Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N). An essential interaction between N and a C-terminal region of P tethers the L/P polymerase to the template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how the polymerase component P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to activity assays and imaging of inclusion bodies in cells. We report a 2.9 Å resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template. To read and transcribe the viral genome, the polymerase complex of human metapneumovirus (HMPV) needs to attach to the nucleocapsid. Here, the authors define the dynamic basis of how the polymerase cofactor P facilitates this attachment. HMPV is thought to have a zoonotic origin following a spillover event from an avian reservoir host species2. The pathogen is now known to cause both upper and lower respiratory tract infections, including both bronchiolitis and pneumonia, with children being the most affected demographic6. It is estimated that 5% to 12% of hospitalizations due to viral respiratory infections in children are caused by HMPV7. Furthermore, infections with HMPV can also be severe in the elderly and in patients with comorbidities8. Serology studies carried out in 2001 found that by the age of 5, virtually all children had been exposed to the virus1. While vaccines against the closely-related respiratory syncytial virus (RSV) have been recently approved for use in pregnant individuals and the elderly12, there are currently no licensed vaccines or specific therapeutics for the treatment of HMPV infections. A better understanding of the molecular mechanisms of the HMPV life cycle are therefore needed.Human Metapneumovirus (HMPV) was first reported in 2001 when it was isolated from children in the Netherlands with symptoms ranging from mild respiratory disease to severe pneumoniaPneumoviridae family13. HMPV possesses a 13.3 kb negative-sense RNA genome containing eight genes: 3’-N-P-M-F-M2-SH-G-L-5’14. The F, SH, and G proteins constitute surface glycoproteins17. The M and M2 genes encode the matrix protein M, and the M2-1 and M2-2 proteins via overlapping ORFs20. The L protein harbors the RNA-dependent RNA polymerase activity (RdRP) and is responsible for the transcription of capped and polyadenylated viral mRNAs as well as replication of the genome21. The viral genome is encapsidated in a sheath of oligomerized copies of the nucleoprotein N, forming a ribonucleoprotein complex termed nucleocapsid23. The nucleocapsid is the template for transcription and replication by L, which is also dependent on the obligate polymerase cofactor, the phosphoprotein P, together forming the active L/P holoenzyme24.HMPV shares many features with RSV, both being non-segmented negative-sense RNA viruses (nsNSVs) belonging to the 25. During RSV and HMPV infections, these inclusion bodies (IBs) have been shown to harbor L/P and N and to be active sites of viral transcription and replication27. Pneumoviridae IBs are spherical membrane-less organelles. Recent studies provide evidence that IBs are biomolecular condensates that form through liquid-liquid phase separation (LLPS)30. HMPV IBs mature in the course of infection and grow via actin-dependent coalescence and fusion of replicative sites27. In a cellular context, the minimal elements for the morphogenesis of pneumoviral condensates are the P and N proteins, which together can form pseudo-IBs, even in the absence of any other viral proteins29. The interaction between N and P has been previously demonstrated to be crucial for the formation of cellular IBs32. However, the HMPV P protein is also able to form liquid condensates through LLPS in the absence of N, at least in vitro32.A hallmark of nsNSVs is the formation of specialized viro-induced cytoplasmic inclusions which concentrate viral RNA and proteins33. In oligomerized and RNA-bound N (N-RNA) the nucleic acid is threaded along an extended groove in between the NTDs and CTDs of laterally connected N protomers. Furthermore, N possesses short N-terminal and C-terminal extensions (termed arms) which contact neighboring N protomers and facilitate oligomerization. In the context of infected cells, nucleocapsids are co-transcriptionally assembled from N0-P – RNA-free N protomers which are kept in a monomeric state by binding to the N-terminal region of P. A crystal structure of the HMPV N0-P complex has been previously reported33. The HMPV P protein is modularly disordered and forms a tetramer via a central coiled-coil domain35. Upstream and downstream of the coiled coil, P possesses extended regions which are mainly intrinsically disordered or present transient secondary structures in the absence of binding partners, but can conditionally fold upon interaction with other proteins. For instance, a recent cryo-EM reconstruction of the HMPV L/P complex showed contextual folding of regions of P lying downstream of the coiled coil through interaction with the polymerase24. However, in the same structure, only 22% of the total residues of tetrameric P present in the construct were visible in the cryo-EM density, with the remainder being disordered. These substantial conformational dynamics make structural elucidation projects involving P challenging.The nucleoprotein N possesses globular N-terminal and C-terminal domains Paramyxoviridae (e.g. measles virus), N proteins possess a long (up to ~170 residues) unstructured tail termed Ntail, which extends far from the nucleocapsid38. A region of the paramyxoviral Ntail interacts with the so-called PXD domain of the phosphoprotein, forming a PXD-NMoRE complex42. Recently, analysis of compensatory mutations after N truncation has indicated that the PXD-NMoRE complex can also attach to the core of N, however high-resolution structural data of this interaction is not yet available43. In the family Rhabdoviridae, which includes rabies and vesicular stomatitis viruses, the C-terminal domain of the P protein is less disordered than in other nsNSVs and attaches to two C-terminal domains of neighboring N-RNA protomers45. Finally, in the Pneumoviridae HMPV and RSV the N-RNA/P attachment occurs via an interaction of the disordered C-terminal region of P (PCT) with N46. The N-RNA/P interaction is thought to be important in multiple steps of the viral replicative cycle: 1) tethering of the L/P complex to the nucleocapsid during transcription/replication, 2) formation of IBs, 3) loading of nucleocapsids into assembling progeny virions through an interaction with the viral matrix47. Even though these constitute crucial functions, the structural basis of this interaction in HMPV has thus far remained elusive.An important function of the C-terminal domain of P in nsNSVs is to bind to assembled N-RNA, forming an N-RNA/P complex. However, the nature of the N-RNA/P interaction differs within nsNSV families. In the family CT (N-RNA/P), revealing the structural basis of the interaction surface. P binds into a groove at the periphery of the NTD of N, between a helix and an extended loop of a beta-hairpin of N (here denoted as NβHL). We observed that the density of NβHL becomes more ordered upon P binding, indicative of a folding-upon-binding event. All-atom explicit solvent MD simulations of an N-RNA/P 5-mer indicated substantial conformational dynamics of the PCT, in line with the notion of a transient interaction. We further investigated pseudo-IB formation via a functional minigenome assay to quantify the effects of mutations within the beta-hairpin loop. Our experiments confirmed that NβHL is important for the binding of P and that perturbations of this interaction disrupt polymerase activity and IB formation. Taken together our data elucidate the structural mechanism of P attachment to N-RNA which retains substantial conformational flexibility in the bound state. Our observations are in line with a dynamic N-RNA/P interaction which allows rapid binding and unbinding during viral transcription/replication, yet is still sufficiently strong to maintain association through the multivalent character of the interaction partners.In this study we report cryo-EM structures of recombinant HMPV N-RNA bound to PPneumoviridae nucleocapsids are helical assemblies of N bound to the viral RNA genome. Our strategy to elucidate the interaction between assembled N-RNA with the polymerase cofactor P was to employ a tractable system of recombinant N-RNA rings, described previously33. Viral N-RNA rings are present in both infected cells as well as in virions, however, their function is currently unclear49. We reasoned that recombinant N-RNA rings could be leveraged as a tool to obtain high-resolution cryo-EM structures of a complex with P, circumventing confounding factors such as the inherent flexibility of helical N assemblies in Pneumoviridae22. As an initial step, we set out to determine the suitability of N-RNA rings for high-resolution single particle cryo-EM. HMPV N (strain NL/00/1) was expressed in E. coli and purified via size exclusion chromatography  indicates that a similar loop is also present in other viral families .The intra-assembly tilting of N begs the question of which N-N interactions at the lateral flanks remain constant and support side-to-side binding throughout the motion. While the importance of the N-terminal and C-terminal arms of N has been well established in this roleing Fig. . Multipl31. Mutagenesis of P308 to alanine resulted in a notable decrease of polymerase activity, while the R27A mutation nearly abrogated polymerase activity  and N-RNA. P is a flexible protein with extensive intrinsically disordered regions, and the N-RNA/P interaction has been suggested to be transient, complicating structural characterization59. Using the deep-learning-based Metapredict server60, we generated disorder-score plots for both HMPV N and P. In agreement with previous work, N disorder scores are globally low, while P contains significantly disordered regions . Cryo-EM analysis of the N-RNA/P complex revealed the presence of the same classes of assemblies as was the case with the apo sample. We carried out 3D reconstruction of 10-mers, 11-mers, and spiral particles of the N-RNA with PCT  on one side and an extended loop of a beta-hairpin of N  on the other, burying a total area of ~1100 Å2 upon complex formation. We observed a noticeable increase in ordered density of NβHL upon P-binding, compared to the apo form , highlighting their importance in clamping onto N .To investigate the dynamics of Pons Fig. . The RNAons Fig. . Comparient Fig. . In content Fig.  and the ent Fig. , D. Notaent Fig. , and re- CT Fig. . These dCT and the flexible NβHL within cells using minigenome assays. We focused our mutations on the residues in positions 100, 103, and 111 of N, which pack against Y290 of the PCT  upon co-expression in bacteria  and BFP-tagged P and imaged the cells by fluorescence microscopy. The P-BFP fluorescence allowed us to observe pseudo-IBs, while labeling with anti-N antibodies allows only the observation of inclusion outlines, likely due to inaccessibility of the interior. Compared to the wt condition, pseudo-IBs formed in the presence of L111E mutant of N were more numerous and smaller, suggesting a defect of nucleation. In line with the pull-down and minigenome assays, the N triple mutant TM-E resulted in a total loss of IB formation, with the P protein being distributed throughout the cytoplasm. Together, these results confirm that NβHL is in contact with P, and that perturbation of this contact leads to a loss of polymerase activity and affects LLPS induced by the interaction of N and P proteins.The expression of P and N is sufficient for the formation of pseudo-IBs in cellsPneumoviridae L/P complexes revealed that the central oligomeric domain of the P tetramer and sections of the four C-terminal domains of tetrameric P interact with L62. These structures also showed that each of the C-terminal domains of P adopts specific and different conformations upon interaction with L, highlighting the structural versatility of P which is required for the proper functioning of L/P. Of note, in the HMPV structure the last C-terminal residues of P, identified as essential for the interaction with the nucleocapsid32, were not resolved. The structures imply that one tetramer of P can interact with both L and the nucleocapsid simultaneously. Residues of HMPV P and N involved in the P-nucleocapsid interaction have been identified32. Previous crystallographical investigations of this N-P interaction in the related pneumovirus RSV utilized a truncated N protein and revealed density for only the last two residues of P 46. Previous attempts to elucidate how L/P attaches to N-RNA thus remain limited, and the impact of P binding on full-length N is still unknown.The viral RNA genome of all nsNSVs is constitutively encapsidated by the nucleoprotein N, forming the nucleocapsid. Packaging within this ribonucleoprotein complex is thought to protect the genome from degradation by cellular RNases but also from the recognition by sensors of the cellular immune system such as RIG-I. However, a consequence