act as a protein–protein interaction module. However, our structural analysis shows that many of the negatively charged residues predicted to be part of the domain are in fact located in the neighbouring unfolded regions and rather being than strongly acidic, the domain actually has an overall charge that is close to neutral. In order to obtain some initial information on the determinants of the N-terminal domain’s functional interactions, we mapped the conserved residues on the domain structure  that are part of the domain’s conserved surface ted Fig.  represented Fig. . These Sted Fig. . Importated Fig.  and disc15N-correlation NMR spectra recorded with different RNA pools confirmed that this conserved surface mediates RNA binding , where the canonical RRM β-sheet RNA-binding surface is extended by the two β-strands of the αββ element. This surface is highly conserved Figs. , and incing Fig. , althougNotably, in the crystal structure the conserved RNA-binding surfaces of the RRM and NURR domain are aligned by the physical coupling between the two domains Fig. , providi10. Indeed inserting this sequence in a non-target miRNA (miR29a-1) leads to its exosomal loading10. Our present study shows that the human NURR domain recognises, with high specificity, a GG sequence that matches the core nucleotides of the hEXO sequence  sequence present in a group of miRNAs, and that this interaction directs the loading of these miRNAs into exosomesnce Fig. , and we To test our hypothesis, we first examined the NURR–hEXO interaction in isolation using NMR and confirmed that the short hEXO RNA binds to the conserved RNA-binding surface of the domain by monitoring chemical shift changes during a titration of the protein with the RNA and mapping the perturbed resonances on the domain structure Fig. . Then we10. First, we compared the binding of the four-domain RNA-binding region of Syncrip (NeR1R2R3) to wild-type full-length miR-3470 and to miR-3470 with a mutated hEXO sequence, and showed that mutation of the hEXO sequence decreases the binding affinity ~6-fold  assays. As a model we chose miR-3470, the miRNA that (i) is most efficiently pulled down by Syncrip after crosslinking and (ii) shows the strongest change in exosomal partitioning upon silencing Syncripold Fig. . This exold Fig. . It is w. Modelling of these changes on the protein structure indicated that the mutated side chains are unlikely to interfere with the protein fold  of miR-3470 and the negative control non-hEXO miR-92a-1-p was performed using extracts of cells silenced for endogenous Syncrip expression after introduction of WT Syncrip, Syncrip ΔNURR or Syncrip R60A G97L by retroviral transduction. As shown in Fig. Finally, we tested whether RNA binding by the NURR domain affects the specific recognition of the RNA targets in the cellular environment. To probe this, we used both a protein construct where the NURR domain had been deleted (ΔNURR) and a variant of the domain where RNA binding has been eliminated through the introduction of two point mutations that do not change the protein structure. These structure-driven mutations changed a positively charged side chain in the RNA-binding surface to alanine (R60A) and introduced a bulky hydrophobic side-chain to disrupt protein-RNA packing (G97L) Fig. . Modelliold Fig. , and we old Fig. . Then, tOur previously published cellular assays identified the hEXO sequence as the only sequence required for exosomal miRNA partitioning. However, the structural work we present here indicates that RRM and NURR binding surfaces are coupled and that this coupling is likely to play some role in the interaction. One question is whether this role is exclusively to increase the affinity of the interaction or whether binding of the RRM domains may contribute to selectivity in a non-sequence-dependent fashion, i.e., by providing some previously hidden structural restraint. To de-convolute the two possibilities we have obtained a mechanistic model for protein–RNA recognition. Kd for the interaction between the NURR domain and a miR-3470-derived 14-nucleotide RNA   Figs. . The fouRM3 Fig. . We disc23, as well as miRNA partitioning in the exosomes10. However, to date we do not have a quantitative and mechanistic understanding of how RNA is targeted and regulated by Syncrip. This understanding is crucial to gain insight into the mechanistic basis of Syncrip-mediated exosomal partitioning of miRNAs.The loading of selected miRNAs into exosomes is a recently discovered mechanism for cell–cell communication that is important to the development and function of a broad range of tissues. Selectivity in miRNA loading is mediated by RNA-binding proteins, which recognise specific sequences on the target miRNAs. Here, we have focussed on the Syncrip protein to investigate the molecular basis of the selection of its miRNA targets. Syncrip is an abundant and conserved RNA-binding protein that binds both mRNA and miRNA targets and regulates mRNA transport, editing and translation degradationThe output of our RBD analysis indicates