The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg Mobile group II introns function as ribozymes to splice and reinsert themselves into DNA, thereby colonizing new genomic regions. Here the authors use single-molecule FRET and molecular dynamics simulations to reveal a structural link between metal ion induced kinetic heterogeneity and the sugar puckers at the exon-intron binding interface. To precisely locate the cleavage site, group I and II introns engage specific RNA recognition elements that base pair with complementary stretches on the exons  or stops after the first step, with the 5′-exon leaving the active site without being ligated. Conversely, if the association of intron and exon is too strong, the ribozyme no longer discriminates between correct and mismatched targets, which may lead to gene disruption and disease if reverse splicing occurs in tumor suppressor genes like p5313. To minimize such errors, most group II introns use two independent exon recognition sites to keep hold of the 5′-exon: exon binding site 1 (EBS1) contributes the most to thermodynamic stability, while the surface-exposed EBS2 is responsible for target selectivity14. Mono- and divalent ions play their part in strengthening the intron–exon interaction both through non-specific charge screening as well as site-directed coordination. A number of such specific binding sites have been identified near active site elements10. Like a padlock, the metal ion packs the strands together, thereby retaining the exon in the active site for a longer time than if no gatekeeping ion was present. As ion coordination is transient though, designated binding pockets are often only partially occupied and exon dissociation is thus kinetically heterogeneous15. Particularly, Mg2+ is known to induce such kinetic partitioning by interacting with RNA directly (inner-sphere coordination) or via a water molecule (outer-sphere coordination)18.In the course of RNA maturation, self-splicing ribozymes catalyze two consecutive reactions, that is, the excision of the intron and the ligation of its flanking exonsons Fig. 2–4. In gyme Fig. 6–8, wher21. This involves ion-induced collapse of RNA secondary structure elements as well as formation of more distant tertiary contacts17. Spectroscopic rulers such as Förster resonance energy transfer (FRET) capture these rearrangements based on a set of predefined distance coordinates. Yet, in some cases, be it due to the choice of the dye positions or the local geometry of the coordination environment, metal ion binding yields no detectable change in resonance energy transfer and results in a degenerate FRET state that comprises multiple kinetic states. Their existence is only inferred from the multiexponential decay rates from the one FRET state.Single-molecule techniques are well suited to detect subpopulations of molecules in different conformational states22. RNA folds hierarchically into a set of interconnected topological modules. The kinetics of their tertiary assembly is often rate determining in the folding process17. Pseudoknots are a recurrent motif in riboswitches, ribozymes, and the ribosome where they interface secondary and tertiary structure. They are often involved in the formation of the catalytic core or the aptamer domain26. In its simplest form, an H-type pseudoknot consists of a hairpin that base pairs intramolecularly with a stretch of nucleotides outside the stem27. In group II introns such a hairpin reaches out to the flanking exon and forms the recognition site for splicing and retrohoming30. Here we use single-molecule FRET in combination with hidden Markov modeling to probe the pairing of EBS1 in the prototypical group II intron Sc.ai5γ with its cognate intron binding site 1 (IBS1). To facilitate the interpretation of the binding free energy landscape, we have isolated the tertiary contact and tagged both interaction partner covalently with a fluorescent dye. In this way, we establish FRET as a reaction coordinate, which directly reports on the binding of exon and intron. Formation of this contact is a prerequisite for subsequent catalysis. While structures of the exon–intron complex before the first step of forward and reverse splicing have been solved recently32, dynamic information on the association and dissociation reaction is still largely missing to date. By comparing how RNA and DNA exons bind to the isolated EBS1 hairpin, we can dissect the kinetics of the recognition step, which precedes forward and reverse splicing.As metal ions direct folding and catalysis, heterogeneity is inherent to many RNAs33. Hence, sugar puckering is an effective way to counteract ion-induced heterogeneity in exon recognition and is a potentially widespread mechanism to modulate macromolecular interactions.We find that RNA–DNA hybrids not only dissociate much faster than their pure RNA counterparts, but they do so in a more kinetically homogeneous manner. Our molecular dynamics simulations show that the origin lies in the conformational fit between the binding partners. Fast switching of the sugar puckers in the DNA exon compromises tertiary contact stability and likely impairs metal ion binding. This smooths out the rugged energy landscape and homogenizes the kinetics2+ concentrations is summarized in Supplementary Fig. 2+, as indicated by the strong depletion of static zero FRET molecules in favor of dynamic molecules when Mg2+ is added  chemical shift mappings with Mg2+ and [Co(NH3)6]3+, as well as Mn2+-induced line broadening35. The affected nucleotides coincide with a patch of negative surface potential near the 5′-splice site. Upon docking of IBS1* or dIBS1*, a cavity is formed, which readily accommodates a Mg2+ ion35.We monitor the binding and unbinding of two seven nucleotides long exon strands, IBS1* (RNA) and dIBS1* (DNA), to surface-immobilized EBS1* hairpins by following the anti-correlated Cy3 and Cy5 emission over several minutes  imaging because of the high background signal  suggests a tighter inner- and/or outer-sphere coordination of Mg2+ at the RNA–RNA interface in line with previous binding affinities calculated from NMR chemical shifts40.Divalent ions are therefore expected to promote the interaction of exon and intron by a combination of non-specific charge compensation and site-specific bindingles Fig. . The relion Fig. . The nor2+ is present. By computing a mean dwell time 〈tzero〉 and 〈thigh〉 for each trace, we can assess how uniformly the immobilized EBS1* hairpins react to IBS1* and dIBS1* binding  also feature long mean dwell times.Among the four molecular classes defined above, dynamic molecules are the most informative ones as they all represent functional RNA tertiary contacts that associate and dissociate within the observation window. To explore the kinetics of the interaction between the exon and intron binding sites, we discretized the FRET trajectories of the dynamic molecules into bound and unbound segments as shown in Fig. ing Fig. . If thering Fig. . On the ing Fig. . There iing Fig. . We obse2+ concentrations . In contrast, addition of 20 mM Mg2+ ion triples the average residence time of the IBS1* exon and prolongates the dwell time of dIBS1* by a factor of 10. This goes along with an overall broadening of the distribution of mean dwell times  the Kd of EBS1*/IBS1* decreases by a factor of 10, resulting in an affinity of 29 ± 8 nM at 20 mM Mg2+ , reflecting the lower stability of the RNA–DNA hybrid compared to the RNA–RNA, consistent with previous surface plasmon resonance (SPR) experiments IBS1* in its bound and unbound state. This analysis gives us a robust estimate of the tertiary contact stability from the ensemble of single molecules. On the downside, variations in dwell time length within one trace are averaged out. Mgβ to account for small deviations at longer times. Unbinding, on the other hand, is not homogeneous, but partitions into two exponential terms in the presence of low millimolar amounts of Mg2+. The relative weight of these two components differs considerably depending on the nature of the complementary strand. At 20 mM Mg2+, the fast off-rate, koff,1, accounts for 40 ± 6% of the RNA–RNA contact decay, whereas its amplitude rises to 96 ± 1% in the RNA–DNA contact, meaning the decay is practically homogeneous for the hybrid interaction, even though a small percentage of longer-lived contacts persists , which coincides with the Kd of Mg2+ to the tertiary contact  in the absence of Mg2+, the mechanism simplifies to a two-state system with one on- and one off-rate 6]3+ (green sphere)35. Alternatively, a Mg2+ may exchange one or more of its coordinated water molecules with RNA atoms and bind deeper within the pocket (dark green sphere). It seems likely that inner-sphere binding of Mg2+ in that tunnel locks the tertiary contact in a rigid and stable conformation, which prevents fast dissociation of IBS1*. In the HMM model, the corresponding