of the encapsidation is that it is not naked RNA but rather the N-RNA complex which serves as a template for the viral polymerase L. The P protein is an essential cofactor of L, which interacts with both L and N-RNA, functioning as a flexible tether. The recent cryo-EM structures of 33. Analysis of our structural data revealed heterogeneity in the relative geometries of assembled N protomers, supporting the notion of a highly flexible nucleocapsid. Furthermore, we identified a key N-N interaction involving the loop ranging from residues 232-239 of the Ni protomer which inserts into the neighboring Ni-1 protomer. We found that residue R27 of the loop was sensitive to mutation and a single exchange to alanine had a major impact on polymerase activity. However, further structural characterization of N-RNA oligomers of R27 mutants are necessary to determine the precise role of this interaction for nucleocapsids.Here, using cryo-EM and full-length N, we were able to determine the structures of assembled and RNA-bound HMPV N in different oligomeric states, including a spiral particle reminiscent of a helical turn, and with improved resolution compared to a previous crystal structureCT-N-RNA interaction. Utilizing an approach that made use of a large molar excess of PCT vs. N, computational symmetry expansion, and local refinement we were able to resolve PCT bound to the NTD of assembled and RNA-bound N. Our structure reveals how P binds by hooking itself around a centrally positioned arginine of the NTD, thereby latching on to N-RNA. The PCT interacts with the partially disordered NβHL region of the NTD, which becomes more ordered upon binding. Furthermore, in the apo-state the PCT itself has been characterized as having high disorder propensity35. Such folding upon binding events of intrinsically disordered regions are thought to facilitate highly specific interactions, yet possess low affinities and rapid dissociation kinetics63. We recognize that our experimental conditions involving ~100-fold molar excess of PCT would not be the case in vivo, but likely assisted in shifting the equilibrium to a bound state, enabling resolving the site.We were successful in obtaining the first structural data on the HMPV PCT/N-RNA complex we turned to all-atom MD simulations of assembled 5-mers. We observed that the presence of the peptide did not have a major impact on the stability of RNA bound to the nucleoprotein cleft. This suggests that PCT attachment alone is not sufficient for inducing the release of RNA from the nucleocapsid – a fundamental requirement to enable L/P access its template. Perhaps it is instead the free CTD-arm of N that is involved in dislodging RNA at the 3’-end of nucleocapsids and thereby assists L/P binding the initial nucleotides. In line with this notion, the CTD-arm has previously been shown to possess a wide spectrum of possible conformations23 and affinity for the RNA-binding cleft of N33. In addition, the simulations revealed substantial conformational flexibility of PCT within the N-RNA/P complex. In one instance we observed the dissociation and rebinding of one of the simulated peptides. This is in line with the disordered character of P, but also with previous reports in RSV characterizing the interaction as giving rise to a fuzzy complex64.To explore the dynamics of the P24. It was previously suggested that the three bundled P protomers interact with N-RNA, while the P protomer at the nucleotide entry channel is more directly involved in polymerase function24. This notion is fully consistent with the fact that in a structure of the related RSV L/P the PCT of the protomer at the nucleotide channel is found to fully attach to L and is thus unavailable for N-RNA binding62. The observation that the 3 remaining P protomers from HMPV only interact with L up to residue 236 implies a long linker to the N-RNA binding site ( ~ 50 residues) potentially able to span large distances between L/P and N-RNA. In addition, this linker region is potentially highly flexible35, suggesting that the N-RNA binding sites of HMPV P can sample large capture radii, and L/P may be able to bind to N-RNA over considerable distances or over multiple turns of the helical nucleocapsid. However, structural data of nucleocapsid-bound L/P is required to substantiate these ideas.In the cryo-EM structure of HMPV L/P, the P tetramer binds L in an asymmetrical manner: the C-termini of 3 protomers of P are bundled together on one side of the polymerase complex and are resolved to around residue 236, while another P protomer on the opposite side is resolved up to 266 of a total of 294 residues and winds around the nucleotide entry channel65 and in the case of L/P this needs to be sufficient to dislodge tetrameric P from any given position on the nucleocapsid while it is being traversed by L/P. In the hypothetical case of a PCT binding too tightly to N-RNA, the pulling force might not be able to overcome this tethering, leading to a stalled complex. At the same time, the interaction needs to be strong enough to recruit L/P for transcription. Several decades ago a cartwheeling model was proposed for the Sendai paramyxovirus66, suggesting that “P is envisaged to “walk” on the template via the simultaneous breaking and reforming of subunit-template contacts”. Our data fully support the applicability of this classic model for the Pneumoviridae L/P complex, in which it scuttles along the nucleocapsid template during processive RNA synthesis using the C-terminal domains of tetrameric P as legs.Taking together the properties of the N-RNA/P interaction, we suggest that its evolution is constrained by a need to balance sufficient tethering of L to the nucleocapsid with being able to unbind/rebind with low energy barriers during transcription. RNA-polymerases exert pulling forces in the tens of Piconewtons rangeCT binding site on RSV N46. Unfortunately, antiviral inhibition through M76 was limited. More recently, the 1,4-benzodiazepine-derived compound EDP-938 shows promising antiviral activity against RSV-A and RSV-B, a relatively high barrier against resistance, efficacy in non-human primates, and was superior to placebo in a randomized, double-blind challenge trial68. As of 2023, EDP-938 is still undergoing phase II clinical trials. Based on the mapping of resistance mutations, it was concluded that the compound targets the RSV N protein. Interestingly, the by-far strongest resistance mutant to EDP-938  is located in a region corresponding to the NβHL of HMPV  and NL99-1 (glycine) differ from the reference strain NL/00/1 used for the structural studies (isoleucine). Point mutations were introduced in the N sequence by site-directed mutagenesis using the Q5 site-directed mutagenesis Kit (New England Biolabs). Primers used for mutagenesis are shown in Supplemental Table The published plasmidsAntisera used in this study included polyclonal rabbit antisera raised against recombinant HMPV N expressed in bacteria. A mouse monoclonal anti-tubulin  and horse-radish peroxidase-coupled secondary antibodies directed against mouse IgG  and rabbit IgG  were also used for immunoblotting. A secondary antibody directed against rabbit IgG coupled to Alexafluor-488  was used for immunofluorescence experiments.69 were grown in Dulbeco Modified Essential Medium (Lonza) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and antibiotics. The cells were grown at 37 °C in 5% CO2 and transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer.BHK-21 cells (clone BSRT7/5) constitutively expressing the T7 RNA polymeraseBSRT7/5 cells at 90% confluence in 96-well dishes were transfected using Lipofectamine 2000 (Invitrogen) with a plasmid mixture containing 62.5 ng of pGaussia/Firefly minigenome, 62.5 ng of pN, 62.5 ng of pP (WT and mutants), 31.25 ng of pL, 31.25 ng of pM2-1, as well as 15.6 ng of pSV-β-Gal (Promega) to normalize transfection efficiencies. Cells were harvested at 24 h post-transfection and lysed in 100 µl of Firefly lysis buffer . Then 50 µL of lysates were mixed to 50 µl D-luciferine  and the Firefly luciferase activity was determined for each cell lysate with an Infinite 200 Pro , and normalized based on β-galactosidase  expression. Transfections were done in quadruplicate, and each independent transfection experiment was performed up to four times. For protein expression analysis, cells were lysed in Laemmli buffer, boiled, and analyzed by Western blotting (WB) using the following primary antibodies: rabbit polyclonal raised in-house against recombinant HMPV N (1:1000 dilution) and mouse monoclonal against tubulin . The following secondary antibodies were utilized: anti-mouse coupled to horse-radish peroxidase  and anti-rabbit coupled to horse-radish peroxidase . Uncropped Western blots are shown in Supplementary Fig. Immunofluorescence microscopy was performed with cells grown on coverslips and previously transfected with pP-BFP and pN (WT or mutants), at a ratio of 1:1. At 24 h post-transfection, cells were fixed with 4% paraformaldehyde (PFA) for 20 min, made permeable and blocked for 30 min with PBS containing 0.1% Triton X-100 and 3% bovine serum albumin (BSA). Cells were then successively incubated for 1 h at room temperature with primary and secondary antibody mixtures diluted in PBS containing 3% BSA and 0.05% Tween20. Utilized antibodies: rabbit polyclonal raised in-house against recombinant HMPV N (1:5000 dilution) and secondary anti-rabbit IgG coupled to Alexafluor-488 . Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen) and observed with an inverted fluorescence microscope (Zeiss Axiovision). Images were analyzed with ImageJ 1.53.E. coli BL21 bacteria (DE3) (Novagen) transformed with pET-N (WT or mutants) alone or with pGEX-PCT were grown at 37 °C for 8 hours in 100 ml of Luria Bertani (LB) medium containing 100 µg/ml ampicillin or ampicillin (100 µg/ml) and kanamycin (50 µg/ml), respectively. The same volume of LB was then added and protein expression was induced by adding 80 µg/ml isopropyl-ß-D-thio-galactoside (IPTG) to the medium. The bacteria were incubated for 15 hours at 28 °C and then harvested by centrifugation. For purification using the GST-tag, bacteria were re-suspended in lysis buffer  supplemented with complete protease inhibitor cocktail (Roche), incubated for 1 hour on ice, sonicated, and centrifuged at 4 °C for 30 min at 10,000 g. Glutathione-Sepharose 4B beads  were added to clarified supernatants and incubated at 4 °C for 3 hours. Beads were then washed with 10 volumes of lysis buffer, then three times with PBS 1X, and then stored at 4 °C in an equal volume of PBS.2+ . Beads were then successively washed in the washing buffer  containing increasing concentrations of imidazole , and proteins were eluted in the same buffer with 500 mM imidazole. For SDS-PAGE analysis, samples were prepared in Laemmli buffer, denatured for 5 min at 95 °C, and separated on 12% polyacrylamide gel colored by Coomassie brilliant blue.For purification of N-6xHis fusion protein purification, bacterial pellets were re-suspended in lysis buffer  supplemented with complete protease inhibitor cocktail (Roche). After sonication and centrifugation, lysates were incubated 30 min with chelating Sepharose Fast Flow beads charged with NiYP_009513265.1) was cloned into the pOPINE bacterial expression vector, also coding for a C-terminal His-tag, using the In-Fusion system 70. The vector DNA sequence was verified by means of Sanger sequencing. Rosetta2 E. coli cells containing the plasmid were grown at 37 °C in terrific broth (TB) containing ampicillin (100 µg/ml). Once an OD600 of 0.8 was reached, the cells were cooled to 18 °C and then induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were harvested after overnight growth . Cell pellets were resuspended in 40 mL of wash buffer  per litre of bacterial culture, and lysed by means of sonication. The resultant lysate was clarified  and the supernatant was filtered and loaded on a column containing Ni2_NTA (nitrilotriacetic) agarose . The column was equilibrated with wash buffer and the bound protein was washed. The protein was eluted in elution buffer , and the resultant eluate was further purified by size exclusion chromatography, using a Superose6 10/300 column , equilibrated in SEC buffer . The protein was then buffer exchanged into 25 mM Tris-HCl, pH 8.0, and 250 mM NaCl using a PD10 column .The full-length N protein gene from human metapneumovirus strain NL/00/1 (GenBank: 4OH to improve solubility. N-RNA preparations were mixed with the resulting peptide solution in a ~ 100-fold molar excess of P peptide to N. The N-RNA and PCT mixture was incubated at 4 °C overnight.A synthetic peptide of P covering the sequence N-EDDIYQLIM-C was obtained from GenScript. The peptide was dissolved in deionised water and dosed with a drop of 5 M NHCT (0.5 mg/ml) sample was placed onto glow-discharged Quantifoil R2/1 Cu300 holey carbon grids  , blotted for 3.5 s before flash-freezing in liquid ethane using a Vitrobot mark IV (FEI) at 95 – 100 % humidity. Cryo-EM data were collected using SerialEM 3.6 on a 300 kV Titan Krios microscope (Thermo Fisher Scientific) fitted with a K2 Summit (Gatan) direct electron detector and GIF Quantum energy filter. Data collection parameters are listed in Supplementary Table 71.Cryo-EM experiments were carried out at the Oxford Particle Imaging Centre (OPIC). 