that the three RRM domains in the central region of Syncrip cross-link to RNA in vivo. Unexpectedly, the N-terminus of Syncrip, which includes an N-terminal domain and a conserved sequence N-terminal to RRM1, also interacts with RNA. As a first step to understanding Syncrip-RNA binding at the molecular level, we determined the crystal structure of this non-canonical RNA-binding region. The structure shows that the N-terminal and RRM1 domains are brought together by an αββ structural element created by the folding of the highly conserved sequence amino-terminal to RRM1. One question is whether and how this coupling is functionally relevant. The αββ motif aligns the two highly conserved RNA-binding surfaces of RRM1 and NURR to create one continuous RNA-binding surface and to establish the direction of how the NURR and RRM(s) position on the RNA target, providing a first indication that the physical coupling between domains is likely to be important to Syncrip RNA binding.10. BLI data confirm that Syncrip specifically recognises the hEXO sequence within the miR-3470 target and that the removal of the NURR domain impairs this Syncrip-hEXO recognition. These assays directly relate hEXO-mediated miR-3470 recognition to the hEXO–NURR interaction, explaining the molecular basis of the reported miRNA target selectivity. This result was tested to the cellular environment with the design of the double mutant where the RNA-binding function of the protein is selectively impaired. When tested using RIP assays abolition of NURR RNA-binding function also resulted in a loss of miR-3470 pulldown confirming that RNA binding by the NURR domain is essential for target recognition in the cell. It is worth highlighting that the sixfold effect on binding affinity we observe in our in vitro two-component system translates into a loss of binding in our cellular assays. This emphasises how relatively modest changes in the affinity for an RNA target may result in drastic changes in the protein–RNA association in the cell, where other proteins and RNAs are competing for interacting partners.Unexpectedly, our work shows that the N-terminal NURR domain of Syncrip is a RNA-binding domain with a highly conserved RNA-binding surface. Further, our SIA assays and titration analyses show that RNA binding by the NURR domain is sequence specific, consistent with a direct role in the selection of Syncrip target RNA target sequence(s). Indeed the NURR domain binds to the hEXO-motif (GGCU), whose recognition by Syncrip mediates exosomal partitioningKd), while high-affinity binding (~2.0 nM Kd) to the target miRNA requires the additional interaction of the RRM domains. That is, the NURR and RRM domains act together to achieve a specific and high-affinity interaction with the Syncrip RNA targets. Importantly, the contacts between the NURR and eRRM1 domains position the RRM domains 5′ of the NURR domain on the bound RNA sequence. This provides a previously hidden structural constraint and implies that for Syncrip to interact with a target miRNA with high-affinity and specificity the hEXO sequence cannot be positioned at the 5′ end of the miRNA. The 5′ end of a miRNA molecule is occupied by the seed sequence, which is the main determinant of target recognition in canonical miRNA–mRNA target interaction, while sequences 3′ of the seed are less functionally constrained. Arguably, this mutational freedom would facilitate establishing a regulatory sequence (the hEXO sequence) common to a set of miRNAs with different targets. Therefore, we revisit the rules of recognition previously drawn and that propose the architecture of the RNA-binding region of Syncrip underpins its ability to recognise a common sequence in miRNAs with very different seed sequences and to efficiently regulate a group of miRNAs with very different target specificities.A second question important to understanding Syncrip-miRNA recognition is whether the NURR domain is a stand-alone high-affinity, high-specificity miRNA recognition element that mediates the interaction with the miRNA targets, or whether and how the RRM domains contribute to the interaction. We show that, in isolation, the NURR domain binds to the miRNA target with moderate affinity  and codon-optimised H. sapiens Syncrip/hnRNP-Q   and the wild-type protein expression vector as template. Mutants of the full-length hSyncrip for eukaryotic expression were prepared using the Quickchange protocol (primers sequences in Supplementary Table 10) expressing the wild-type gene as template. The sequences of all constructs were confirmed by DNA sequencing prior to expression.The genes encoding Uniprot AV364 and E. coli strain BL21 (DE3) Gold (Agilent) and purified using the same protocol. Bacterial cultures were grown at 37 °C to a density of OD600 = 0.5–0.8 and protein expression then induced by addition of 0.5 mM isopropyl-β-d- thiogalactoside (IPTG). Prior to induction, cultures were cooled and maintained at 20 °C for protein expression overnight. Cell pellets were harvested by centrifugation for 7 min at 8600 × g and