exchange rate kex,12, which describes the Mg2+-induced stabilization of IBS1*, increases at the expense of the dissociation rate koff,10 over the course of the Mg2+ titration  on all dynamic FRET tracesent Fig.  and prioate Fig. ; (ii) asS1* Fig. : an oute46. The 2′-hydroxyl group contributes to the structural diversity of RNA by forming hydrogen bonds that make up tertiary structure elements such as the ribose zipper or the A-minor motif48. In helical regions, the puckering mode of the sugar is critically influenced by the presence or absence of the 2′-OH group. The most common helical topologies in nature are the A and B forms. While DNA mostly adopts the B-form (C2′-endo), RNA prefers the A-form (C3′-endo), in which the 2′-OH is in a sterically and electronically more favorable axial position  are a result of interconverting conformations averaged out in the NMR measurement, we run molecular dynamics simulations for both tertiary contacts in their docked form. We first simulated the two contacts in the presence of K49. As a result, the RNA/DNA hybrid adopts neither a pure A-form nor a B-form geometry55. The conformational exchange at the RNA–DNA interface probably weakens the binding, thus contributing to the lower stability of hybrid contacts in general and in our case to faster dissociation rates of dIBS1*38.Because the dihedral angles of the sugar ring are all interdependent, the pucker dynamics propagate onto the backbone where they alter the distance between neighboring phosphates and the shape of the helix2. However, common to all is the EBS1, which forms a duplex directly adjacent to the 5′-splice site. Here, we have investigated the thermodynamics and kinetics governing the stability of this tertiary contact using single-molecule FRET in combination with molecular dynamic simulations.Group II introns have developed a highly specific recognition mechanism to cut and paste themselves from one part of the genome to another. Each of the three classes, IIA, IIB, and IIC, relies on a slightly different set of tertiary interactions to position the target strand in the active site for cleavage+ and Mg2+ concentrations. Consistent with previous experiments, IBS1 (un)docking and Mg2+ coordination are coupled, resulting in Mg2+ exchange rates on a timescale of milliseconds to seconds, thus accessible to camera-based detection16. We find that Mg2+ association and dissociation from the EBS1*/IBS1* contact are linked to very subtle conformational changes within the duplex, indiscernible by FRET. As a spectroscopic ruler, the FRET efficiency fails to resolve these kinetic states. In the time dimension, however, the states can be distinguished with regard to their dwell times IBS1* using a combination of dwell time analysis and hidden Markov modeling on an ensemble of FRET trajectories.Our FRET trajectories capture binding and unbinding events across various Kmes Fig. . Hence, 2+ binding sites are occupied or not. While outer-sphere Mg2+ ions are typically thought to exchange on a millisecond timescale56, our previous single-molecule Mg2+ pulse experiments suggest that some ions remain associated with the RNA for seconds or even minutes15. These ions are probably chelated by the RNA18 and buried in a binding pocket formed upon docking of the 5′-exon34. The said cavity is shallower in the hybrid EBS1*/dIBS1* structure and Mg2+ binding to RNA–DNA contacts is generally weaker35. Yet, Mg2+ is required to stabilize the intrinsically labile RNA–DNA contact. Loss of a coordinated Mg2+ ion is therefore likely to be followed by immediate strand dissociation. Such a concerted mechanism of a Mg2+ and exon dissociation is consistent with the observed reduction in kinetic heterogeneity from IBS1* to dIBS1*.Based on our model system, we propose that the 5′-exon dissociates at different rates depending on whether the Mg13. To achieve such high selectivity, ribozymes make use of kinetic partitioning. Mg2+ prolongates the residence time of the exon given that there is a suitable binding site. Selective stabilization of the structurally and catalytically important exon–intron tertiary interaction forms the basis of its heterogeneous kinetics. A depletion of Mg2+ homogenizes the rates. Furthermore, splicing is downregulated, because K+ alone at physiological concentrations is neither able to stabilize the exon–intron contact sufficiently nor activate the phosphodiester bond for cleavage57.How does ribozyme catalysis benefit from heterogeneity? Group II introns have evolved to find just the right balance of keeping a correct substrate in the active site long enough for the two transesterifications to occur, while releasing mismatched sequences before the first chemical step can take place2+ is present in very low millimolar concentrations, Ca2+ is tightly regulated, and other divalent metal ions are generally scarce, intron-encoded or host supplied proteins are recruited to stabilize the weak interactions and promote catalysis. The group IIB intron Sc.ai5γ from which the EBS1*/IBS1* model interaction is derived, is a splicing-only intron, and resides in the housekeeping gene COX1 of yeast mitochondria, where it is assisted by splicing factors such as the ATP-dependent helicase Mss11660. Mobile group II introns, in contrast, still have their own open reading frame encoding a maturase that stabilizes the exon–intron contact62. Only recently, cryo-EM structures of a maturase–intron complex from Lactococcus lactis and Thermosynechococcus elongatus have revealed how the exon is sandwiched between the protein’s thumb domain and EBS1/232. This ternary complex between intron, exon, and protein might be of particular importance to convey stability to the labile RNA–DNA interaction during reverse splicing. Notably, the intron-encoded protein (IEP) has been found to bind more tightly to an intron lariat with the 5′-exon added in trans than to a spliced intron lacking the EBS/IBS interactions61. The exon–intron recognition complex and the IEP thus seem to cooperate to resolve instabilities in DNA target binding.In the cell, where Mg63. These rather slow puckering modes are further reminiscent of the cis/trans isomerization in peptide bonds containing proline residues, which were recently discovered to be a source of heterogeneity in intrinsically disordered protein interactions64. Unlike proline isomerization and secondary structure rearrangements, sugar puckering in dIBS1* happens on a timescale of pico- to nanoseconds and is thus much faster than exon release. Consequently, the intron is prone to let go of its DNA substrate unless the contact is properly stabilized by Mg2+ and/or a protein. We see this facile reversibility of EBS1/dIBS1 binding as a sort of quality control mechanism that ensures correct integration site selection as the ribonucleoprotein (RNP) scans along the exon65.Our molecular simulations provide an atomic view on the exon–intron binding interaction and suggest that fast sugar puckering dynamics may be responsible for the lower affinity of the intron towards DNA exons. The simulations show that sugar puckers of dIBS1* are dispersed across the Eastern half of the pseudorotation cycle. In this energy basin the sugars are free to adopt different conformations. Does this switching have any regulatory function? On a microsecond to millisecond timescale, exchange between 3′-endo and 2′-endo sugars was found to be associated with helical to non-helical transitions, leading to alternative secondary structures, a feature common in riboswitches66. For instance, to probe the same reaction coordinate as in our EBS1*/(d)IBS1* model construct, a dye needs to be placed adjacent to EBS1, which is located around nucleotide position 330 in Sc.ai5γ. Making such a FRET construct would require multiple fragments to be ligated enzymatically. Recent advances in co- and post-transcriptional nucleic acid labeling now provide more flexibility in choosing dye positions, which are close to functionally important elements, and thus provide a basis for studying the kinetics of large RNA–protein assemblies on a single-molecule level68.To test this hypothesis, the EBS1/IBS1 interaction needs to be put back into the context of the full intron. Such large and architecturally complex RNAs and RNPs have long eluded a detailed single-molecule kinetic analysis mainly because the site-specific introduction of suitable reporters, like fluorophores, has proven to be challenging2+ ions delay strand dissociation, sugar switching effectively promotes it. Off-rates are therefore the determining factor for contact stability, in line with previous studies on RNA kissing loops and various kinds of RNA, DNA, and hybrid duplexes69. A question that remains is whether sugar puckering attenuates heterogeneity by directly interfering with metal ion coordination. To explore the relative binding affinities of Mg2+ to pure RNA and RNA–DNA hybrid contacts, MD