3 µL of N-RNA 1.0 mg/ml) or N-RNA/P.0 mg/ml 5FVC. We also attempted an approach with 10-fold symmetry expansion and local refinement of a N-RNA monomer, however this yielded worse maps, presumably because a single monomer is not of sufficient size for local refinement (less than 50 kDa). Total particle numbers and numbers of particles used for the reconstruction of every map can be seen in Supplementary Figs. CT, respectively, and in Supplementary Table All single-particle reconstructions were carried out in cryoSPARC 2.1. Particle picking was carried out using a combination of the cryoSPARC blob picker and template picker. The 2D classification was utilized to separate particle subsets of different oligomeric states and remove bad particles. Particles were further cleaned through heterogeneous refinement. The final maps were obtained from the non-uniform refinement job type. Symmetry was imposed only when it was clearly visible in template-free 2D class averages, as was the case for the 10-mer and 11-mer classes . No symmetry was imposed for the refinement of spiral particles. To improve the density of N-RNA protomers we carried out 5-fold symmetry expansion of the 10-mer particles, followed by local refinement of a N-RNA dimer. Mask creation for the local refinement was based on a N-RNA dimer from PDB ID: 5FVC). Iterative cycles of manual refinement and building in COOT 0.9.772 and real-space refinement in PHENIX 1.1873 were used to optimize geometry and fit the density. PHENIX validation tools were used to monitor geometry and density fit parameters. For maps with imposed symmetry (10-mer and 11-mer maps), we refined a single protomer copy and generated symmetry mates for figure preparation. The N-RNA models (from maps without P) encompassed residues 3 to 365 of the N protein, with a gap from residues 100 to 111 where the density was not sufficient for building a model. Moreover, we observed only very incomplete and diffuse density after residue 365 (the C-arm) and thus refrained from continuing to build the model after this point. For the models of the spiral particles we limited ourselves to rigid body fitting of N-RNA protomers (from the high-resolution refinements) with flexible N-and C-termini truncated, as the spiral particle maps were only of intermediate quality, presumably due to low particle numbers and heterogeneity. For the N-RNA/PCT complex, the local refined map showed much-improved density in the region of residues 100-111 and thus allowed building a continuous model up to residue 365. The density for the P-peptide had the best quality in the N-RNA/PCT local refinement, so this was used to model P residues 288 to 294. The N-RNA/PCT model from the locally refined dimer maps was then used as a basis for the model refinement of N-RNA/PCT 10-mers and 11-mers. For the spiral map we again only refined the overall positions of N-RNA/PCT protomers with truncated termini via rigid-body, due to limited map quality. Model refinement statistics are given in Supplementary Table The model of HMPV N obtained from X-ray crystallography was used as a starting point for further refinement (PDB ID: CT complexes. A 5-mer of N-RNA complex, with or without the 5 bound PCT peptides, was extracted from the N-RNA/PCT cryo-EM structure. The MD systems were set up using the solution builder tool from CHARMM-GUI input generator74. Each system was solvated in a rectangular periodic box which size was determined by the biomolecular extent, resulting in the addition of approximately 200k TIP3P water molecules. The systems were neutralized by adding 150 mM NaCl. The two systems were then energy minimized, equilibrated in NVT ensemble, and simulated for ~180 ns in 2 independent trajectories in GROMACS202175 by making use of the CHARMM-GUI webserver provided scripts76. The CHARMM36m force field was used for all simulations77. The trajectories were analyzed using GROMACS tools to extract RMSDs and a number of hydrogen bonds as a function of simulation time.Classical MD simulations were used to study the dynamics of the N-RNA and N-RNA/P79. Structure figures and movies were prepared in ChimeraX. Multiple sequence alignments were prepared using the PROMALS3D webserver and Jalview 2.11.2.081, using the Clustal X color scheme. Structure interfaces were analyzed using the COCOMAPS server82.Structures were inspected and analyzed using tools within UCSF Chimera 1.14 and ChimeraX 1.1Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary


Saccharomyces cerevisiae telomerase RNA, TLC1, is an 1157 nt non-coding RNA that functions as both a template for DNA synthesis and a flexible scaffold for telomerase RNP holoenzyme protein subunits. The tractable budding yeast system has provided landmark discoveries about telomere biology in vivo, but yeast telomerase research has been hampered by the fact that the large TLC1 RNA subunit does not support robust telomerase activity in vitro. In contrast, 155–500 nt miniaturized TLC1 alleles comprising the catalytic core domain and lacking the RNA’s long arms do reconstitute robust activity. We hypothesized that full-length TLC1 is prone to misfolding in vitro. To create a full-length yeast telomerase RNA, predicted to fold into its biologically relevant structure, we took an inverse RNA-folding approach, changing 59 nucleotides predicted to increase the energetic favorability of folding into the modeled native structure based on the p-num feature of Mfold software. The sequence changes lowered the predicted ∆G of this “determined-arm” allele, DA-TLC1, by 61 kcal/mol (−19%) compared to wild-type. We tested DA-TLC1 for reconstituted activity and found it to be ~5-fold more robust than wild-type TLC1, suggesting that the inverse-folding design indeed improved folding in vitro into a catalytically active conformation. We also tested if DA-TLC1 functions in vivo, discovering that it complements a tlc1∆ strain, allowing cells to avoid senescence and maintain telomeres of nearly wild-type length. However, all inverse-designed RNAs that we tested had reduced abundance in vivo. In particular, inverse-designing nearly all of the Ku arm caused a profound reduction in telomerase RNA abundance in the cell and very short telomeres. Overall, these results show that the inverse design of S. cerevisiae telomerase RNA increases activity in vitro, while reducing abundance in vivo. This study provides a biochemically and biologically tested approach to inverse-design RNAs using Mfold that could be useful for controlling RNA structure in basic research and biomedicine. Telomeres are the protein-bound repeated segments of DNA at the ends of linear chromosomes that help to define the natural DNA ends. Because canonical DNA polymerases are unable to fully replicate the ends of the chromosome, telomeres shorten with each successive generation . If leftSaccharomyces cerevisiae, the 1157 nt telomerase RNA is TLC1, which adopts an overall Y-shaped secondary structure with three long arms radiating out from the central catalytic core ) ) . After sXhoI, electrophoresed through a large 1.1% agarose gel at 70 V for 17 h, and transferred to Hybond-N+ Nylon membrane (GE). The blot was then probed for yeast telomeric sequence and a 1627 bp non-telomeric fragment of chromosome IV. Average Y′ telomere fragment length was quantified using the weighted average mobility (WAM) method described previously -dGTP. Products were electrophoresed through a 10% polyacrylamide/TBE/urea gel and imaged using phosphor screens and a Typhoon 9410 Variable Mode Imager. As an internal control for product recovery and loading, ~1 nM [γ-32P] end-labeled primer was added before the telomerase reaction. Activity levels were normalized to the internal control.Linearized “run-off” dsDNA templates for T7 transcription of telomerase RNAs were made using PCR (with low-error DNA polymerase) of the gene with a T7 promoter included at the 5′ end of the forward primer. In vitro telomerase activity assays were performed as described previously . Briefly


We conduct meta-regressions to identify demographic and clinical characteristics that contribute to cross-study heterogeneity in UNC findings. UNC FA was reduced in individuals with depression compared to healthy individuals. UNC RD was comparable between individuals with depression and healthy individuals. Comorbid anxiety explained inter-study heterogeneity in UNC findings. Depression is associated with perturbations in UNC microstructure, specifically with respect to UNC FA and not UNC RD. The association between depression and UNC microstructure appears to be moderated by anxiety. Future work should unravel the cellular mechanisms contributing to aberrant UNC microstructure in depression; clarify the relationship between UNC microstructure, depression, and anxiety; and link UNC microstructure to psychological processes, such as emotion regulation.Aberrant microstructure of the uncinate fasciculus (UNC), a white matter (WM) tract implicated in emotion regulation, has been hypothesized as a neurobiological mechanism of depression. However, studies testing this hypothesis have yielded inconsistent results. The present meta-analysis consolidates evidence from 44 studies comparing fractional anisotropy (FA) and radial diffusivity (RD), two metrics characterizing WM microstructure, of the UNC in individuals with depression ( Major depressive disorder (MDD), a leading cause of disability worldwide , a metric derived from diffusion tensor imaging (DTI) that correlates positively with the directionality and coherence of fibers within a WM bundle . A second strategy is to track fibers between relevant gray matter regions . Notably, a recent meta-analysis using the ENIGMA approach  did not find atypical UNC FA in individuals with MDD . First-degree relatives (REL) of individuals with MDD are at an increased risk for MDD themselves  whether other DTI metrics such as AD and MD also differ between individuals with and without MDD, and (b) whether atypical UNC microstructure is also present in first-degree relatives of individuals with MDD.The present meta-analysis has been pre-registered in PROSPERO ID #CRD42021276200). To identify relevant studies, we used the search engine PubMed with the following search terms: depression and WM. We included all original research articles published before July 9th, 2022, that used a TOI approach to compare FA of the left and right UNC between individuals with a lifetime diagnosis of depression or REL and HC. Depression was broadly defined as a lifetime diagnosis of MDD, persistent depressive disorder, or other specified depressive disorders. REL were defined as first-degree relatives of an individual who either (1) met criteria for MDD, or (2) scored higher than the clinical cut-off on a dimensional depression rating scale. We excluded book chapters, conference abstracts, review articles, and case reports. We identified 49 different studies that met our inclusion criteria . In addi276200. TnMDD = 4930), sex ratio , mean illness duration , the percentage of participants using psychotropic medication , and the percentage of subjects with lifetime comorbid anxiety disorder . Further, we extracted Hamilton Depression Rating Scale , given that it was the most frequently reported measure of depressive symptom severity. In line with prior work, we standardized scores across different versions of the HDRS . We calculated the effect sizes for each study as standardized mean difference values (Cohen's d) and used the effect sizes to conduct random-effects inverse-variance weighted meta-analyses. We tested whether lower FA or higher RD in the bilateral UNC differentiated MDD or REL from HC, and considered effects significant at p < 0.025, correcting for tests in two hemispheres. As an exploratory analysis, we also tested whether there were differences in MD and AD in the bilateral UNC when comparing MDD and HC. Publication bias was determined using Egger's test for asymmetry, and the robustness of results was assessed using jackknife sensitivity analyses. Using a meta-analytic fixed-effects model, we compared FA effect sizes in the left and right hemispheres in individuals with MDD. Using this approach, we also indirectly compared FA effect sizes between REL and individuals with MDD to determine whether the effect size was similar in these study groups.All analyses used the metafor package (version 3.4-0) for R software . Recommendations suggest that meta-regression analyses should include at least 20 studies to produce robust findings. Thus, we examined sources of heterogeneity for studies comparing FA between MDD to HC (n = 44), but not for studies comparing RD between MDD and HC (n = 15) or studies investigating FA between REL and HC (n = 6). We also did not examine sources of heterogeneity for studies comparing MD between MDD and HC (n = 17) or for studies comparing AD between MDD and HC (n = 12). Data and code supporting this study's findings are publicly available on the Open Science Forum (https://osf.io/hdp57/).We conducted meta-regression analyses to investigate whether age, sex, illness duration, depressive symptom severity, medication use, comorbid anxiety, and DTI processing pipeline contributed to heterogeneity across studies. Using Bonferroni correction to correct for multiple comparisons, we set a final statistical threshold for significance of n = 43), sex (n = 42), mean illness duration (n = 24), mean HDRS scores (n = 23), medication use (n = 35), and comorbid anxiety (n = 30). All studies provided information on the DTI processing pipeline. There were missing data on demographic characteristics  and clinical characteristics .Our initial literature search encompassed 2803 studies . After sz = 0.79, p = 0.432; z = −0.01, p = 0.993; p = 0.003; p = 0.059; p = 0.735), suggesting no significant difference between the two hemispheres. In the right UNC, jackknife sensitivity analysis found that no single study drove these results. For the left UNC, jackknife sensitivity analysis showed that the effect became significant (p = 0.013) when leaving out one particular study, which consisted of individuals with MDD who experienced moderate anxiety symptoms  but not right UNC . Specifically, comorbid anxiety attenuated the group difference between individuals with MDD and HC in the left UNC. We found no evidence that age, sex, duration of illness, the severity of depressive symptoms, medication use, or the DTI processing pipeline contributed to variance in UNC findings in individuals with MDD .Comorbid anxiety explained variability in findings in the left . In the right UNC, Egger's test suggested no publication bias . Thus, for the final analysis, we excluded one study  or right UNC  when comparing individuals with MDD to HC. RD effect sizes were comparable between the left and right UNC (p = 0.416).For the left UNC, Egger's test indicated one outlier that showed greatly elevated RD in individuals with MDD . However, indirect comparisons of UNC FA between REL and individuals with MDD yielded significant results in the right UNC  and marginally significant results in the left UNC . In other words, individuals with MDD showed reduced UNC FA compared to REL. Separately, we found no differences in MD or AD in the left or right UNC when comparing individuals with MDD to HC .We found no FA differences in the left or right UNC when comparing REL to HC . Indirect comparisons of UNC FA between hemispheres also yielded no significant results (NMDD = 1087) and without MDD (NHC = 1532). We also found no support for the hypothesis that aberrant UNC FA represents a vulnerability for MDD. However, the available sample of REL was small , and thus must be cautiously interpreted.The present meta-analysis, which integrates data from 5016 individuals with MDD and 18 425 HC, associates MDD with reduced FA in the uncinate fasciculus, suggesting perturbed coherence of this tract. We also provide preliminary evidence for a laterality effect potentially related to comorbid anxiety. In contrast to our hypothesis, this effect was not related to alterations in RD, which was comparable between individuals with  did not report these variables. It will be important that future studies report participants' racial and ethnic background, as these demographic features may relate to traumatic experiences (Kirkinis, Pieterse, Martin, Agiliga, & Brownell, While we were well-powered to examine our main question, findings on RD, MD, AD and REL must be considered preliminary, given the substantially smaller number of studies used in these analyses. Unfortunately, we could not determine whether findings generalize to diverse racial and ethnic backgrounds as the majority of studies (The present meta-analysis found reduced FA in the UNC in individuals with, but not those at risk for, MDD. However, comorbid anxiety may weaken associations between depression and UNC microstructure. Future work needs to investigate when and how perturbations in UNC microstructure develop and should aim to link it to psychological processes previously implicated in depression, such as impaired emotion regulation.


RNA molecules play many functional and regulatory roles in cells, and hence, have gained considerable traction in recent times as therapeutic interventions. Within drug discovery, structure-based approaches have successfully identified potent and selective small-molecule modulators of pharmaceutically relevant protein targets. Here, we embrace the perspective of computational chemists who use these traditional approaches, and we discuss the challenges of extending these methods to target RNA molecules. In particular, we focus on recognition between RNA and small-molecule binders, on selectivity, and on the expected properties of RNA ligands. However, it has become increasingly evident that RNA has many functional and regulatory roles in all domains of life. For instance, RNA molecules can regulate gene expression directly or by interacting with small organic molecules  how to describe the target’s structure, and in particular how to account for its intrinsic flexibility and structural dynamics, and ii) how to assess quantitatively the binding poses. These aspects are already critical in the context of protein targets but may be even more crucial for RNA targets. Concerning the former aspect, any docking campaign requires the target’s structure as the starting point. Experimental methods to reconstruct the 3D structures at the atomistic level include X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy  algorithm in the Rosetta software suite  with overall favourable interactions between the two binding partners. We here briefly outline some popular docking software packages and refer the interested reader to a recent comprehensive overview  knowledge-based, when the scoring is built upon information extracted from known three-dimensional structures of target-ligand complexes; ii) physics-based, when the scoring is based on force fields or simplified empirical functions of the target-ligand interactions and iii) machine learning (ML)-based, when the scoring is evaluated through ML models trained on available experimental data , this complex folding can nevertheless produce distinctive cavities reminiscent of protein-like binding pockets  future. Indeed, as a further level of complexity, recently discovered potent and selective ligands of an RNA ribozyme contained chemical groups that would usually be classified as PAINS  the prominent role of target flexibility in predicting small-molecule binding; ii) the importance of achieving binding selectivity; and iii) the knowledge of the chemical space expected for RNA-binding drugs.We have discussed how the currently available computational procedures, which have been optimised and refined over decades of efforts directed to protein targets, can be used in the novel and partially unexplored context of RNA targets, and how to take appropriate precautions. For example, recent scoring functions were specifically developed to describe the interaction of small molecules with RNA, which means that well-established docking protocols can be easily adapted to RNA targets. Moreover, we discussed how MD simulations, which are extensively used for lead optimisation in protein-based drug discovery, will become an essential tool for considering RNA target flexibility in the earlier stages of screening campaigns. Furthermore, experimental data can increase the reliability of RNA conformational ensembles reconstructed via MD. Interestingly, we note how experimental information often has a dual function when targeting RNA, since the experimental data can be used as the source of starting structures for the simulations and as a guide to refine the conformational ensembles. Moreover, MD simulations  may help identify suitable pockets to focus on in the search for specific interactions. In this respect, by showing a greater amount of structural complexity, RNA motifs such as riboswitches, ribozymes, multi-junctions and pseudoknots are intrinsically more inclined to pocket formation, and thus they are better suited for RNA-targeted discovery of small molecule drugs. Given the above and relative to traditional protein-based drug discovery, computational medicinal chemists may need a more skilled background in statistical mechanics and simulative methods in order to take full advantage of these approaches.et al.,Once the chemical space of RNA-targeting drugs has been defined, machine learning and artificial intelligence, which are revolutionising several aspects of conventional drug discovery , such as the synthetic oxazolidinone linezolid that acts by binding a highly structured pocket, and the very recent Roche and PTC Therapeutics’ risdiplam, used in the treatment of spinal muscular atrophy (SMA), which acts by stabilising the interaction between an RNA splice site and a small nuclear ribonucleoprotein (snRNP)  as pharmaceutical targets, which are currently in the market/clinical trials for major unmet medical needs. Remarkable examples thereof are the recently approved risdiplam for SMA and the Novartis’s branaplam, under clinical trial for both SMA and Huntington disease, which act as splicing modulators by binding to pre-mRNA , and what new targets might be of interest for the future, and why these are particularly promising, given the very limited number of RNA targeting therapies compared to DNA and proteins. I was also curious as to whether the RNA specific docking tools perform “better” for RNA compared to generic methods, and how this might be quantified. The authors may consider expanding their review to cover these points. There are a couple of minor language and typographical errors, so careful proof-reading is also needed before resubmission. Please could they define their “ADMET” abbreviation in the text for non-medicinal chemists. Comments to Author: The manuscript by Bernetti et al. provides an overview of the challenges and opportunities in the field ofcomputational drug discovery of small molecules targeting RNA. This is an emergent field that is attracting a lot of attentionfrom both the academic and industry perspectives. I found the manuscript concise, yet extremely clear and complete.The major challenges facing computational researchers are discussed at an appropriate level of detail. I havejust a few comments that I invite the authors to address.1) The manuscript is focused on non-coding RNA. However, also other types of RNA might be interesting targets.For example, one of the 3 (to the best of my knowledge) FDA-approved small molecule targeting RNA, Risdiplam,binds to a pre-mRNA and affects mRNA splicing. I invite the authors to comment on this point.2) Some of tools that the community developed to target proteins with small molecules have not been extensively tested with RNA targets. For example, I am not sure whether the software mentioned at page 8 to detect binding pockets have been used extensively to detect pockets in RNA molecules. Could the authors elaborate on this point?3) Could the authors give more details bout how the two examples of RNA pockets in Fig. 4 were chosen?4) Methods to use experimental data to improve the accuracy of MD structural ensembles have been developed by several groups.I invite the authors to provide at least 2 or 3 additional references  The manuscript is focused on non-coding RNA. However, also other types of RNA might be interesting targets.For example, one of the 3 (to the best of my knowledge) FDA-approved small molecule targeting RNA, Risdiplam,binds to a pre-mRNA and affects mRNA splicing. I invite the authors to comment on this point.2) Some of tools that the community developed to target proteins with small molecules have not been extensively tested with RNA targets. For example, I am not sure whether the software mentioned at page 8 to detect binding pockets have been used extensively to detect pockets in RNA molecules. Could the authors elaborate on this point?3) Could the authors give more details bout how the two examples of RNA pockets in Fig. 4 were chosen?4) Methods to use experimental data to improve the accuracy of MD structural ensembles have been developed by several groups.I invite the authors to provide at least 2 or 3 additional references , and what new targets might be of interest for the future, and why these are particularly promising, given the very limited number of RNA targeting therapies compared to DNA and proteins. I was also curious as to whether the RNA specific docking tools perform “better” for RNA compared to generic methods, and how this might be quantified. The authors may consider expanding their review to cover these points. There are a couple of minor language and typographical errors, so careful proof-reading is also needed before resubmission. Please could they define their “ADMET” abbreviation in the text for non-medicinal chemists. No accompanying comment. No accompanying comment.