then resuspended in lysis buffer  containing , and 1 µL mL−1 of Triton X-100) and lysed by sonication. The cell lysate was then centrifuged for 45 min at 23,600 × g, the supernatant was collected and applied onto a Ni-Sepharose resin column (bed size 3 mL per litre culture). The column was washed with 40 mL wash buffer I  and 20 mL wash buffer II . Bound proteins were eluted with 40 mL of elution buffer . Subsequently 350 µL of human rhinovirus 3C protease was added per litre culture to remove the His6-SUMO or HIS tag while the sample eluent was dialysed overnight at 4 °C against wash buffer I. Next day, the dialysed sample was passed through a further Ni-Sepharose column to remove cleaved tags and/or any uncleaved fusion protein. Samples of the fractionated eluent were applied to 12% NuPage BIS-TRIS denaturing gels to assess sample purity and the purified protein was concentrated using VivaSpin 20 centrifugal concentrators with a molecular weight cut-off (MWCO) of 10 or 5 kDa (depending on size) and stored at −20 °C in small aliquots. For samples used for NMR assignments, NMR titrations, BLI and crystallography a further size exclusion chromatography step was included to remove residual high-molecular-weight aggregates. For these purposes either a Superdex200 (16/60) or a Superdex75 (16/60) prep grade column was equilibrated with gel filtration buffer  and 5 mL purified protein was injected. The fractions containing the protein were collected and concentrated using VivaSpin 20 with a MWCO of 10 or 5 kDa (depending on size).The human and Drosophila Syncrip proteins were expressed in the 15N- and 13C- labelled proteins for NMR assignment, cells were grown in minimal medium  containing 1 g per litre 15N-ammonium sulphate and 2 g per litre 13C-glucose as sole sources of carbon and nitrogen.To produce 600 = 0.5 at 37 °C in M9 minimal medium. Then the culture was cooled to 20 °C. After 15 min, an amino-acid supplement  was added to inhibit endogenous methionine biosynthesis and initiate SeMet incorporation. After a further 30 min protein expression was induced by the addition of 0.5 mM IPTG, and cells were grown for a further 18 h. Incorporation of selenomethionine was assessed by mass spectrometry, which showed that the incorporation was >95%.To produce seleno-methionine (SeMet)-labelled N-terminal-RRM1 construct, cells were grown to an ODProtected RNA oligonucleotides were purchased from GE Healthcare, Dharmacon, and deprotected as required using the standard protocol provided by the company. Ribo-oligonucleotides were: hEXO-motif: AGGCU, full-length miR-3470: UCACUUUGUAGACCAGGCUGG, 14mer miR-3470: GUAGACCAGGCUGG and chimera miR-3470: UCACUUUGUAGACCAAGUGUU.1H frequencies, except for relaxation experiments on the dSyncrip protein, which were recorded on a Varian Inova spectrometer operating at 600 MHz 1H-frequency. Temperature was maintained at 298 K. Data were processed using the NMRPipe/Draw suite of programmes38 or Topspin. Assignment of protein resonances was performed in CARA39 and CCPNMR analysis40, titrations were analysed in Sparky41 and CCPNMR analysis.All NMR experiments were carried out using Bruker Avance NMR spectrometers operating at 600, 700, 800 and 950 MHz 1H, 15N, 13Cα and 13Cβ, and 13C′ chemical shifts of the dSyncrip N and NeR1 constructs were obtained from standard HNCACB, CBCA(CO)NH, HNCO, HNCA and HN(CA)CO experiments recorded on a 0.6 mM protein sample in 5 mM Tris-HCl, 50 mM NaCl, 1 mM TCEP-HCl, 0.04% NaN3 at pH 7.4. For hSyncrip NeR1 and eR1 the backbone assignments for 1H, 15N, 13Cα and 13Cβ chemical shifts were obtained from HNCA and CBCA(CO)NH experiments recorded on 0.6 mM protein samples in the buffer and conditions above. The 1H and 15N amide resonances of hSyncrip N-terminal domain and of the dSyncrip eR1 were assigned based on the superimposition of the spectra of the individual domains and the di-domains.Backbone resonances assignments for 15N T1 and T2 values and 15N heteronuclear NOE values were obtained from standard experiments42 recorded at 600 MHz 1H frequency on 0.3 mM samples of dSyncrip NeR1 and eR1 proteins. 10, 100, 200, 400, 500, 750, 1000, 1250, 1500 ms delays and 9, 18, 27, 36, 45, 63, 81, 100 ms delays were used, respectively, for T1 and T2 experiment recorded on dSyncrip NeR1. 10, 100, 200, 300, 400, 700, 800, 1000, 1500 ms delays and 8, 16, 24, 32, 40, 48, 72, 80, 96, 120, 144 ms delays were used, respectively, for T1 and T2 experiment recorded on dSyncrip eR1. Heteronuclear NOE values were obtained from standard experiments42.15N-labelled NeR1 in NMR buffer and filamentous phage Pf1 . The RDC values were obtained by subtracting the reference value in isotropic solution. All data were processed with NMRPipe and the spectra were analysed in CCPNMR analysis. Fitting of the measured RDCs to those calculated from the crystal structure was performed using the program Module43 using either one tensor or two tensors corresponding to the two domains.RDCs were obtained for the dSyncrip NeR1 construct. In-Phase and Anti-Phase (IPAP) experiments were recorded