simulations with enhanced sampling may prove useful70. Such simulation might also detect small structural changes induced upon Mg2+ coordination that FRET struggles to resolve. Altering the degree of residual dynamics at binding interfaces is a common type regulation in biomolecules. By balancing flexibility and rigidity, sugar puckering and metal ions control RNA dynamics and form the molecular basis of kinetic heterogeneity.Here, we have used instead a minimal construct, which is chemically synthesized and covalently labeled, giving us a more straightforward access to studying its binding kinetics. We have shown that ion binding and sugar puckering are two interacting processes that shape kinetic heterogeneity. While site specifically coordinated Mg71. Sequences are derived from the group IIB intron Sc.ai5γ and feature a slightly elongated stem as well as two A-to-C transversions in the loop. These were previously introduced at residues 15 and 17 to stabilize the exon–intron interaction (see Supplementary Methods). For surface immobilization on the microscopy slides, a single-stranded overhang was added that contains four uracils and a biotin at the 3′ terminus15. We refer to these mutated and labeled constructs as EBS1*/IBS1* (RNA–RNA) and EBS1*/dIBS1* (RNA–DNA) to differentiate them from the original sequence in the group II intron.Oligos were purchased from IBA Lifesciences  or Microsynth  with cyanine labels at the 5′ end (Cy3-EBS1* and Cy5-IBS1* or Cy5-dIBS1*) and a biotin at the 3′ end. Photophysical parameters  of the labeled oligos have been previously characterized in detailN-morpholino)propanesulfonic acid (MOPS) at pH 6.9. The imaging solution further contained 100 mM KCl, 1% glucose (w/v), 1 mM Trolox, an oxygen-scavenging system , and varying amounts of MgCl2 (0–20 mM) or alternatively 1 M KCl with 100 µM EDTA to remove any traces of divalent ions. The biotinylated EBS1* RNA was immobilized on a quartz surface via a streptavidin linkage72. For this purpose, the microfluidic channel was first coated with biotinylated bovine serum albumin (5 min), treated with streptavidin (10 min), and finally incubated with biotinylated EBS1* RNA . IBS1* or dIBS1* strands were then flushed into the chamber at concentrations of 35 or 50 nM, respectively. Molecules were imaged on a custom-built TIRF microscope using a water-immersion objective  at a camera frame rate of 5 or 10 Hz (Andor iXon DU-897). Single molecules with anti-correlated donor/acceptor emission were selected and corrected for background and donor bleedthrough into the acceptor channel using the freely available software package MASH-FRET (https://github.com/RNA-FRETools/MASH-FRET.git)73. The FRET efficiency was then calculated from the corrected donor and acceptor intensities after donor excitation according to74.RNA hairpins and their RNA or DNA complements were buffered in 50 mM 3-IBS1*−1. Dissociation constants, Kd, were computed from the on- and off-rates for each decay component m or alternatively for each individual molecule n. In case of the latter, cumulative distributions of Knd, were built and fitted to a normalized logistic functionIndividual single-molecule traces were discretized based on a threshold criterion to generate sets of dwell times in the well-separated zero and high FRET state. A mean dwell time, 〈R and temperature T. The energy barrier separating the bound and unbound state was computed according to transition state theory askon, Planck’s constant h, and Boltzmann’s constant kB. Our calculations of κ = 1, thus providing a lower limit for the difference in free energy between the reactants and the transition state75.Error estimates on all variables were computed from a set of 100 bootstrap samples. For a more detailed mathematical description of the kinetic analysis, we refer the reader to the Supplementary Information. The Gibbs free energy of the bound state with respect to the free hairpin was determined using2+- bound RNA Θ is computed using association constants, K1 and K2, derived from NMR chemical shift perturbations15The fraction of Mg76. In a second step, the predetermined emission probabilities were fixed and the start and transition probabilities of a global HMM were optimized collectively on the entire ensemble of dynamic FRET traces. Kinetic models with different connectivities and degeneracies were tested and evaluated based on a BIC and biochemical significance (see Supplementary Discussion). To validate the obtained rate system, degenerate FRET traces were re-simulated in a subroutine of MASH-FRET77 from the underlying rate matrixFor each salt condition, a HMM was first trained on a trace-by-trace basis as implemented in the software package SMACKSDwell time histograms were built analogously to the experimental traces.78 for PyMOL. Partial charges and VdW radii were assigned with PDB2PQR79 using parameters from AMBER-ff99. Calculations were run at 300 K with 100 mM monovalent and 20 mM divalent ions.The electrostatic surface potential of EBS1* alone and EBS1* in complex with either IBS1* or dIBS1* were computed with the APBS Electrostatics plugin80. The simulations use the AMBER-ff99 force field81 with parmbsc082 and χOL384 corrections (corresponds to AMBER-ff14). Monovalent ions were modeled with parameters from Joung and Cheatham85, while those for Mg2+ were taken from Li et al.86. The molecules were solvated in a dodecahedral box filled with TIP3P water and randomly interspersed with K+ and Cl− ions to neutralize the net charge and reach a concentration of about 100 mM. In a second set of simulations, Mg2+ ions were placed at specific locations suggested by NMR chemical shifts and NOE restraints to [Co(NH3)6]3+. The nucleic acids were then charge neutralized and the K+ and Mg2+ concentrations set to 100 mM and 20 mM, respectively (concentrations are estimates because of the limited box size). In an equilibration phase , the temperature was adjusted to 300 K using the velocity rescale thermostat and the pressure was kept constant at 1 bar by the Parrinello–Rahman barostat. Bonds were constrained by the LINCS algorithm with an integration time step of 2 fs. Nonbonded interactions use the Verlet scheme with a cut-off of 1.4 nm, while long-range electrostatics were treated with the particle mesh Ewald algorithm. Simulations of EBS1*/IBS1* and EBS1*/dIBS1* (both with and without Mg2+) were run for 2 μs each. Convergence was assessed by computing the root mean square deviation over the nucleic acids and time binned histograms of the sugar puckers, showing that the repuckering time is much shorter than the overall simulation length. The pseudorotation angles of the riboses were calculated with PLUMED 2.5.287 and assigned to one of ten puckering modes (phase angle increments of 36°)49 according to the definition by Huang et al.88  were used as starting points for molecular dynamics simulations with GROMACS 5.11H-1H]TOCSY spectra were calculated using NMRFAM-Sparky90 by fitting a set of two-dimensional Gaussians to the assigned peaks.Linewidths and peak volumes in [Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5′-hydroxylated RNA fragments requiring 5′-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs. In the No-Go decay mRNA surveillance pathway, mRNAs containing stalled ribosomes are cleaved by an endoribonuclease. Here the authors show the endonucleolytic cleavage on the artificial No-Go decay target mRNAs, revealing downstream decay process by Trl1 kinase and the 5′ to 3′ exonuclease Dxo1. NGD occurs when translation elongation is blocked by the presence of stable intramolecular or intermolecular RNA structures, enzymatic cleavage, chemically damaged sequences or rare codons8. This mRNA degradation process is dependent on translation and involves an endoribonuclease that cleaves just upstream of the stall sequence9. Other mRNA surveillance pathways can also ultimately lead to NGD. For instance, transcripts synthesized without a stop codon due to premature polyadenylation have stalled ribosomes that are initially detected by the non-stop decay (NSD) pathway10. NSD-targeted mRNAs are cleaved by an uncharacterized mechanism and become targets of NGD when ribosomes reach the new 3′-end and stall12. NGD thus plays a key role in resolving translational issues potentially detrimental to cellular homeostasis. When mRNAs are truncated, the stalled ribosomes are rescued in a process mediated by the Dom34/Hbs1 complex that dissociates the ribosomal subunits5. Their association with the 60S subunit is recognized by the ribosome quality control (RQC) pathway, leading to the rapid degradation of the nascent peptide15. However, despite extensive study, the precise location of NGD cleavage and the mechanism of degradation of the resulting RNA fragment remain elusive.The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stalled ribosomes5 and that simultaneously blocking the 5′–3′ and 3′–5′ exoribonuclease decay pathways is synthetically lethal16. It has been shown, however, that the stability of such mRNAs is largely dependent on the Dom34/Hbs1 complex17. In dom34 mutant cells, ribosomes stalled at the 3′-end of truncated mRNAs inhibit the degradation by the exosome and facilitate the detection of sequential endonucleolytic cleavages upstream of the ribosomal stall site5. Interestingly, dom34 and xrn1 mutations (inactivating the main 5′–3′ exonucleolytic degradation pathway) are not synthetic lethal1. Moreover, NGD endonucleolytic cleavages still occur in the absence of Dom343. The limited 3′–5′ degradation of specific mRNA targets (in the absence of Dom34) combined with 5′–3′ exoribonuclease mutants thus allows an accumulation of RNA fragments resulting from endonucleolytic cleavages whose extremities can be mapped accurately. We have created truncated mRNAs in vivo by insertion of a hammerhead ribozyme sequence (Rz)18 known to generate NGD-targeted mRNAs5. This construction mimics chemically or enzymatically cleaved mRNAs, or those resulting from abortively spliced mRNAs that are processed by the NGD pathway19. As anticipated, these designed truncated 3′-ends block ribosomes at determined positions and, because ribosomes guide NGD mRNA cleavages20, we were able to detect 3′-NGD RNA fragments of specific sizes. By analysing these RNAs in detail, we show the importance of the 5′–3′ exoribonuclease Xrn1 in the production of 3′-NGD fragments. We also perfectly match a 3′-NGD cleavage product with a 5′-NGD cleavage fragment in the region of the third stalled ribosome. We have mapped this site and show that a unique endonucleolytic cleavage occurs eight nucleotides (nts) upstream of the first P-site nt within the third stacked ribosome. The two leading ribosomes are apparently not competent for this cleavage. We demonstrate that this 3′-NGD RNA has a hydroxylated 5′-extremity and show that 5′-phosphorylation by the Trl1 kinase21 is required to allow degradation by 5′–3′ exoribonucleases. Interestingly, in the absence of Xrn1, the alternative 5′–3′ exoribonuclease Dxo1 takes over22. We additionally analysed mRNAs containing rare codons and demonstrate that at least three stacked ribosomes are also required for endonucleolytic cleavage of these mRNAs. We show that 5′ ends observed in regions covered by disomes are the result of 5′–3′ trimming by Dxo1.In this paper, we focus on the fate of NGD-cleaved mRNAs, with an initial goal of mapping the sites of mRNA cleavage with accuracy. Two major obstacles to achieving this objective are that NGD fragments are rapidly attacked by 5′–3′ and 3′–5′ exoribonucleases after ribosome dissociation18 in the 3′-sequence of the URA3 gene ORF (mRNA1Rz). This results in the production of an mRNA that lacks a stop codon and a polyadenylated tail, called mRNA1 in Fig. 5. We first verified that we could detect NGD cleavages in the 3′-proximal region of mRNA1, by northern blotting with a probe corresponding to the 3′-end  produced a different ladder of 3′-NGD RNAs compared to WT or the ski2 mutant. This was confirmed by a higher resolution PAGE analysis followed by northern blotting  was detected in all four strains  and the leading ribosome protecting a half footprint to the site of mRNA truncation . An additional ribosome would thus be expected to protect a ~77-nt RNA. Accordingly, the B5 RNA (77 nts) was found to associate with two and three ribosomes, in fractions 15 and 16, respectively. The 71-nt B4 RNA was also associated with the disome and trisome peaks  or a hydroxyl group (5′-OH) at its 5′-end by treating RNA purified from dom34 cell extracts with T4 polynucleotide kinase to see whether this would stimulate attack of B4 by Xrn1 in vitro. Remarkably, the B4 RNA was completely degraded by Xrn1 only after 5′-phosphorylation by T4 kinase in vitro  is a specific 3′-product of NGD cleavage in our constructs Fig. . While Btro Fig. , demonstfer Fig. . It has ski2 and ski2/dom34 mutants were ligated to a pre-adenylated oligonucleotide linker using truncated RNA ligase to perform 3′-RACE get Fig. 5. SimilaNAs Fig. . As prevNAs Fig.  using thNAs Fig. . By probund Fig. . The proive Fig. . These r4-mRNA, using 3′-RACE for the detection of the 3′-ends of 5′-NGD RNAs in ski2 and ski2/dom34 mutant cells 4-mRNA and truncated mRNA1 are NGD-targeted in a highly similar process that results in cleavage within the footprint of the third ribosome, 71 nts upstream of the stall site for the leading ribosome.The detection of short RNA species by prH probe suggested that endonucleolytic cleavages occurred just downstream, in a region located ~70 nts upstream of the cluster of rare codons  are not competent for the activation of the endonuclease activity , vortexing, and adding 500 µl of phenol previously equilibrated in buffer AE. After vortexing, the tubes were incubated at 65 °C for 4 min and then frozen in a dry ice–ethanol mix, followed by a 15-min centrifugation at room temperature. A second extraction with 500 µl of phenol–Tris–EDTA–chloroform was performed. RNA from 360 µl of supernatant was precipitated with 40 µl of 3 M Na acetate (pH 5.3) and 2.5 volumes of ethanol and centrifuged at 4 °C for 30 min. The pellet was washed in 80% ethanol, dried at room temperature and resuspended in water. After electrophoresis, RNA was blotted onto a Hybond-N + membrane (Amersham) and hybridization to end-labelled oligodeoxynucleotide probes was carried out at 42 °C overnight in Roti-Quick solution (Carlroth). The Hybond-N + membrane was washed successively at 42 °C in 5 × SSC–0.1% SDS and twice in 1 × SSC–0.1% SDS. Blots were exposed to PhosphorImager screens, scanned using a Typhoon FLA 9500 (Fuji), and quantified with ImageJ software.8 ODTotal RNA was resolved by 8% TBE-urea polyacrylamide or 1.4% TBE-Agarose gels, and to dissociate 5′-hydroxylated from 5′-phosphorylated RNAs, total RNA was resolved by 12% TBE-urea polyacrylamide gels. All gels were followed by northern blot analysis.260nm of cell extracts were performed by using 1 unit of Xrn1 (Biolabs) in NEB buffer 3 at 25 °C during 30 min unless otherwise indicated. NEB Buffer 3 was replaced by T4 PNK buffer (NEB) in kinase assays in the presence or absence of Xrn1  were incubated with 0.5, 1, or 2 µl of RNase I  30 min at 25 °C. For total RNA treatment, 5 µg of RNA were digested 30 min at 25 °C. All RNase treatments were followed by RNA extraction and northern blot analysis as described above.RNA digestion of 20 ODn1 Figs. . For RNa600 at 28 °C and the 200 ml cell culture was quickly cooled in ice water and then centrifuged and washed in 20 ml of buffer A containing 10 mM Tris pH 7.4, 100 mM NaCl and 30 mM MgCl2, and 0.5 mg/ml heparin. Heparin was only omitted for RNase I treatment24. Cycloheximide was not used to prevent any drug-induced ribosome positioning. The cell pellet was resuspended in 0.6 ml of buffer A. A 1.4 g amount of glass beads was added, and cell lysis was performed by bead beating twice for 30 s each time (with intermittent cooling). After lysis, the cell extract was clarified for 5 min at full speed in a microcentrifuge. The equivalent of 20 OD260nm units of cell extract  was then layered onto linear 10–50% sucrose density gradients. Sucrose gradients  were prepared in 12 × 89 mm polyallomer tubes (Beckman Coulter). Polysome profiles were generated by continuous absorbance measurement at 254 nm using the Isco fraction collector. Gradients were collected in 20 fractions and processed for northern blotting as described above.Yeast cells were grown exponentially to 0.8 OD4-mRNA) were used and Maxima H Minus reverse transcriptase (ThermoFisher) was used to synthesize a single-stranded DNA toward the 5′-end of the RNA. The size of the labelled single-stranded DNA was determined relative to a sequencing ladder (ThermoFisher Sequenase sequencing kit) on 5% TBE-urea polyacrylamide gel. Oligonucleotides were radio-labelled with [γ-32P]ATP with the T4 polynucleotide kinase (NEB).Radiolabelled primers 31 with minor modifications: Total RNA preparations were first 3′-dephosphorylated using T4 PNK for 1 h at 37 °C without ATP and pre-adenylated linker  ligation was performed during 4 h at 22 °C in the presence of truncated RNA ligase 2 (NEB)30. Reverse transcriptase reactions were performed using primer prE complementary to the linker sequence. PCR primer prF specific to mRNA1, or primer prK specific to (CGA)4-mRNA, were used with primer prE in PCR reactions (Supplementary Figs. Mapping was performed according to the 3′-RNA ligase-mediated RACE methodFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the pre-mRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN∆7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal nuclear accumulation in euchromatin domains of poly(A) RNA granules (PARGs) enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were accompanied by changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. PARG-containing MNs were observed at presymptomatic SMA stage, increasing their number during the symptomatic stage. Moreover, the massive accumulations of poly(A) RNA granules in MNs was accompanied by the cytoplasmic depletion of polyadenylated mRNAs for their translation. We suggest that the SMN-dependent abnormal accumulation of polyadenylated mRNAs and Sam68 in PARGs reflects a severe dysfunction of both mRNA processing and translation, which could contribute to SMA pathogenesis.Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the  An analysis of the human transcriptome revealed that >90% of protein-coding genes undergo alternative splicing, which generates multiple mRNA variants that encode the vast proteomic repertoire required for the protein interactome3. Another fundamental modification of most mRNAs is the addition of a polyadenosine tail at the 3′ end. Polyadenylation is a modification necessary not only for the stability of mRNAs but also for their nuclear export4. Poly(A)-binding protein nuclear 1 (PABPN1) plays a critical role in polyadenylation by strongly enhancing the processivity of poly(A) polymerase4. Upon the completion of co-transcriptional processing, mRNAs are exported to the cytoplasm as parts of messenger ribonucleoprotein particles for translation and degradation. During their nuclear journey, mRNAs associate with several RNA-binding proteins that contribute to the regulation of their processing and exportation.The processing of mRNAs includes three essential modifications, capping, splicing and polyadenylation, which mainly occur at the sites of transcription 7. These nuclear compartments store, assemble and recycle splicing factors and are also enriched in poly(A) RNAs and several mRNA processing factors, including PABPN111.On their nuclear route, some normal and incompletely spliced mRNAs are trafficked and may be retained in nuclear speckles12. Thus, some abnormal mRNA transcripts may accumulate in nuclear inclusions under pathological conditions, such as oculopharyngeal muscular dystrophy, myotonic dystrophy type 1 and fragile X-associated tremor/ataxia syndrome15. Moreover, we have previously reported that the dysfunction of nuclear RNA processing in the sensory ganglion neurons upon proteasome inhibition induces the nuclear aggregation of polyadenylated mRNAs and the RNA-binding protein Sam68 (src-associated protein in mitosis of 68kD) into a new nuclear structure called the “poly(A) RNA granule” (PARG)17. The sequestration of crucial RNA-binding proteins in nuclear inclusions or granules may prevent their normal function, and contribute to disease pathogenesis18.Defects in pre-mRNA splicing or polyadenylation often prevent the recruitment of mRNA export factors, resulting in the nuclear retention of immature or aberrant pre-mRNAs20. SMA is caused by a homozygous deletion or mutation in the survival of motor neuron 1 (SMN1) gene that results in decreased levels of the full-length SMN protein22. SMA patients carry a nearly identical SMN1 gene paralogue named SMN2, which differs from SMN1 by a C to T transition in exon 723. Although both the SMN1 and SMN2 genes encode the SMN protein, approximately 90% of the SMN2 mRNA transcripts generate an alternatively spliced isoform that lacks exon 7 and encodes a truncated form of the SMN protein (SMN∆7) that is rapidly degraded24. Therefore, SMN2 expression cannot fully compensate for the deficiency of the full-length SMN protein. The best-known function of SMN is the biogenesis of spliceosomal snRNPs . Linked to this function, SMN deficiency in SMA produces alterations in this snRNP repertoire and causes widespread splicing defects that result in a severe dysregulation of mRNA metabolism in MNs31. Splicing alterations in the spinal cord in murine SMA models include widespread intron retention, particularly of minor U12 introns, as well as a time-dependent differential expression of a number of exons associated with neurodevelopmental and cell stress pathways32. In addition to the dysfunction of pre-mRNA processing, as a pathogenic factor in SMA, recent studies identified ribosome biology and translation efficiency as key processes affected by SMN depletion, which may also contribute to MN degeneration and SMA pathogenesis34.Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the progressive degeneration and loss of spinal cord and brainstem motor neurons (MNs)37. The formation of PARGs associates with variations in the alternative splicing regulation of several mRNAs, including the Sam68-dependent Bcl-x and Nrxn-1 mRNAs. Moreover, the massive accumulation of PARGs in MNs is accompanied by cytoplasmic depletion of polyadenylated mRNAs, supporting the existence of a severe dysfunction of both mRNA processing and translation, which may be an important pathogenic factor in SMA.The aim of this work is to analyze the subcellular reorganization of polyadenylated mRNAs and certain RNA-binding proteins induced by the SMN-dependent dysfunction of pre-mRNA splicing in MNs from the SMN∆7 mouse model of SMA. Our results demonstrate the nuclear accumulation of poly(A) RNAs in PARGs enriched in the RNA-binding protein Sam68, which is an alternative splicing regulator of pre-mRNAs4. Therefore, in situ hybridization of poly(A) RNAs with an oligonucleotide poly(dT) probe, which recognizes the poly(A) tail, is a good approach for studying the perikaryal distribution of global mRNAs in MNs.With the exception of histone mRNAs, all mRNAs are polyadenylated and have a poly(A) tail that is essential for their exportation and stability38  RNAs in dissociated WT MNs. Poly(A) RNAs, in addition to being diffusely distributed throughout the nucleus, excluding the nucleolus, were concentrated in nuclear speckles , a cytochemical fluorescent staining for nucleic acid detection, dissociated MNs from wild-type (WT) mice exhibited a prominent nucleolus and large RNA-rich cytoplasmic areas of the Nissl substance, a classic name used to designate the neuronal protein synthesis machinery  RNA in some SMN∆7 MNs during both the late-presymptomatic  and symptomatic (P14) stages, compared with the staining pattern of WT MNs  RNA, which were identified as PARGs16  RNAs. MNs Figs , 2A–F. Ts16 Fig. .Figure 2The PARGs, which ranged from 0.1 to 1.5 µm in diameter, were distributed throughout the nucleus, excluding the nucleolus. They frequently appeared in close proximity to the nucleolus and nuclear speckles Fig. . Two str40 was not observed in SMN∆7 MNs. Changes in the relative concentration of poly(A) RNA in the cytoplasm were validated by a densitometric analysis of the fluorescence hybridization signal intensities at P14. Several measurements of the poly(A) RNA signal intensities were performed in the peripheral cytoplasm, where the Nissl substance is largely distributed in MNs. A significant reduction of the relative poly(A) RNA concentration was detected in the Nissl substance of PARG-containing MNs, compared with PARG-free MNs, from both SMN∆7 and WT mice  RNA hybridization signal in the cytoplasm Fig. . Moreoveice Fig. .34. Ultrastructural analysis confirmed the presence of two categories of PARGs: rounded, compact electron-dense bodies  RNA and coilin confirmed the depletion of typical Cajal bodies in SMN∆7 MNs. Nevertheless, small coilin-positive and poly(A) RNA-negative residual Cajal bodies were occasionally found adjacent to PARGs, but as two clearly distinct nuclear structures  RNA in combination with molecular markers of nuclear compartments, such as coilin , SMN (gems), the TMG-cap (nuclear speckles) and the proteasome 20S (clastosomes). In a recent study, we demonstrated that the reduced levels of SMN in MNs from SMN∆7 mice were associated with a severe depletion of Cajal bodiesres Fig. . SimilarRGs Fig. . MoreoveRGs Fig. . FinallyRGs Fig. .Figure 4SMN2 alternative splicing46, we investigated its nuclear reorganization in SMN-deficient MNs from the SMN∆7 mice. In WT MNs, co-staining for Sam68 and poly(A) RNA revealed a predominant nuclear localization of Sam68, which excluded the nucleolus and poly(A) RNA-positive nuclear speckles  or the proapoptotic Bcl-x(s) variants48. Real-time PCR quantification of these two variants in spinal cord extracts showed a significant decrease of the Bcl-x(s)/Bcl-x(L) ratio in SMN∆7 mice compared with the WT mice /Nrxn1 4(+) ratio in SMN∆7 mice , which includes exon 20 in the Nrxn1 alternatively spliced segment 4 (AS4).Previous studies have demonstrated that Sam68 regulates the alternative splicing of two important genes for neuronal function, ice Fig. . Similarice Fig. , which rHaving demonstrated the concentration of Sam68 in PARGs, we then proceeded to investigate the possible accumulation in these granules of other RNA-binding proteins involved in nuclear mRNA processing and export, such as PABPN1, TDP43 (TAR DNA-binding protein 43), hnRNP (heterogeneous nuclear ribonucleoprotein) A2/B1, hnRNP