Breakthroughs in innovative techniques and instruments have driven the exploration of non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Whenever the data collected is at a transcriptional-wide scale, it becomes crucial to utilize computational power to identify ncRNAs. Moreover, the information extracted from this experimental data needs to be annotated to associate it with existing knowledge and aid in the further detection of new ncRNAs. After transforming these data into meaningful information, further data processing is essential to share this acquired knowledge with a broader community, thereby enabling more scientific discoveries. In this Special Issue, experimental techniques, and tools for ncRNAs, as well as computational approaches to process, annotate, classify, and distribute this information to shed more light on the role of ncRNAs, are shared.MicroRNAs (miRNAs) are small, single-stranded RNA molecules, approximately 22 nucleotides long. They play critical roles in RNA silencing and the regulation of gene expression following transcription. Given that a single miRNA can bind several hundred messenger RNAs (mRNAs), they impact numerous signaling pathways. Moreover, disruptions in miRNA regulation have been associated with the development of diverse diseases, underlining their significance in maintaining the normal physiological functions of an organism. As such, there are various bioinformatic tools available to analyze miRNA sequences, perform target prediction/validation, associate miRNAs to diseases, perform pathway analysis, and discover genetic variants within miRNAs. Since there are many useful tools available, it is a daunting task to search and investigate each tool. To address this point, Buitrago et al. nicely summarize the currently available online tools for miRNAs .https://github.com/Fadeel/miRinGO, accessed on 4 August 2023), to detect biological processes indirectly targeted by miRNAs transcriptionally through transcription factors, even if there is no physical interaction between miRNA and the regulated genes [As we accumulate biological experimental data, more information about miRNAs can be obtained. Having more data does not necessarily mean we know more about miRNAs, unless these data are cross-validated with other existing data. As the main objective of bioinformatic approaches is to combine different types of data to discover something new, it is no exception that new algorithms and methods are being continuously developed for miRNA research. From this perspective, Sayed and Park introduced a R Shiny application, miRinGO , which predicts pathogenic miRNAs by inferring biological characteristics of pathogenicity, including network influence and evolutionary conservation [Dysregulation of miRNAs has been linked to various diseases. Thus, there is an intense interest in studying the association between miRNAs and diseases. As there are over 2000 miRNAs in humans, it is often difficult to pinpoint which miRNA is important for a particular disease. To solve this problem, Wang and McGeachie introduced a novel computational method, DisiMiR , was introduced.Besides miRNAs, there is a growing interest in studying much longer ncRNAs, namely lncRNAs, whose lengths are longer than 200 nucleotides. Compared to protein-coding genes, the number of lncRNAs is several times higher as they are expressed in a certain cell type but not the others. Also, they are often dysregulated under stressed conditions and diseases, suggesting their potential involvement in the pathogeneses of various diseases. Roughly half of lncRNAs own poly A tails, which enables them to be discovered from RNA sequencing (RNA-seq) data as this technique captures gene expression at a particular time point in an unbiased manner. If the most abundant transcripts in a cell ) are depleted in prior to sequencing library construction, lncRNAs without poly A tails can also be detected. As the price for performing RNA-seq experiment has dropped significantly in recent years, RNA-seq assay has been used as a primary screening tool to analyze the transcriptome of various cell types and tissues, developmental stages, and diseases, including patients’ samples. Unfortunately, most of these RNA-seq data are analyzed only for protein-coding genes but not for lncRNAs. However, many of these RNA-seq data are available online in public domains ) as scientific community pushes for the sharing of generated data after publication of the original studies. Thus, such RNA-seq data are gold mines for discovering lncRNAs that are dysregulated in a particular disease with well characterized clinical data that have been peer-reviewed by editors and reviewers of prominent journals. To utilize such RNA-seq data from patients suffering from diseases, Ilieva et al. conducted a secondary analysis of the published RNA-seq data from patients suffering from nonalcoholic fatty liver disease (NAFLD) to discover dysregulated lncRNAs . To dissMAP3K4-AS1 might play a protective role against doxorubicin toxicity. To further disseminate the analyzed data, Distefano et al. built a web database DoxoDB .Significant progress in cancer treatment has led to the emergence of cardio-oncology, which aims to minimize cardiovascular risks while maximizing cancer therapy effectiveness. However, treatment-induced cardiotoxicity remains a prominent health risk. Among these cancer drugs, doxorubicin is notoriously known to cause cardiotoxicity, which may ultimately lead to death or cardiac transplantation. Thus, numerous approaches, including RNA-seq, have been employed in various cell types and tissues to uncover the side effects of doxorubicin treatment. To further understand the impact of doxorubicin, Distefano et al. conducted a systematic analysis of published RNA-seq data to discover doxorubicin-induced lncRNA genes . By perfAlthough discovering lncRNAs from the existing RNA-seq data is important, it is much more challenging to uncover the functional roles of lncRNAs. Slowly but surely, more lncRNAs have been functionally and mechanistically studied, including their functions achieved via binding to DNA, RNA, and proteins to regulate epigenetic, genomic, transcriptomic, epitrancriptomic, and proteomic events. Within the nucleus, the engagement of lncRNAs with DNA and the resulting creation of atypical nucleic acid configurations appear to be especially pertinent. Apart from interactions between single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), like R-loops, ssRNA can also associate with double-stranded DNA (dsDNA) to constitute DNA:DNA:RNA triplex structures. A current challenge in studying DNA:DNA:RNA triplexes is the identification of the precise RNA component interacting with specific regions of the dsDNA. To this end, computational methods are useful to predict such sequences, which Warwick et al. nicely summarized .Taken together, this Special Issue highlights the importance of computational methods and tools for studying miRNAs and lncRNAs. Besides introducing new algorithms and bioinformatic tools, including databases, this Special Issue includes review articles that provide tables of useful bioinformatic tools to further advance the field of ncRNAs, which researchers could use to screen a set of disease-relevant lncRNAs in silico and to design further biological experiments based on computational predictions.