on a 0.3 mM sample of 15N SOFAST NMR spectra of protein–RNA complexes at the concentrations and ratios below. A 50 µM sample of dSyncrip N-terminal domain was titrated with four and five-nucleotide fully randomised oligonucleotide pool (4N and 5N RNAs) at 1:4 protein:RNA ratio. A 50 µM sample of hSyncrip N-terminal domain was titrated with 4 N RNA pool up to a 1:4 protein:RNA ratios. A 50 µM sample of hSyncrip N-terminal domain was also titrated with hEXO RNA at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 protein:RNA ratio. Fifty micromolar samples of hSyn and dSyn extended RRM1 were titrated with NNNANN, NNNCNN, NNNGNN and NNNUNN RNA (for reasons of availability we used an oligo series) at 1:4 and 1:16 protein:RNA ratios. One-hundred and twenty micromolar samples of hSyn NeR1R2R3 were titrated with either hEXO  or full-length miR-3470 (at 0 and 1 protein:RNA ratios). Fifty micromolar samples of hSyncrip NeR1 wild-type and R60A G97L mutant were titrated with hEXO RNA at 0, 1 and 2 protein:RNA ratios.NMR titrations were performed in 5 mM Tris_HCl, 50 mM NaCl, 0.5 mM TCEP-HCl, at pH 7.4. buffer recording 34 were performed on 25 µM samples of human and Drosophila N-terminal proteins. Protein–RNA ratios employed were 1:4 and 1:8, respectively, to better match optimal shift changes. Experiments were performed as described44. Briefly, NMR data were acquired using a Bruker Avance III NMR spectrometer operating at 700 MHz and equipped with a 5 mm TCI cryoprobe. Samples were stored in 3 mm NMR tubes at 4 °C within a Bruker Samplejet auto-sampler and loaded automatically after a short pre-heating 25 °C step. Locking, tuning, matching and shimming were performed automatically.SIA experiments−1 solution of NeR1 in in either Gel filtration buffer or NMR sample buffer was mixed in a 1:1 ratio with mother liquor. Initial conditions were optimised and drops were seeded to produce fewer, larger crystals. The best NeR1 crystals formed in 2.2 M ammonium sulphate, 0.1 M sodium citrate pH 6 with seeding and incubation at 18 °C for 5–7 days. Crystals were cryoprotected with 20% (v/v) glycerol and frozen by direct immersion in liquid nitrogen. The crystals belong to the space group P21 with two copies of NeR1 present in the asymmetric unit (ASU). Seleno-methionine (Se Met) derived protein was crystallised under the same conditions. The diffraction quality of the tested crystals was highly variable, often being highly anisotropic in one direction.Drosophila Syncrip NeR1 was crystallised using sitting drop vapour diffusion. Typically, a 5 mg mL45 was used to process the data which extended to 3.1 Å but with anomalous signal only extending to 4.2 Å. Determination of the number of selenium atoms present in the ASU was performed using SAD methods in PHENIX46 and the initial map phased using ten Se sites. The backbone of the secondary structure elements of RNA15 (PDB code:  2X1B) was used as an initial model in the automated model-building function in PHENIX, which yielded a map-model CC of 0.67. Manual, real space building/re-building of the model was performed using COOT47.Diffraction data sets were collected at 100 K at the I03 beamline at Diamond Light Source . The structure of NeR1 was solved by SAD using a fine-sliced data set (0.15°) of Se-Met derived protein recorded at 0.9795 Å. X-ray Detector Software (XDS)48. Iterative rounds of reciprocal space and real space refinement in PHENIX or REFMAC49 and COOT were performed to complete the model. TLS groups were calculated using TLSMD50 and only used in the final round of refinement. The model was refined to a final Rwork/Rfree of 0.174/0.206 and has good geometry as determined by PROCHECK51. Details of the crystal parameters and refinement statistics are presented in Supplementary Table The native data  were processed using XDS with data cut at 2.2 Å and a single chain from the SAD model was used to perform molecular replacement (MR) in PHASER32 and standard parameters with the exception that only 5% of the input data were discarded. As experimental input parameters, all residues showing CSPs > 0.02 ppm were marked as active with HADDOCK determining the passive residues automatically. The RDC data were input with different tensor values for the NURR-domain (D −17.7 and R 0.22) and the extRRM1 domain (D −18.0 and R 0.31) as obtained from the best fit of the RDC data to the crystal structure with the program PALES52. Two-hundred structures were calculated that grouped into three clusters  representing 97.5% of the structures. Cluster 1 is by far the largest and also has the lowest overall Haddock score (−71.4 +/− 3.2), with cluster 2 having a less favourable Haddock score (−66.8 +/− 1.0) and cluster 3 considerably lower score (−50.9 +/− 2.5). The results compare well with the tutorial example given in ref. 53.The HADDOCK runs were performed using the GURU interface of the WeNMR/West-Life GRID-enabled web portal for HADDOCK 2.2−1 bovine serum albumin (BSA) pH 7.4 on an Octet Red 96 instrument  operating at 30 °C. 5′-Biotinylated miR-3470 RNA variations  (1 ng µL−1 