M3, Y14 and REF/Aly.4 and is required for efficient poly(A) RNA export from the nucleus49, we investigated changes in its nuclear pattern and protein levels in WT and SMN∆7 mice. PABPN1 was concentrated in nuclear speckles in both WT and SMN∆7 MNs  tail of polyadenylated RNAsMNs Figs , 7B–D, bMNs Figs , suggestMNs Figs .Figure 650. This protein is a component of the hnRNP particles that regulate the splicing of a variety of pre-mRNAs52. TDP43 immunolabeling showed a diffuse nuclear localization, excluding nuclear speckles and the nucleolus, in both WT and SMN∆7 MNs, but the protein was not detectable in pathological PARGs  and chondrolectin (Chodl), which are processed by the major U2-dependent spliceosome, and the non-muscle myosin II isoforms IIA (Myh9) and IIC (Myh14), which are processed by the minor U12-dependent spliceosome. Splicing efficiency was analyzed by estimating the percentage of the unspliced (exon-intron sequence) forms of the Chodl, Chat, Myh9 and Myh14 pre-mRNAs by qRT-PCR in spinal cord RNA extracts. Importantly, we found that in relation to WT samples, samples from P5 SMNΔ7 animals had a significant increase in the proportion of unspliced forms of the Chat and Myh14 mRNAs, while the accumulation of the unspliced forms was extended to the four examined pre-mRNAs  during the late symptomatic stage (P14),  pre-mRNAs were detected when samples from WT and SMN∆7 mice were compared at P5 or P14 , Fig. . No signP14 Fig. .Figure 8Chat, Chodl, Myh9 and Myh14 mRNAs. The accumulation of poly(A) RNAs in PARGs appears to be an early (P5) cellular manifestation of the nuclear mRNA metabolism dysfunction in SMA MNs, which could contribute to SMA pathogenesis. Moreover, MNs that contain numerous PARGs frequently exhibit other signs of neuronal dysfunction. These signs include the segregation of the granular component of the nucleolus and cytoplasmic reduction of polyadenylated mRNAs, which support defective ribosome biogenesis and translation, according to previous studies in SMA mice34. Collectively, the nuclear accumulation of poly(A) RNAs in PARGs is consistent with the splicing alterations reported here and in previous studies in SMA MNs31.The present study demonstrates that the SMN deficiency in SMN∆7 MNs affects the nuclear distribution of polyadenylated mRNAs, resulting in their progressive accumulation in Sam68-positive PARGs. During the symptomatic stage, this neuronal response is associated with an increased proportion of intron-containing SMN1 gene are affected differently. Our results confirm this heterogeneity, showing that at both P5 and P14, affected MNs carrying PARGs coexist with other MNs that have an apparently normal pattern of poly(A) RNA distribution. In this regard, differences in the pathogenic vulnerability of MNs have recently been correlated with individual differences in SMN protein levels in MNs from a particular SMA mouse58.A poorly understood aspect of SMA is why MNs that carry the same deletion or mutation of the 17, the present work provides the first demonstration that these structures are present in SMA MNs. The PARG represents a distinct nuclear entity that is clearly distinguishable by its structure and molecular composition from other nuclear compartments, such as nuclear speckles9, Cajal bodies60, gems44 and clastosomes45. PARGs share with nuclear speckles the presence of poly(A) RNAs; however, PARGs lack spliceosomal snRNPs and display an ultrastructural configuration as sharply defined nuclear bodies that is clearly different from the interchromatin granule clusters of nuclear speckles43. Cajal bodies, the nuclear structures involved in spliceosomal snRNP biogenesis42, are depleted in SMN∆7 MNs34; additionally, the Cajal body marker coilin was not detected in PARGs. Similarly, gems, SMN-positive and coilin-negative nuclear bodies of unknown function44, have been reported in fetal MNs61, but they are absent in postnatal and mature MNs from both WT and SMN∆7 mice34. Moreover, we did not detect SMN in PARGs. Finally, these RNA granules also lack the 20S proteasome, a molecular marker of clastosomes, which are nuclear proteolytic factories enriched in catalytic proteasomes, ubiquitylated proteins and proteasome substrates62.Although we have previously reported the presence of PARGs in sensory ganglion neurons upon proteasome inhibition63. We propose that the formation of PARGs in SMN∆7 MNs reflects a stress-related dysfunction of RNA metabolism, essentially in pre-mRNA splicing. Consistent with this view, pre-mRNA splicing has emerged as a well-known and important target of several stressing agents, resulting in alternative splicing dysregulation and splicing inhibition64. In the case of SMA, several studies demonstrated that the hyperactivation of the endoplasmic reticulum stress pathway and widespread defects in splicing underlie the neurodegeneration observed in SMA MNs65. Moreover, we have previously reported the formation of PARGs following proteasome inhibition-induced proteotoxic stress in rat sensory ganglion neurons, an experimental condition that produces a dysfunction of RNA metabolism and a disruption of the protein synthesis machinery17.PARG-containing MNs were already observed at P5, but they increased in number during the late symptomatic stages (P14), presumably reflecting the well-established asynchrony at the beginning of the MN degeneration in SMN∆7 mice68. As a splicing regulator, Sam68 may promote exon inclusion or exclusion in certain neural pre-mRNAs, including SMN2, Bcl-x and Nrxn 147. Regarding SMN genes, Sam68 is a physiological regulator of SMN2, but not of SMN1, splicing. Thus, Sam68 directly binds to the SMN2 pre-mRNA and acts as a splicing repressor of exon 7 inclusion in SMN2 transcripts69. In the present study, we detected no significant changes in the mRNA and protein levels of Sam68 in the spinal cords of SMN∆7 mice compared with the WT animals. Although the accumulation of Sam68 in PARGs should reduce its nucleoplasmic levels, potentially facilitating the inclusion of exon 7 in SMN2 transcripts69, its partial relocation in PARGs does not correct the defective SMN2 splicing in SMN∆7 mice. In fact, SMN protein levels are dramatically reduced in the spinal cords of the SMN∆7 mice during the symptomatic stages (present results and34). However, our results show changes in the alternative splicing regulation of the Sam68-dependent Bcl-x and Nrxn-1 mRNAs in the spinal cords of SMN∆7 mice. It is well-established that Sam68 binds Bcl-x mRNA, and its intracellular levels regulate the balance of alternative splicing to produce pro-apoptotic Bcl-x(s) transcripts or anti-apoptotic Bcl-x(L) transcripts48. In particular, whereas the upregulation of Sam68 enhances Bcl-x(s) splicing, its downregulation promotes the production of Bcl-x(L) transcripts47. In this context, our finding of a reduction of the Bcl-x(s)/Bcl-x(L) ratio in the SMN∆7 mice, compared with WT littermates, is consistent with a downregulation of Sam68.An important finding in this study is the accumulation of Sam68 in PARGs. Sam68 is a member of the STAR  family of RNA-binding proteins that bind both RNA and DNA and are involved in signal transduction, transcription and alternative splicing regulationNrxn1 transcripts, changes in alternative splicing regulation result in a decreased Nrxn1 4(−)/Nrxn1 4(+) ratio, with a relative increase in the isoform that includes exon 20 in Nrxn1 alternatively spliced segment 4 (AS4). Importantly, the neurexin protein 4(+) and 4 (−) variants show different interactomes with proteins that are key mediators of synaptic formation and maintenance, including neuroligins . Thus, previous studies have demonstrated that neurexin 4(+) exhibits weak binding with the neuroligin-1B postsynaptic receptor, which affects synaptic adhesion71. In this context, the Sam68-dependent relative abundance of neurexin 4(+) in SMN∆7 mice might contribute to the synaptic dysfunction observed in SMA MNs25. Collectively, our results suggest that the recruitment of Sam68 into PARGs impacts on its normal function in splicing regulation. In this line, recent studies have shown that Sam-68 deficient preadipocytes exhibit alternative splicing imbalances in components of the mTOR signaling pathway that lead to defective adipogenic differentiation72.Regarding 12. We believe that PARGs may contain incorrectly processed polyadenylated transcripts bound to Sam68, but lack other RNA-binding proteins involved in packing nascent mRNA (hnRNPA2/B1), splicing regulation (hnRNPM3/M4 and TDP43) and polyadenylation (PABPN1). Consistent with the nuclear accumulation of poly(A) RNA in PARGs, REF and Y14, two RNA-binding proteins that link pre-mRNA splicing to nuclear export74, are not detectable in these granules. Moreover, we observed a reduction of the levels of PABPN1, a protein that, in addition to polyadenylation, is also involved in mRNA export from the nucleus49. Therefore, a