Ziziphus jujuba fruit powder at rates of 0%, 3%, 5%, and 10%. The Physicochemical and antioxidant as well as the antibacterial activity and sensory qualities of Z. jujuba fruits were also investigated. The highest levels reached the values of 245.15 mg GAE/g DW (phenol) and 180.23 mg RE/g DW (flavonoids). Pulp extracts were also subjected to HPLC analysis in order to identify and quantify the sugar composition. This technique allowed us to identify Mahdia as the richest provenance especially in glucose (136.51%) and sucrose (113.28%) contents. The antioxidant activities investigated using DPPH assay decreased slightly from 175 μg/mL (Sfax) to 55 μg/mL (Mahdia). Furthermore, the antibacterial activity indicated that the S. aureus was the most inhibited especially by Sfax powder extracts (from 12 to 20 mm). Our results showed that the incorporation of Z. jujuba powder ameliorated the physicochemical and rheological characteristics  of the dough. Sensory analysis showed that consumer scores were increased with the increasing supplementation powder levels. Highest scores attributed to the cake supplied with 3% jujube powder collected from Mahdia provenance and confirmed that Ziziphus fruit could be advised a part of our diet. These results could validate a novel method to conserve Z. jujuba fruits in order to avoid their soilages for a long period.This study aimed to improve the effects on the physical, textural, and rheological characteristics of cake supplied with  Ziziphus jujuba fruits could be an active ingredient in the food industry by transforming it to cake and increase the consummation of Z. jujuba fruit that could solve the problem of conservation for long periods. So the disease‐free and identical in shape jujubes were selected for the sun drying process in order to evaporate water from fruits by sun and conserve their biochemical and biological activities . It was combined with 10 mL of hydrochloric acid and then filtered into 50 mL graduated flasks. The solution was then stored in vials until it was analyzed. The mineral element contents were determined using a flame spectrophotometer .The mineral composition  of 2.2Z. jujuba powder fat contents were achieved by extracting samples in a Soxhlet apparatus using the hexane as solvent. The extractions were carried out in duplicate.Physicochemical analysis focused specifically on the water, fat, and protein contents. The water content was determined after desiccation of the fresh material at a temperature of 103 ± 2°C in an isothermal oven ventilated at atmospheric pressure to a nearly consistent measurement. The protein contents were evaluated according to Kjeldahl methods . Water l2.32.3.1Ziziphus fresh pulp (1 g) was macerated in 10 mL of Ultra High Quality (UHQ) water for 30 h. After centrifugation and filtration, the supernatant extracts were kept in the freezer before HPLC analysis.2.3.2−1with constant Helium purge. The identification and the quantification of the compounds were performed in comparison with the commercial standards. A column wash between two successive injections was made with methanol.Sugars were analyzed in triplicate using HPLC Dionex (P680‐ASI 8100‐Agilent) equipped with a Carbo Pac PA1 column (250 × 4 mm). The postcolumn injection was used with a volume of 25 μL. The HPLC worked under suitable programs: First it was stabilized with 1 mM of KOH during 10 min, and then 100 mM KOH solution was used as a mobile phase during the analysis. Evaluation was done at a flow rate of 1 mL min2.43 (2%) in darkness. The flavonoid was used to prepare the curve calibration and the absorbance was measured at 430 nm. Results were expressed as mg quercetin equivalent per gram dry weight (mg QE/g DW). All analyses were run in triplicate. Tannin condensed contents (TC), expressed as mg Catechin Equivalent per gram dray weight (mg CE/g DW), werealso quantified  were carried out by the Folin Ciocalteau's reagent  was determined according to the following formula0 is the absorbance of the blank sample and a1 is the absorbance in the presence of the sample.The antioxidant activity was investigated according to Ben Hmed et al.  method w2.5.2Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 070101121 and 2 g‐negative bacteria: Kleibsella Pneumoniae ESA 8 and Aeromonas hydrophila.The antibacterial activities were evaluated using 2 g‐positive bacteria: These species were provided by the Veterinary Epidemiology and Microbiology, Bacteriology and Development Groups Biotechnology Laboratory (Tunisian Institute of Pasteur).Z. jujuba were dissolved in sterile water. Before use and for the detection of the antimicrobial activity, each extract was diluted to 50 μg/mL and sterilized by filtration through a 0.2 μm pore size filter.The various methanol extracts from 7 CFU/mL for bacteria and 105 cell/mL for fungus were inoculated onto the surface of agar plates. Afterward, wells 6 mm diameter, were punched in the inoculated agar medium and 30 μL of the extract was added to each well. Negative controls consisted of using 30 μL water. The plate was allowed to stand for 40 min at 4°C to permit the extract diffusion followed by incubation at 37°C for 24 h for bacteria. The antibacterial activities were evaluated by measuring the zones of inhibition (clear zone around the well) against the test microorganisms. All tests were repeated three times  of each extract was determined by using the microdilution broth method. MIC values were estimated visually by the absence of turbidity as previously reported by Khemiri et al. .2.62.6.1The cake was prepared using the Expert Design V 6 in order to formulate a product that meets customer and industrial approval.1). A second mixture (M2) was prepared by adding jujube powder or wheat flour with vanilla. Finally, the two mixtures were combined. The baking operation was then carried out in an oven  at 180°C for duration of 30 min.Cakes were prepared according to Najjaa et al.  methods.After cooling, each sample was packed in polyethylene bags for evaluation of different characteristics and stored at ambient temperature.2.6.2The dough was characterized rheologically by a Chopin Alveograph . This method was used to describe dough resistance against the biaxial deformation. Different stages of the Chopin Alveograph test were resumed in Figure 2.6.3Z. jujuba fruits. The studied parameters were taste, color, texture, volume, and global appreciation.Sensory analysis defined as texture, flavor (taste), aroma, and visual aspect was achieved according to international standardized (NF ISO 5492) hedonic method . Each data presented was mean ± standard deviation of three replicates. Multiple mean comparisons were performed using the Student–Newman Keuls test with a significance level of 33.1Jujube fruit was known for many uses as a vital food due to its richness on many components such as ash, protein, fat, and carbohydrate Table . This coThe analysis of the protein levels varied between 0.40% and 0.64%. The Mahres provenance was the richest one, and the Choutrana was the poorest one Table .Z. jujuba fruits.The humidity levels varied between provenances. It ranged from 9% (Mahres) to 10.1% (Mahdia). This variability could be due to some conditions such as climatic conditions  and geographic location…. This low percentage facilitated the storage of Z. lotus .Ash analysis varied between 1.71% DW and 2.05% DW for Mahdia and Mahres respectively. Such levels were more important than those (0.82% DW) for 3.2Z. jujuba fruit.Total sugar levels as mentioned in Figure 3.2.1Chromatogram analysis showed three essential sugars classified in order of decreasing value: sucrose 24.3 min), glucose (16.6 min), and galactose (44.8 min)  and sucrose 113.28%) contents  decreased slightly from 175 μg/mL (Sfax) to 55 μg/mL (Mahdia). Such results confirmed that the Mahdia provenance was the richest one and exceeded those obtained for the BHT  used as a standard (77.86 μg/mL).3.4.2Due to the multi‐resistance microorganisms, there were great demands to research new molecules from alternative sources like plants.S. aureus was the most inhibited microorganism especially when they were treated by Sfax extracts. The diameters of zone inhibition expressed in mm varied from 12 to 20 mm  of the cake formulated with jujube powder.This study proved that edible and delicious 3.6Past tenacityRheologic behavior briefly could predict dough processing parameters and the quality of the final product. Some rheological parameters, investigated in this study, were past tenacity, extensibility, swelling capacity, baking strength index, elasticity index, and falling number.Z. jujuba powder, especially those of the Mahdia provenance powder. In fact, this parameter decreased from 87 to 37 mm H2O for the control and the Z. jujuba powder of Mahdia provenance, respectively.ExtensibilityRheological properties of dough and gluten contents were greatly affected during the mixing process by the flour compositions (low or high protein contents), the processing parameters  and the ingredients . As shown in Figure Z. jujuba powder increased the extensibility of the past  to 58.7; 40.8; 50.2; 45.7 and 39.7 of Sfax, Choutrana, Mahres, Mahdia, and Regueb Ziziphus powder provenances, respectively added at levels of 5%  were also studied….Z. jujuba powders reduced the wet, index, and dry gluten contents of the pastry flour. This reduction was observed for Mahres provenance . This characteristic could inform us about the quality of the flour and its rheological parameter. As showed in this study, the incorporation of Z. jujuba incorporation, dry gluten percentages ranged between 6% and 9.4%. This inveterate the conservation of heat‐resistant protein, so our flours keep their nutritional qualities.In fact, higher amounts of gluten proteins increased the dough elasticity and prevent hamper molding of the dough Hanee, . In addiZ. jujuba fruits and avoid their soilage for a long period.According to this result we can validate a novel method to conserve 3.7Z. jujuba powder cake was evaluated through taste, color, texture, volume, and global appreciation  for Mahdia provenance added at rates of 1% and 3%, respectively. Such variability could be attributed to high levels of sugar fruits.3.7.2The color of the cake expressed the sensation the cake produced on the eyes. The difference between the mean of the provenance was quite significant. In fact, the comparison between provenances showed that the color of Mahdia provenance had the highest mean (11). The Sfax provenance had a low mean relative to the other provenances (2.4).3.7.3Z. jujuba powder. This increase could be explained by the high content of polysaccharides, which may lead to the dilution of gluten and extensive gluten structure , the texture levels ranged between 6.9 and 9 for Sfax and Mahdia provenances, respectively, incorporated rates of 1% and 3%. The result indicates that the texture of the Mahdia provenance was preferred to other samples. In addition, as shown in Figure 3.7.4The difference in volume of all samples was easily detected among consumers. However, the volume of the control cake was low with the mean scores of the cakes evaluated equal to 4. This could be explained by the absence of cells in this cake.The comparison between provenances showed that the cake enriched with Mahdia jujube powder (1% et 3%) had scored the highest volume mean (8.4) by panelists, while cake sample supplemented with Sfax jujube powder at 1% and 3% were less accepted than all other provenances, but more important than those made only with wheat flour (3.5). Such variability in volume varied not only according to the provenances but also the rates of their additions to the cakes.3.7.5This parameter expresses how the panelists accept the jujube cake. As shown in Figure 4Ziziphus jujuba fruit characterizations showed that protein contents varied between provenances and attempt the value of 0.60%. Such results were higher than those cited by El Aloui , geographic location… This low percentage facilitated the storage of Z. jujuba fruits. Our results were in comparison to other species such as Z. mauritiana and Z. spina‐christi with levels of 46% and 85%, respectively  than those reported in i et al. . Resultsi et al.  who confSecondary metabolites analysis enhanced the richness of our extracts on phenolic compounds. Levels were higher than those found (10.43 to 15.85 mg/ L) by Elaloui et al. . Other wZ. jujuba powder increased the ash contents to 70% especially for the Mahdia provenance. These results were not in accordance with the findings of John et al.  or also for the diluting gluten . The extensibility was also reduced after this incorporation. This decline could be attributed to the diminution in the gluten level in particular the gliadins level that gave rise to larger cake volume during baking. Such variation reflected an increase in the gas retention property within the dough bubble and thus the retention of carbon dioxide within the cake dough. From these results, the dough which will be obtained from the basis of the mixture of two flours (wheat and Z. jujuba) will exhibit a decreasing density with the rate of incorporation and a cake of increasing volume.In addition, the In addition, the increase in falling number demonstrated a decrease in α‐amylase activity that created sticky dough. This idea was also cited by Tozatti et al. . In oppoZ. jujuba powder ameliorates the physicochemical and rheological characteristics of the dough by reducing its humidity; gluten yields. So Z. jujuba powder could be recommended by nutritionists and industrial to formulate cake supplement with Z. jujuba powder. According to overall acceptability of results, the cake enriched with Mahdia jujube powders used at dose of 3% and 1% were the most acceptable, by panelists. Other studies carried out on biscuits supplemented with jujube flour had as acceptable quality as the control  of the dough. In addition, cakes prepared with 3% (w/w) fruit powders of Mahdia provenance had comparatively the best nutritional and sensory characteristics over other provenances. According to these results we can validate a novel method to conserve Z. jujuba fruits especially these of Mahdia provenance and avoid their soilages for a long period. Ziziphus fruit was advised to be to be part of our diet.This study proved the benefits of No potential conflict of interest was reported by the author.