solutions) were immobilised on streptavidin-coated biosensors and incubated with varying concentrations of hSyncrip NURR  and Syncrip NeR1R2R3 . Dissociation constants for RNA–protein interactions were determined from plots of the increase in BLI signal as a function of the protein concentration and fitting using non-linear regression using in-house software, as described54.BLI experiments were performed in 5 mM Tris-HCl, 50 mM NaCl, 1 mM TCEP, 0.5 mg mL55(accessible at http://www.ebi.ac.uk/Tools/msa/tcoffee/). All the alignment figures were generated by using Jalview/ClustalX56. The models of the human eRRM1 and NeR1 proteins were built using the SWISS-PROT modelling server57, with default parameters and the corresponding region of the two-domain Drosophila protein as an input template.All sequence alignments were carried out using T-Coffee multiple sequence alignment program10, with the changes described below. A total volume of extract containing 3 mg of protein was precleared with protein G Dynabeads  for 30 min at 4 °C with end-over end rotation and then incubated with 5 µg mouse anti-hnRNPQ antibody  or mock antibody  for 16 h at 4 °C with end-over end rotation. Twenty microlitres of Protein G Dynabeads , blocked with 3% BSA, were added for 4 h, followed by five washes with denaturing wash buffer58. In brief, co-immunoprecipitated miRNAs were extracted using Total RNA miniKIT  reverse transcribed and analysed by quantitative PCR (qPCR). miRNA fold enrichment in immunoprecipitated samples was expressed as fold with respect to IgG isotypic control.RIP experiments were performed as in Santangelo et al.Structure Factors and co-ordinates for Syncrip NeR1 have been deposited in the protein data bank with accession number 6ES4. All relevant data are available from the corresponding authors upon reasonable request.Supplementary Information


RNAs (lncRNAs), and a cadre of small peptides. These ever‐growing catalogs require high‐throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse. Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA‐based functionality. We applied MPRNA to identify RNA‐based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine‐rich motif. These nuclear enrichment sequences are highly conserved and over‐represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single‐molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA‐based functionalities.In the post‐genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding  This was optimized to develop a robust statistical method that leverages the interdependencies and variances of each 110mer to identify larger RNA domains enriched in the nucleus.Toward this goal, we have developed and optimized such a massively parallel RNA assay (MPRNA). Briefly, we developed a construct that expressed and appends thousands of 110mer RNA sequences—each uniquely barcoded—to a cytosolic‐localized reporter transcript: a noncoding, frame‐shifted variant of Sox2 see . By sequin situ hybridization  to broadly diffuse cytosolic patterns . As previously shown , we used fsSox2 instead of regular Sox2 to avoid any unwanted translation artifacts. The oligo‐pool was expressed in HeLa cells, followed by subcellular fractionation, and targeted RNA‐seq of unique barcodes to determine the enrichment of each fsSox2 variant in the nucleus relative to the total barcode representation in total RNA  act as potent nuclear localization signals  originating from 29 distinct lncRNAs that were significantly enriched in nuclear fractions relative to whole‐cell lysates . Two of D et al, . By contet al, P < 1/106, Mann–Whitney test; We further analyzed the evolutionary conservation, length distribution, and sequence content of DRs for putative nuclear localization sequences. We used phastCons  occurring 18 times exclusively in XIST but not elsewhere in the human genome  was found in 52 DRs of 21 different lncRNAs  in nuclear localization of transcripts containing the C‐rich motif across all 11 ENCODE TIER 2 cell lines . Appending the C‐rich motif did not significantly affect the localization of fsSox2  in HeLa cells, and compared their subcellular localization to that of the fsSox2‐only transcript by smFISH. As expected, we found that the MALAT1 “Region M” significantly increased nuclear enrichment of fsSox2 . Similarly, the TUG1 DR and XIST DR (which harbors the XIST motif) also promoted nuclear enrichment of fsSox2  as the drivers of nuclear enrichment. This includes known regions required to retain Deeper analysis of potential common sequence motifs underlying these longer RNA nuclear enrichment regions uncovered a C‐rich sequence motif that is over‐represented in these regions. Supporting this finding, C‐rich motifs are modestly enriched in total RNA sequencing of nuclear versus cytoplasmic samples provided by ENCODE. Also, a similar motif was identified in an independent study using a similar approach  with MPRNA, one could assay thousands of sequence and structural variants in parallel to determine common binding motifs or structures of RNA–protein