deficiency of this export factor in SMN∆7 MNs could also contribute to the accumulation of poly(A) RNA in PARGs.The nuclear accumulation of immature or aberrant pre-mRNAs can be triggered by a wide range of errors in mRNA processing, which prevent the recruitment of mRNA export factors75, and missplicing events are particularly prominent in neurodegenerative diseases77. Previous studies in mouse models of SMA have demonstrated SMN-dependent downregulated spliceosomal snRNPs and altered alternative splicing, preferentially in U12-dependent introns31. Our results extend the splicing defects in SMA MNs. Thus, we demonstrate here the accumulation of incompletely spliced (intron-containing) Chat, Chodl, Myh9 and Myh14 pre-mRNAs during the late symptomatic stage (P14), when a higher incidence of PARG-containing MNs occurs. Interestingly, the accumulation of intron-containing mRNAs impacts the transcripts processed by both the minor (Myh9 and Myh14) and major  spliceosomes, suggesting a global splicing dysfunction. Importantly, reduced Chodl expression has been demonstrated in SMA mouse MNs and linked to MN outgrowth defects78.Alternative splicing defects account for nearly 50% of human inherited diseases79, spliceosome dysfunction plays a critical role in SMA pathogenesis . We propose that the abnormal accumulation of polyadenylated RNAs in PARGs reported here, together with the previously demonstrated depletion of canonical  Cajal bodies81 are two cellular manifestations of the global splicing dysfunction in SMA MNs. Notably, an enhancement of the nuclear poly(A) RNA signal, which is associated with the formation of cytoplasmic stress granules, has been reported in a cellular model of C9orf72 amyotrophic lateral sclerosis82, suggesting that the nuclear accumulation of polyadenylated mRNAs may be a more general mechanism in the pathogenesis of MN diseases.Although MN functions, such as axonal RNA transport and translation control at the neuromuscular junction, are altered in SMA34. Furthermore, a recent study on the transcriptome and translatome in SMA mice, by means of next-generation sequencing, provides an important catalogue of the mRNAs with altered translation efficiency33. The negative impact of the reduced levels of cytoplasmic poly(A) RNAs on translation might be reinforced by the failure of SMN∆7 MNs to recruit these RNAs into cytoplasmic stress granules, which is a protective cellular mechanism for the transient storage and stabilization of mRNAs during the stress-induced inhibition of translation40.The reduction of the cytoplasmic poly(A) RNA signal in MNs containing a large number of PARGs supports a decrease in the translation efficiency, with reduced availability of polyadenylated mRNAs for protein synthesis. Consistent with this notion, by ultrastructural and immunocytochemical analyses, we have previously reported nucleolar alterations and a severe disruption of the protein synthesis machinery in MNs from the SMN∆7 miceIn conclusion, SMN deficiency in SMN∆7 MNs causes an abnormal nuclear accumulation of polyadenylated RNAs in PARGs and the cytoplasmic depletion of these RNAs. This neuronal response suggests a defective mRNA processing, export and translation and is also consistent with the widespread splicing defects reported in SMA MNs. The present study provides additional support for the hypothesis that the dysfunction of nuclear mRNA metabolism plays a critical role in MN degeneration and consequently in SMA pathogenesis.Smn+/−, SMN2+/+, SMN∆7+/+, heterozygous knockouts for mouse Smn , which were purchased from The Jackson Laboratory , were crossed to generate Smn−/−, SMN2+/+, SMN∆7+/+ (hereafter referred to as SMN∆7) mice and Smn+/+, SMN2+/+, SMN∆7+/+ mice that were wild-type for Smn (hereafter referred to as WT). SMN∆7 mice exhibit a severe postnatal SMA phenotype with a mean lifespan of approximately two weeks83. Animal care and handling were performed in accordance with the Spanish legislation  and the guidelines of the European Commission for the Accommodation and Care of Laboratory Animals (revised in Appendix A of the Council Directive 2010/63/UE). The experimental plan was examined and approved by the Ethics Committee of the University of Cantabria and the Committee for Animal Care and Use of the University of Lleida. On postnatal day (P) 0, the identification of WT and SMN∆7 mice was carried out by genotyping with PCR. DNA was extracted from the tail, as previously described63. Age-matched WT littermates of mutant animals were used as controls.The 84. The samples were sequentially treated with 0.5% Triton X-100 in PBS for 45 minutes, 0.1 M glycine in PBS containing 1% bovine serum albumin (BSA) for 30 minutes and 0.05% Tween 20 in PBS for 5 minutes. Then, the samples were incubated for 3 hours with the primary antibody containing 1% BSA at room temperature, washed with 0.05% Tween 20 in PBS, incubated for 45 minutes in the specific secondary antibody conjugated to FITC, TexasRed or Cy3, or Cy5  and counterstained with PI for the detection of nucleic acids. The slides were then washed in PBS and mounted with the ProLong Anti-Fading Medium (Invitrogen) or Vectashield .For immunofluorescence, four mice per group (WT and SMN∆7) at P5 and P14 were perfused, under deep anesthesia with pentobarbital (50 mg/kg), with 3.7% paraformaldehyde (freshly prepared) in phosphate-buffered saline (PBS). The spinal cords were rapidly dissected, removed, post-fixed for 6 hours and washed in PBS. Transverse sections (160 µm thick) of the spinal cord were obtained with a vibratome, and small tissue fragments from the anterior horn were dissected out. The samples were transferred to a drop of PBS on a positively charged slide , and squash preparations of dissociated MNs were generated following the previously reported procedureConfocal images were obtained with an LSM510  laser scanning microscope using a 63x oil (1.4 NA) objective. To avoid overlapping signals, images were obtained by sequential excitation at 488 nm, 543 nm and 633 nm, to detect FITC, TexasRed or Cy3, and Cy5, respectively. Fluorescence profiles of confocal intensity signals across a line were generated to analyze the spatial association between PARGs and the nuclear speckles (TMG-Cap), Cajal bodies (coilin), gems (SMN), clastosomes (20S proteasome) and the RNA-binding proteins PABPN1, Sam68, TDP43, hnRNP A2/B1, hnRNP M3/M4, REF and Y14. Images were processed using Photoshop software.85), and anti-TDP43 , and the goat polyclonal antibody anti-hnRNPA2/B1 .The following primary antibodies were used for immunofluorescence: the mouse monoclonal antibodies anti-SMN , anti-TMG-cap , anti-REF , anti-Y14  and anti-hnRNPM3/M4 , the rabbit polyclonal antibodies anti-PABPN1 , anti-proteasome 20 S subunit α5 , anti-Sam68 , anti-coilin 204/10 (204.3 serumin situ hybridization (FISH). MN perikarya were then dissociated as described above. Preparations of MNs were permeabilized with TBS-E-SDS for 15 minutes at 37 °C, washed three times in 6x SSPE-0.1% Tween 20 for 15 minutes, and incubated with the probe containing tRNA for 3 hours at 42 °C in a humidified chamber. An oligo dT (50)-mer, 5′-end labeled with biotin  was used as a probe for fluorescence in situ hybridization (FISH) to poly(A) RNA. The hybridization mixture contained 80 ng of oligo dT(50), 2xSSC, 1 mg/ml tRNA, 10% dextran sulfate and 25% formamide. After hybridization, the MNs were washed in 6xSSC for 15 minutes, and then washed in 4x SSC-0.1% Tween 20 for 15 minutes at room temperature. The hybridization signal was detected with FITC-avidin for 30 minutes. For the amplification of the hybridization signal, neuronal samples were incubated with avidin-biotin for 30 minutes, washed in 4x SSC-0.1% Tween 20 for 15 minutes and then incubated with FITC-avidin for 30 minutes. All samples were mounted with Vectashield . Some samples were also processed for double- or triple-labeling experiments combining poly(A) RNA detection with immunofluorescence for TMG-cap, PABPN1, Sam68, TDP43, hnRNP A2/B1, hnRNPM3/M4, REF or Y14.Tissue fragments from the ventral horn of the spinal cord fixed with 3.7% paraformaldehyde were processed for fluorescence The quantitative analysis of the proportion of SMN∆7 MNs containing PARGs was performed in squash preparations processed for FISH with the poly(dT) probe. The proportion of neurons containing these granules was estimated by direct examination of the different focal planes throughout neuronal nuclei, using a 40 × objective. Quantification was performed on at least 100 MNs from three WT and SMN∆7 mice.The fluorescence intensity of poly(A) RNAs in the Nissl substance was quantified using the confocal Zeiss LSM 5 image analysis system. At least 30 confocal images of MNs per animal from WT (n = 3) and SMN∆7 (n = 3) mice were recorded using a 63x oil objective. For each MN, four measurements of poly(A) RNA signal intensity in the peripheral cytoplasm, where