The 7SK ribonucleoprotein (RNP) is a dynamic and multifunctional regulator of RNA Polymerase II (RNAPII) transcription in metazoa. Comprised of the non-coding 7SK RNA, core proteins, and numerous accessory proteins, the most well-known 7SK RNP function is the sequestration and inactivation of the positive transcription elongation factor b (P-TEFb). More recently, 7SK RNP has been shown to regulate RNAPII transcription through P-TEFb-independent pathways. Due to its fundamental role in cellular function, dysregulation has been linked with human diseases including cancers, heart disease, developmental disorders, and viral infection. Significant advances in 7SK RNP structural biology have improved our understanding of 7SK RNP assembly and function. Here, we review progress in understanding the structural basis of 7SK RNA folding, biogenesis, and RNP assembly. Although differences are observed among studies, the 7SK RNA secondary structure generally consists of four stem-loop domains  with intervening single-stranded RNA (ssRNA) linker sequences. The first secondary structure model was reported in 1984, where 7SK RNA was extracted from rat cells and subjected to nuclease digestion . Four re.Sequence homology studies showed that SL1 and SL4 are highly conserved across both vertebrate and invertebrate 7SK RNA sequences  Gruber . The cenin vitro and in cellulo  has a near-identical secondary structure for experimentally and evolutionarily determined secondary structures . Several63 forms a base triple to either G42-C67  or U44-A65 . A detailed comparison of X-ray and solution NMR structural differences is discussed elsewhere , C75 (ASM1), and A77 (ASM1) which may explain the observed structural differences.All SL1 distal structures have globally similar conformations, although differences are observed in helical compaction, loop backbone conformation, and interhelical bending . Moleculormation . Small-a320 in the C320U321 loop was found to form a base triple with the G303-C323 base-pair   . In addi3 (Bin3) . MePCE c3 (Bin3)  (Cosgrov3 (Bin3) . The MTa3 (Bin3) . An X-ra3 (Bin3) , identif3 (Bin3) .via a “triphosphate binding tunnel” comprised of residues in α0, α7, α5′, and α6′  in complex with an RNA UUU trimer showed a similar RNA recognition mode to genuine La  Figure ; Table 2 7SK RNA  , providing a binding surface for MePCE−Larp7 interaction. In addition to protein-RNA interactions, there is an extensive MePCE−Larp7 interaction interface at MePCE α3-β3 loop, Larp7 RRM1 α3, and α4/MID . For X-rThe prominence of P-TEFb in human health and disease has made it a promising therapeutic target for wide-ranging diseases such as cancer , cardiac158 by PKC abolishes interactions with 7SK RNA, likely through electrostatic repulsion (HEXIM1 is a promiscuous double-stranded RNA (dsRNA) binding protein  that regepulsion . As a diepulsion , 4, 8  intercalate into the major groove and interact with ASM1, ASM2, ASM3, and ASM4; In contrast, only two arginine residues in HEXIM1 peptide (R155 and R156) interacted with ASM1 and ASM2  or Tat ARM variants (residues 44–60) ; Table 2and ASM2 . In bindand ASM2 , consistand ASM2 .via leucine zipper  . Structue zipper . In addie zipper . An X-rae zipper .186 is phosphorylated (186 (186 must be re-phosphorylated upon P-TEFb release.While domain structures of HEXIM1 ARM or coiled-coil motifs have been determined, structural information on HEXIM1 binding to P-TEFb and recruitment to 7SK RNP are lacking. HEXIM1 domains are involved in binding both CycT1 and CDK9. The stammer motif in the C-terminal TBD was shown to be important for CycT1 binding, and deletion resulted in a 5-fold reduction in HEXIM1−CycT1 association compared to wild-type . A modelorylated . Severalted (186  as part In addition to HEXIM1 requirement for P-TEFb assembly, 7SK SL4 and Larp7 are also required for stable association of P-TEFb onto 7SK RNP. In the HIV-1 TAR element, CycT1 and Tat cooperate to bind to the TAR apical loop . A similAdvances in 7SK RNP structural biology over the past decade have significantly improved our understanding of 7SK RNP assembly and maturation. A consensus model has started to develop that describes early 7SK RNA biogenesis. Likewise, a nucleotide-resolution model is beginning to emerge that describes 7SK RNA secondary structure switching in response to different functional states. High-resolution structures of individual domains, RNA-protein complexes, and ternary complexes have increased understanding of HEXIM1 recognition of 7SK RNA, as well as assembly into a stable 7SK core RNP. Rich biochemical data have provided insights into P-TEFb sequestration and kinase inactivation and have revealed alternate pathways of 7SK RNP function in RNAPII transcription regulation. However, despite these advances, several outstanding questions remain unresolved, particularly regarding the structural basis for P-TEFb assembly, kinase inactivation, and release.What is the 7SK RNA secondary structural landscape, and how is 7SK RNA secondary structure remodeling achieved? Several secondary structure models have been proposed using biochemical or bioinformatics approaches. While some 7SK RNA regions have common secondary structures across models, residues spanning SL1-SL2 vary substantially. Several helicases have been loosely identified to bind to the central region of 7SK RNA . It has 186, changes as part of the release mechanism from 7SK RNP. The basis for P-TEFb kinase inactivation, and re-activation upon 7SK RNP release, remains unclear. Additional studies are needed to gain an atomistic understanding of P-TEFb assembly with 7SK RNP, P-TEFb inactivation when bound to 7SK RNP, and re-activation upon release from 7SK RNP.How is P-TEFb assembled onto 7SK RNP, and what is the mechanism of kinase inactivation and re-activation? A picture is starting to emerge regarding HEXIM1 recognition of 7SK RNA, and HEXIM1 recognition of P-TEFb. However, a lack of structural information has impeded detailed understanding of 7SK RNP sequestration and inactivation of P-TEFb. In addition to HEXIM1, evidence suggests that P-TEFb interacts with Larp7 . MoreoveHow do accessory proteins bind 7SK RNP to direct P-TEFb release and alternate functional pathways? Numerous proteins have been identified to be involved in P-TEFb release from 7SK RNP, including RNA processing factors, transcription factors, and helicases . Rather Although substantial progress has been made in 7SK RNP structural biology, the sparsity of determined structures has remained an obstacle toward gaining a detailed understanding of how 7SK RNP assembles and functions in transcription regulation. The inherent structural plasticity of 7SK RNA and heterogeneity of 7SK RNP particles has posed a significant challenge toward structural elucidation. Overcoming these challenges will be critical to advance understanding of 7SK RNP structure, particularly for macromolecular complexes. Ultimately, structural biology coupled with biochemical, -omics, and cellular approaches will enable a comprehensive understanding of 7SK RNP cellular function and role in human health and disease.


The results showed that two independent Golgi-targeting domains (GTDs) with different Golgi localization characteristics in dGM130, together determined the precise localization of dGM130 in both the soma and dendrites. GTD1, covering the first coiled-coil region, preferentially targeted to somal Golgi rather than GOs; whereas GTD2, containing the second coiled-coil region and C-terminus, dynamically targeted to Golgi in both soma and dendrites. These findings suggest that there are two distinct mechanisms by which dGM130 targets to the Golgi apparatus and GOs, underlying the structural differences between them, and further provides new insights into the formation of neuronal polarity.GM130 is a matrix protein that is conserved in metazoans and involved in the architecture of the Golgi apparatus. In neurons, Golgi apparatus and dendritic Golgi outposts (GOs) have different compartmental organizations, and GM130 localization is present in both, indicating that GM130 has a unique Golgi-targeting mechanism. Here, we investigated the Golgi-targeting mechanism of the GM130 homologue, dGM130, using  In vertebrate Golgi, these stacks can be further linked by ribbons  is the hub of the secretory pathway in eukaryotic cells. The Golgi usually forms a stacked cisternal structure, which is polarized into  ribbons . Via thiications .Golgi matrix protein 130 (GM130) was the first matrix protein found to maintain the organization of Golgi, and is broadly conserved among metazoans . Once syStructurally, GM130 is predicted to have a coiled-coil-rich central portion, with short non-coil N- and C-terminal regions . The N-tDrosophila neurons, different compartments of GOs are even disconnected in dendrites  of dGM130, providing opportunities for further exploring the Golgi-targeting mechanisms in soma and dendrites.Here, we investigated the Golgi-targeting mechanism of dGM130 in the dendritic arborization neurons (da) in 2.2.1.The amino acid sequence of dGM130 was obtained from NCBI databases . The coiled-coil and conserved Golgin domains were identified from InterPro.2.2.dGM130 cDNA was amplified from UAS-EBFP-dGM130 (dGM130. Then it was transferred into the vector pJFRC-10 × UAS-IVS-CD8-GFP (Addgene plasmid #26214), where the CD8-GFP fragment was replaced between the NotI and XbaI sites.To generate UAS-EGFP-dGM130 construct, P-dGM130  and EGFPdGM130 cDNA. These truncated cDNAs were then transferred into UAS-EGFP-dGM130 to replace dGM130 cDNA between the BamHI and XbaI sites.To generate constructs of dGM130 truncations, appropriate primers for each deletion site were synthesized  and usedAll constructs were verified by sequencing.2.3.To make the transgenic lines of UAS-EGFP-dGM130 and truncations, embryos of PBac{y[+]-attP-3B}VK00033 and P{CaryP}attP40 were collected and microinjected with constructs at 18°C.19–12 (109(2)80 80 , UAS-ManGM130∆23 .All stocks and crosses were raised in a 25°C incubator with 40–60% humidity.2.4.Confocal imaging was performed using an Olympus FV1000 microscope equipped with a 60 × oil objective lens (NA = 1.42).3 voxel for dendritic shaft, 0.04 × 0.04 × 0.5 μm3 voxel for branch point and 0.02 × 0.02 × 0.5 μm3 voxel for soma. In time-lapse imaging of ∆N530 in C3da neurons, images were collected at 10 min intervals for 5 frames. The Z-stack images were processed for maximum projection using Fiji software  for further analysis  software, followed by Tukey or Sidak corrections. Comparison between two groups were used unpaired Student’s t-test. Data presented are in the form of mean ± SEM.3.3.1.To determine the Golgi-targeting region in dGM130, we first analyzed the structure of this coiled-coil protein. dGM130 consists of 795 amino acids. Nine coiled-coil domains and two conserved domains of Golgi proteins were identified from the Pfam database . One coiBesides the coiled-coil structures, we further aligned the amino acid sequences of dGM130 and rat GM130 in the N- and C-termini as well as the proline-rich hinge region between the coiled-coil regions. We found that they are highly conserved in the N-terminal region, and have PDZ ligand motifs at the C-terminus . The corIn summary, four structural regions of dGM130 were inferred from the structural analysis: the highly conserved N-terminus, a non-coil C-terminus with PDZ ligand motif, and two coiled-coil regions in the central portion, linked by a proline-/histidine-rich region .3.2.in vivo, fluorescent protein EGFP was tagged at the N-terminal of dGM130 and its truncations, and the Golgi was labeled by Mannosidase II tagged with TagRFPt (ManII–TagRFPt), which is a medial-Golgi marker, in the following studies  located in ∆C550 and the C-terminal region might make distinct contributions to Golgi-targeting in the soma and dendrites.3.3.In vivo imaging showed that only ∆N100-∆C550 had a similar distribution to that of ∆C550: it formed large units and colocalized with ManII in the soma, but diffused in dendrites  in the C-terminal region. We re-designed the ∆N550 construct to extend it to ∆N530 considering that the extended acidic glutamic-acid-rich region might help protein fold correctly, and generated two more truncations, ∆N608 and ∆N636, based on the coiled-coil region and conserved domain . The expdGM130 null neurons were inspected, including ∆N530, ∆C550, ∆C765, ∆C100, and ∆N735. All truncations remained the same distribution states as wild-type neurons , the National Natural Science Foundation of China (61890951 and 31871027 to WZ), and Fundamental Research Funds for the Central Universities , CAMS Innovation Fund for Medical Sciences  and the director fund of the WNLO.