interactions. We also envision developing additional MPRNA constructs to screen across diverse aspects of RNA biology, such as sequence requirements for splicing, gene regulation, or enhancer and suppressor activity.Collectively, we have demonstrated that MPRNA is a robust and reproducible strategy to identify activity in RNA sequences. Notably, MPRNA can be applied to any assay where a separation of active versus inactive RNAs can be achieved. Overall, MPRNA can be combined with classic biochemical approaches to achieve the needed genome‐scale to address many pressing RNA biology questions.We designed 153‐mer oligonucleotides to contain, in order, the 16‐nt universal primer site ACTGGCCGCTTCACTG, a 110‐nt variable sequence, a 10‐nt unique barcode sequence, and the 17‐nt universal primer site AGATCGGAAGAGCGTCG. The unique barcodes were designed as described previously, while the variable sequences were obtained by tiling lncRNA sequences. The resulting oligonucleotide libraries were synthesized by Broad Technology Labs.The synthesized oligo‐pool was amplified by emulsion PCR , according to the manufacturers’ instructions. The ePCR primers were designed to add the AgeI/NotI restriction sites to the synthesized oligos for subsequent cloning . To determine the oligos representation of the ePCR‐amplified oligo‐pool (based on the unique 3′ barcode of each oligo), 1 ng of the amplified oligo‐pool was used as input for library preparation (see below) and sequenced on a MiSeq .fsSox2. Similar to a previous publication  was transformed into 10× DH5α tubes (ThermoScientific). A total of 20 ampicillin LB plates were inoculated with the 10 transformation reactions and incubated overnight at 37°C. All bacterial colonies were then scraped in 5 ml of LB per plate and pooled, and the plasmids were purified with the endotoxin‐free Qiagen Plasmid Plus Maxi kit (Qiagen). The cloned oligo‐pool was then sequenced on the MiSeq to determine the oligo representation as described above.A minCMV promoter (5′‐TAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGT GAACCGTCAGATCGC‐3′) was cloned upstream of et al, HeLa nuclear and cytoplasmic fractions were isolated as previously described (Hacisuleyman RNA was isolated by TRIzol (ThermoScientific)—chloroform extraction, followed by isopropanol precipitation, according to standard procedures. 2 μg of BioAnalyzer‐validated RNA was digested with recombinant DNase I  at 37°C for 30 min, followed by heat inactivation at 75°C for 10 min. Reverse transcription was performed with SuperScript III cDNA synthesis kit (ThermoScientific). Quantitative RT–PCR was performed using the FastStart Universal SYBR Green Master mix (Roche) on an ABI 7900. Primers were as follows: NEAT1 forward TGATGCCACAACGCAGATTG, reverse GCAAACAGGTGGGTAGGTGA, and SNHG5 forward GTGGACGAGTAGCCAGTGAA, reverse GCCTCTATCAATGGGCAGACA. After processing the raw data by qPCR Miner  and primers designed to anneal to the universal primer site flanking the oligos and to add sequencing index barcode for multiplexing: forward caagcagaagacggcatacgagatCGTGATgtgactggagttcagacgtgtgctcttccgatctACTGGCCGCTTCACTG, reverse AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG ATCT  the index for the library, and (ii) the region complementary to the universal primer site). PCR amplification  was carried out for 30 cycles followed by triple 0.6×, 1.6×, and 1× SPRI beads  cleanup. The quality and molarity of the libraries was evaluated by BioAnalyzer, and the samples were sequenced in a pool of 6 on the Illumina HiSeq2500, full flow cell, single‐read 100 bp. To ensure the transfection was successful, we required that at least 70% of the oligo‐pool was represented back  in the sequencing sample Fig .To find a unique mapping location for the read, we ensured an exact match between the first 10 read nucleotides and a unique oligo barcode. To ensure that the correct oligo was identified using this barcode match, we allowed only two mismatches between the remaining 65 nts of the read sequence and the upstream oligo sequence corresponding to the unique barcode  identifying potential candidate regions, and (ii) assigning a gion Fig D. We ideMEME  cells were electroporated with 2 μg of construct using the Amaxa® Cell Line Nucleofector® Kit R using program I‐013, and cultured for 48 h in LabTek v1 glass chambers. smFISH was performed using Biosearch Technologies Stellaris® probes, as described previously . Images were obtained using the Zeiss Cell Observer Live Cell microscope at the Harvard Center for Biological Imaging. For each field of view, at least 40 slices (each plane: 0.24 μm) were imaged, and z‐stacks were merged with maximum intensity projections (MIP). fsSox2 foci were computationally identified using the spot counting software StarSearch. To ensure robustness, the analysis was blinded and the person counting the spots did not know the identity of the samples. For each construct, fsSox2 foci within at least 150 cells were counted in biological duplicate.Briefly, 70–80% confluent 1 × 10https://github.com/cshukla/oligoGames.All the analysis in this paper was carried out using a custom package developed for the experiment called oligoGames. The package is currently hosted on GitHub—All analyzed sequence data have been deposited in NCBI GEO under accession GSE98828.JLR conceived the project. PGM, ALM, and CG designed and carried out experiments. CJS, KDK, and RAI conceived and implemented the statistical method to detect Differential Regions. CJS and RAI designed and carried out informatic analyses. MNC designed the oligo‐pool and DMS assisted with the experiments. JLR, CJS, and PGM wrote the manuscript. RAI and JLR supervised and funded the project.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Review Process FileClick here for additional data file.


Non-coding RNA sequences outnumber the protein-coding genes in the human genome, however our knowledge of their functions is still limited. RNA-binding proteins follow the transcripts, including non-coding RNAs, throughout their life, regulating not only maturation, nuclear export, stability and eventually translation, but also RNA functions. Therefore, development of sophisticated methods to study RNA-protein interactions are key to the systematic characterization of lncRNAs. Although mostly applicable to RNA-protein interactions in general, many approaches, especially the computational ones, need adjustment to be adapted to the length and complexity of lncRNA transcripts. Here we critically review all the wet lab and computational methods to study lncRNA-protein interactions and their potential to clarify the dark side of the genome. Over the past decade extensive efforts have been made to refine our understanding of the most complex mystery of life: the genome. With the recent advent of deep sequencing methodologies, it has become clear that the full range of the protein-coding and non-protein-coding transcriptome is still vastly underestimated . The class is very heterogeneous and despite the progresses made in annotating them, lncRNA diversity makes it impossible to draw sweeping generalizations or predict their molecular mechanisms. LncRNAs can localize to nuclear  their protein partners.During and after transcription, RNAs are subjected to multiple processing and regulatory steps that are coordinated by dynamic interactions with other nucleic acids and by a variety of RNA-binding proteins (RBPs) Moore, . RBPs ofDevelopment of new sophisticated methods to study RNA-protein interactions are enabling the systematic characterization of lncRNAs. These approaches can be generally classified in either RNA-centric or protein-centric Figure . While tRNA immunoprecipitation (RIP) and cross-linking and immunoprecipitation (CLIP) have been important allies to identify the RNA substrates of RBPs, systematically and comprehensively.To unravel the mechanisms by which RNA binding proteins (RBPs) affect RNA processing and ncRNA functions, many technologies such as The so-called protein-centric methods employ antibodies to immunoprecipitate the endogenous protein of interest and the associated RNAs. Many variants of these methods have been developed, but their essential difference is the condition of immunoprecipitation, either native or crosslinked.RNA immunoprecipitation (RIP), eventually followed by high-throughput sequencing (RIP-seq)   method was initially combined with the Sanger Sequencing method  technology enabling the investigation of genome-wide RBP–RNA binding at single base-pair resolution. Genome-wide CLIP (CLIP-seq)  combined aspects of both RIP and CLIP to quantify the binding strength of different RNA binding sites, during dynamic biological processes. It can measure enrichment scores for RNA-protein binding, both at the whole target transcript level (RIP-Seq-Like or RSL) and at the binding site level (BSL) using continuous metrics, overcoming the above mentioned limitations of simple RIP approach  was developed as a genome-wide tool to map protein-RNA binding sites in vivo, by RNA-seq. For the library preparation a 3′ RNA adaptor needs to be ligated to the RNA fragments in order to allow reverse transcription using probes complementary to the 3′ adaptors . The main difference is that cells are cultured in the presence of nucleotide analogs such as 4-thiouridine (4-SU) or 6-thioguanosine (6-SG) in the media, which is incorporated into the newly synthesized RNAs. UVA light (365 nm) exposure will crosslink the modified residues of the RNAs and the interacting proteins. During the reverse transcription step the nucleotide analogs are converted into cytosine (C) for 4-SU, or adenine (A) for 6-SG, and can be used to specifically mark the exact binding site at single nucleotide resolution  approach managed to overcome the loss of truncated fragments during library preparation by employing a self-circularization strategy and barcoding, thus strongly improving the quality of the quantitative information obtained from the sequencing  improves the library preparation step and optimizes the efficiency of the circular ligation step of the iCLIP by adding adapters in two separate