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recent advancements in small RNA sequencing have unveiled a previously hidden world of regulatory small noncoding RNAs (sncRNAs) that extend beyond the well-studied small interfering RNAs, microRNAs, and piwi-interacting RNAs. This exploration, starting with tRNA-derived small RNAs, has led to the discovery of a diverse universe of sncRNAs derived from various longer structured RNAs such as rRNAs, small nucleolar RNAs, small nuclear RNAs, Y RNAs, and vault RNAs, with exciting uncharted functional possibilities. In this perspective, we discuss the emerging functional principles of sncRNAs beyond the well-known RNAi-like mechanisms, focusing on those that operate independent of linear sequence complementarity but rather function in an aptamer-like fashion. Aptamers use 3D structure for specific interactions with ligands and are modulated by RNA modifications and subcellular environments. Given that aptamer-like sncRNA functions are widespread and present in species lacking RNAi, they may represent an ancient functional principle that predates RNAi. We propose a rethinking of the origin of RNAi and its relationship with these aptamer-like functions in sncRNAs and how these complementary mechanisms shape biological processes. Lastly, the aptamer-like function of sncRNAs highlights the need for caution in using small RNA mimics in research and therapeutics, as their specificity is not restricted solely to linear sequence. The mosget RNAs . This RNget RNAs , also knget RNAs  or tDRs get RNAs ) have beget RNAs , 10, 11,e.g., tRNAs   have distinct roles in inhibiting retrotransposition of endogenous retroviruses (ERVs) in cell culture  can have different modes of action depending on their length. For example, synthetic 3′tsRNA-Lys of differing lengths  translational initiation factors, while a shorter version (21 nt) fails to exert this function , likely Apart from tsRNAs, new types of noncanonical sncRNAs have also been continually discovered. Starting from late 2000s to the early 2010s, various types of small RNAs were identified as originating from the fragmentation of longer structured RNAs, including rRNAs , 16, 94,1A, m1G, m3C, and m22G, can block the RT process during the generation of cDNA libraries for sequencing  that are distinct from those of siRNAs/miRNA   . These se.g., sequential use of T4PNK and AlkB) to remove RNA modifications and convert termini and/or using special RT enzymes  that can switch template during RT and read through the modifications. These advances have increased the detection of tsRNAs and substantially updated the sncRNA landscape of other noncanonical sncRNAs, especially rRNA-derived small RNAs (rsRNAs), which are found to be dominant in many cell/tissue types (A). With the emergence of new methods, it is worthwhile to re-examine previous sncRNA profiles that were generated using traditional small RNA sequencing. For instance, PANDORA-seq has enabled novel discoveries about the sncRNA landscape in mature sperm . Additionally, variations in tRNA gene expression at the isodecoder level (tRNAs having the same anticodon sequence) lead to changes in tsRNA, but not in mature tRNA levels  to act on the target RNA/DNA. However, their other properties such as the potential to fold into various structures and the ability to bind with a wider range of proteins may represent a more fundamental characteristic that is essential for RNAi-independent regulatory functions in tissues and cells.Currently, sncRNAs are mostly known for their RNAi-based functionality, which relies on base pairing with their complementary sequences and recruiting binding proteins  is fragmented into smaller pieces, both the parent and daughter RNAs may be subject to functional selection during evolution. Through this process, numerous possibilities have been tested, with the extant sncRNAs representing the successful outcomes of evolutionary trial and error.RNA is a highly structured polymer molecule by nature, capable of forming dynamic secondary, tertiary, and higher-order structures. These complex structures can result from self-folding based on minimal free energy  or interIn fact, we argue that the emergence of RNAi only represents a later invention in evolution, which ensures the usage of sncRNAs’ linear sequence for base pairing. In this context, RNAi could be considered a specialized form of sncRNA function, and it may have an origin that predates the need for antiviral activity. We discuss these points below.aptus, meaning fit, and the Greek meros, meaning part or region  , a membeThe interaction of let-7 with TLR7 in microglia and macrophages depends on its GU-rich sequence, rather than the 5′ let-7 seed sequence required for posttranscriptional silencing. The binding of let-7 to TLR7 is akin to the GU-rich sequence found in the 20-nt ssRNA40 derived from HIV, a well-known natural RNA activator for TLR7/8 . This eve.g., miR-150) in HMDMs, and it can bind to and strongly activate endosomal TLR7 (but not TLR8), thus acting as an immune activator. 5′tsRNA-HisGUG also contains a GU-rich sequence like ssRNA40, suggesting that the GU-rich sequence is a key feature for the binding. However, full-length tRNA-HisGUG cannot stimulate endosomal TLR7 , which is not present when being folded into a tRNA structure. Collectively, although we currently lack specific structural evidence, these examples suggest that let-7 and 5′tsRNA-HisGUG may function in an aptamer-like fashion. Indeed, activation of TLR7/8 by other miRNAs and sncRNAs have also been reported  discovered that 5′tsRNA-HisGUG can be significantly elevated in HMDMs and their secreted extracellular vesicles upon lipopolysaccharides stimulation, through ANG cleavage . The abumal TLR7 , indicatreported , 133, suIn addition to binding to TLR7, let-7 has also been found to interact with the TRPA1 ion channel to activate nociceptor neurons and elicit pain , suggeste.g., tsRNAs and rsRNAs) in the cell, they may easily surpass the AGO loading capacity and possibly even the mRNA pool for direct binding. The logical conclusion is that these sncRNAs may either exert base pairing with their target mRNA (and other RNAs) using linear sequence or fold into aptamer structures that exert complementarity-independent functions (A and C). Both of these possibilities have been demonstrated. Functions based on AGO-independent base pairing include: (I) tsRNA-mediated unfolding of duplexed mRNA structures, thus regulating translation rates on rates , 147; anon rates . In thesImportantly, the aptamer-like function of sncRNAs could extend beyond the folded structure of a single sncRNA and may also involve intermolecular structures formed through the interactions between different sncRNAs. For example, in the case of 5′tsRNA-Ala–mediated translational inhibition , it has C). Finally, just like the function of an RNA aptamer can be improved or modified by incorporating modified nucleotides during their synthesis .In contrast to the aptamer function, which represents a “default” feature of RNA folding, the emergence of RNAi marks a later evolutionary milestone, which ensures the effective use of sncRNAs’ linear sequences for base pairing–mediated targeting by binding with AGO. This development resulted in a more programmable and simple regulatory system that fully exploits the linear sequences of sncRNAs to bind a diverse range of RNA/DNA targets. In this context, the emergence of RNAi could be considered as a specialized form of an aptamer-like function, wherein AGO binding ensures that sncRNAs do not fold into 3D structures, but maintain a "straightened up" configuration optimal for linear sequence binding B.The evolutionary origin of RNAi was widely considered to be driven by the defense against transposable elements and RNA viruses , thus a e.g., RNase H-like) domain capable of DNA/RNA cleavage, forming a prototype of RNAi that may have emerged without the pressure of invading sequences, which initially function in intracellular gene regulation at either the DNA or RNA level. When confronted with invading sequences (DNAs or RNAs), this prototype loaded with linear DNA/RNA may have afforded an advantage for recognition of the invader with sequence complementarily. Further incorporation of specialized components, such as Dicer , to clekaryotes , 155 andkaryotes  pAGO preThis proposed origin of RNAi aligns with the dual role of RNAi in both antiviral defense and endogenous gene regulation. We posit that RNAi may have emerged among one of the numerous sncRNA–protein interactions through evolution, parallel to other functional RNPs based on sncRNA–protein interaction. The RNAi modes of action may have been under selection and enhanced in species living in environments with high viral loads, such as soil-dwelling organisms including worms and plants, in which robust and diversified antiviral defense capabilities were demonstrated .Tetrahymena, it has been shown that tsRNA can bind to an AGO/PIWI family protein to form nuclear RNPs, which facilitates nuclear rRNA processing and cell growth . For instance, in the CRISPR/Cas system, Cas protein binding with guide RNAs ensures the use of spacer sequences in linear form for base-pair targeting, while the flanking sequences fold in an aptamer-like manner to anchor with the Cas protein  and the dynamic sncRNAs derived from their site-specific cleavage, advanced tools to simultaneously decode sncRNA sequences and site-specific RNA modifications are in great need  have similar concerns, potentially generating nonspecific effects (The modern pharmaceutical industry largely relies on a reductionist approach, simplifying complex systems to harness key principles for treating diseases. This approach is evident in the successful application of the well-defined RNAi mechanism in creating new therapeutics based on targeted gene silencing through sequence-complementarity, as pioneered by Alnylam Therapeutics . However effects  through “...the only simplicity to be trusted is the simplicity to be found on the far side of complexity.”Now, as we delve deeper into the vast sncRNA universe beyond miRNAs  and 2, iThe authors declare that they have no conflicts of interest with the contents of this article.