steps. (i) An indexed 3′ RNA adapter is ligated to the crosslinked RNA fragment on beads during the immunoprecipitation, (ii) a 3′ single-stranded DNA adapter is ligated after reverse transcription, to determine whether two identical sequenced reads come from two unique RNA fragments or from PCR duplicates of the same RNA fragment . By use of infrared-dye-conjugated and biotinylated ligation adaptors, this platform allows a rapid and at the same time quantitative analysis of in vivo captured protein–RNA interactions, using even less material than required by the other standard CLIP protocols  UV radiation allows harsh washings but carries also disadvantages: it can physically and chemically alter the ribonucleoprotein network, leading to results that are far from being physiological. UV cross-linking efficiency is a major issue as it shows sequence bias at the level of both RNA and protein. Generally greatest photoreactivity is observed between pyrimidines and amino acids such as Cys, Tyr, Trp, Phe, and Lys  was initially developed as a hybridization-based purification strategy to map the genomic binding sites of endogenous RNAs and it was first used to determine the genome-wide localization of roX2, a 600-nt ncRNA that regulates dosage compensation in Drosophila  that can be combined to deep sequencing and allow unbiased high-throughput discovery of RNA-bound DNA and proteins  can iteratively find the minimal set of probes targeting the RNA of interest, map at the domain-level RNA–RNA, RNA-protein, and RNA-chromatin interactions as well as identifying genomic binding sites with high sensitivity. Biotinylated antisense 20-mer oligonucleotides are designed to hybridize with non-overlapping and non-redundant sequences on the RNA of choice. Cells are chemically crosslinked as in the standard CHART protocol, chromatin is sheared in small fragments and divided into equal samples to which oligonucleotide pools, all targeting different regions of the RNA of interest, are hybridized under stringent conditions. RNA, DNA and proteins can be separately recovered and analyzed, providing simultaneously information on RNA-, DNA-, protein-interacting domains of an RNA  was initially developed by the Guttman lab to investigate the mechanisms of the Xist lncRNA localization during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation  and the probe design, but depending on the RNA of interest, they can be all adapted, mixed and matched. For example probe design is generally a critical step because the accessible areas of the target RNA might be unknown or hidden by secondary structures. Probes for CHART are empirically determined after RNase H assay to map the accessible regions on the RNA of interest. These regions will be targeted by specifically designed probes  analogs used in the so called Protein-centric and RNA-centric approaches are not redundant methods but complementary to each other, and can be great allies to map the RNA-protein interaction network. Comparison of PAR-CLIP and SILAC-based RNA pull-downs by Scheibe et al., underscores this concept very well: a strong overlap between two completely different approaches would validate them both  to tag the RNA of interest, followed by stable isotope labeling with amino acid in cell culture (SILAC)-based quantitative mass spectrometry. The RNA of interest is tagged with the MS2 stem-loop cluster that does not hamper the normal processing and translation of the RNA , to precisely identify the RNA interface of RBP and its localization on the target RNA   the authors combined a rigorous cell fractionation, with serial purification steps of the RNA of interest  for example, is specifically designed for the detection of regulatory signals in non-coding sequences, and considers mainly the sequences of binding sites. Other tools can model binding sites in a supervised or unsupervised way, considering or ignoring the RNA structure and/or the RNA context , an algorithm developed to evaluate the interaction propensities of polypeptides and nucleotide chains, using their physicochemical properties instead of sequence similarity searches . It was developed modeling experimental data obtained from a small scale proteomic study where 12 Halo-Tagged RBPs were pulled down and their protein interactors analyzed with Multidimentional Protein Identification Technology (MudPIT) mass spectrometry. By comparing an RNAse treated vs. an untreated sample they could distinguish RNA-dependent interactions, therefore SONAR can discover uncharacterized candidate RBPs by leveraging protein-protein interactomes . Moreover, multiple lncRNA-protein complexes might co-exist in different cells. Hence, being able to study lncRNA-protein interactions in single cells with spatial resolution, would undoubtedly improve our knowledge of lncRNA biology. A combination of immunocytochemistry and spatial transcriptomic (Ståhl et al., Mounting evidence from JB wrote the text. EL proposed the topic, selected the literature and reviewed the text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.