the RNA-rich Nissl substance is largely distributed, were made. The values were corrected for background staining by subtraction of a blank measurement taken outside the cell.For conventional ultrastructural examination of MNs, WT and SMN∆7 mice (n = 3 per group) were perfused under deep anesthesia with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Transverse sections (500 µm thick) were obtained with a vibratome, and anterior horn tissue fragments were dissected out, rinsed in 0.1 M phosphate buffer, postfixed in 2% osmium tetroxide, dehydrated in acetone and embedded in araldite . Ultrathin sections stained with uranyl acetate and lead citrate were examined with a JEOL 201 electron microscope.For immunoelectron microscopy of Sam68, WT and SMN∆7 mice were perfused with 3.7% paraformaldehyde in 0.1 M phosphate buffer. Tissue fragments of the ventral horn were washed in 0.1 M cacodylate buffer, dehydrated in increasing concentrations of methanol at −20 °C, embedded in Lowicryl K4M at −20 °C and polymerized with ultraviolet irradiation. Ultrathin sections were mounted on nickel grids and sequentially incubated with 0.1 M glycine in PBS for 15 min, 5% BSA in PBS for 30 min and the primary antibody for 2 h at 37 °C. After washing, the sections were incubated with the specific secondary antibodies coupled to 10 nm gold particles . Following immunogold labeling, the grids were stained with lead citrate and uranyl acetate and examined with a JEOL 201 electron microscope. As controls, ultrathin sections were treated as described above without the primary antibody.Five SMNΔ7 (n = 5) and 3 WT (n = 3) mice were used for qRT-PCR studies. The mice were decapitated after being anesthetized and the lumbar spinal cord was quickly removed and frozen in liquid nitrogen. RNA was isolated with Trizol following the manufacturer’s instructions  and purified with the RNeasy kit .Sam68 (Sam68), Actb (ß-actin), Chat (choline O-acetyltransferase), Chodl (chondrolectin), Myh9 (Myosin-9) and Myh14 (Myosin-14), as well as the long and short isoforms of Bcl-x (Bcl-XL and Bcl-XS), and Nrxn1 4(−) and Nrxn1 4(+) variants (neurexin 4(+) and neurexin 4(−)) was determined by qRT-PCR using gene-specific SYBR Green- based primers (Takara). RT-qPCRs was performed with three biological replicates and technical triplicates/duplicates of each cDNA sample. The threshold cycle (Ct) for each well was determined. The results were normalized to the 45 S ribosomal gene transcripts, which had no significant variation in expression across the sample sets and was therefore chosen as a normalizer in our experiments. Relative gene expression was calculated according to the 2−(ΔΔCt) equation86. Each value in this work represents the mean ± SD of independent samples obtained under the same conditions and compared to two replicated qRT-PCR analyses. The SYBR Green-based specific primers for murine RNAs were as follows: for the Sam68 mRNA 5′-CTCCAGCTAGGCCAGTGAA-3′ and 5′-TTGTGGGTAAAGCAACAGGA-3′; for the unspliced pre-mRNA of Actb containing intron 1, 5′-GACTCCCAGCAC ACTGAACT-3′ and 5′-CTCAGGGCAGGTGAA ACTGT-3; for the spliced mRNA of Actb 5′-CAGCCTTCCTTCTTGGGTATG-3′ and 5′-GGCATAGAGGTCTTT ACGGATG-3′; for the unspliced pre-mRNA of Chat containing intron 3, 5′-CTTGGGGCCAGTCTGATAGC-3′ and 5′-GGACACATGGCTAGAAGGGG-3′; for the spliced mRNA of Chat, 5′-CTGGCTTACTACAGGCTTTACC-3′ and 5′-GTGGCCGATCTGATGTTGT-3′; for the unspliced pre-mRNA of Chodl containing intron 3, 5′-GCTGTTGTCTCCCGCATCTT-3′ and 5′-AAGTGGAAGCGTTTGGGA TT-3′; for the spliced mRNA of Chodl, 5′-GAAGCAGGCATAATTCCCAATC-3′ and 5′-TTCCCAAAGCA ACCAGTATCA-3; for the unspliced pre-mRNA of Myh9 containing intron 6, 5′-CACTTGTGTGGCATTGGGAC-3′ and 5′-AAGAAGGACCT CTCCTCCGA-3′; for the spliced mRNA of Myh9, 5′-TGGTGCCAACATTGA GACTTAT-3′ and 5′-CCAGACAGCAG GTAGTAGAAGA-3′; for the unspliced pre-mRNA of Myh14 containing intron 5, 5′-TCACCCTGAAAAGATGCCCC-3′ and 5′-TCGAAGGTCCAGACAGGCAT-3′; for the spliced mRNA of Myh14, 5′-GGAGCA AACATCGAGACCTATC-3′ and 5′-GGTAGAAGATATGGAAGCTGCATT-3′. For the long form of Bcl-x (Bcl-x(L)), 5′-GAGTTACCGGCGACCCA-3′ and 5′-CGACAGCAA GCAGGTACAA-3′; for the short form of Bcl-x (Bcl-x(s)), 5′-GCTGCCTACCAGAACCTTATC-3′and 5′-GGCTCAACCAGTCCATTGT-3′; for the exon 20 containing Nrxn1 variant (Nrxn1 4(+)), 5′-TAGTTGATGAATGGCTACTCGACAAA-3′ and 5′-GACTCAGTTG TCATAGAGGAAGGCAC-3′; for the exon 20 skipped Nrxn1 variant (Nrxn1 4(−)), 5′-GCTACCCTGCAGGGCGT-3′ and 5′-GAGGTGGACATCTCAGACTGCAT-3′.One microgram of RNA was reverse-transcribed to first-strand cDNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) using random hexamers as primers. The cDNA concentration was measured in a spectrophotometer (Nanodrop Technologies ND-1000) and adjusted to 0.5 μg/μl. The expression of the mRNAs candidates 2, 1 mM dithiothreitol, and 10% glycerol, and supplemented with EDTA-free complete protease inhibitor cocktail and PhosSTOP (Roche). The spinal cord samples were sonicated for 5 cycles of 30 seconds ON/OFF at 4 °C using a Bioruptor Plus (Diadode) and left on ice for 20 min. The samples were then cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. The proteins were separated on 4–20% NuPage TG SDS–PAGE gels (Invitrogen) and transferred to nitrocellulose membranes using standard procedures. Mouse monoclonal anti-Tubulin ß-III (Sigma T8660) and rabbit polyclonal anti-PABPN1 and anti-Sam68 were used. Protein bands were detected with an OdysseyTM Infrared-Imaging System (Li-Cor Biosciences) according to the OdysseyTM Western-Blotting Protocol. Immunoblots were developed with anti-mouse IRDye800DX or anti-rabbit IRDye680DX  secondary antibodies. For the quantitative analysis of the blots, ImageJ software was used .Spinal cords from WT (n = 3) and SMNΔ7 mice (n = 5) were lysed at 4 °C in a buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 2% Nonidet P-40, 1 mM MgClt-test. Significance was established at p ≤ 0.05.For comparisons between WT and SMN∆7 samples, data were analyzed using the GraphPad Prism 7 software and an unpaired Student’s Supplementary Figures


Environmental factors are critical in the development of age-related cognitive decline and dementia. A western diet (WD) can cause nutrient deficiency and inflammation that could impact cognition directly. It is increasingly recognized that innate immune responses by brain myeloid cells, such as resident microglia, and infiltrating peripheral monocytes/macrophages may represent an essential link between a WD, cognitive decline, and dementia. Our previous data demonstrated that chronic consumption of a WD induced inflammation through brain myeloid cells in aging mice and a mouse model of Alzheimer’s disease (AD). However, the subtypes of myeloid cells that contribute to the WD-induced inflammation remain unclear.Ccr2RFP/+Cx3cr1GFP/+), and Ccr2-deficient mice (B6.Ccr2RFP/RFP) were fed a WD or a control chow diet (CD) from 2 to 6 or 12 months of age. CD11b+CD45lo and CD11b+CD45hi cells from WD- and CD-fed B6 or Ccr2-deficient mice were characterized using flow cytometry, RNA-sequencing, and immunofluorescence.C57BL/6J (B6), myeloid cell reporter mice . Finally, we show a significantly greater number of OPN+IBA1– cells in WD- compared to CD-fed mice that we propose are activated neutrophils based on ImmGen data. OPN+IBA1– cells are not significantly increased in Ccr2-deficient WD-fed mice.These data further support the model that peripheral myeloid cells enter the brain in response to diet-induced obesity. Elucidating their contribution to age-related cognitive decline and age-related neurodegenerative diseases should offer new avenues for therapeutic intervention in Alzheimer’s disease and related dementias, where diet/obesity are major risk factors.The online version of this article (10.1186/s12974-019-1527-z) contains supplementary material, which is available to authorized users. Obesity continues to be a major health issue in the USA, with the past decade demonstrating a significantly increasing trend nationwide. From 2015 to 2016, the prevalence of obesity was 39.8% in adults and 18.5% in youths . ObesityAPP/PS1 mice , with an absolute fold change (FC) larger than 1.5 for comparisons between CD11b+CD45lo and CD11b+CD45hi cells or with an absolute FC larger than 1.0 for comparisons between WD and CD in either CD11b+CD45lo and CD11b+CD45hi cells. The lower stringency allows for the detection of any subtle effects of diet on both cell populations. All DE gene analyses and quality control were conducted in R (version 3.5.1). All DE genes were uploaded into Ingenuity Pathway Analysis (IPA) software for canonical pathway analysis. The resulting canonical pathways were ranked by Fisher’s exact test p value, and the top 15 revealed all had Fisher’s exact test p value less than 1 × 10−4. The top DE genes enriched in CD11b+CD45lo cells were defined as those with expression levels above 100 cpm and at least two-fold higher compared to CD11b+CD45hi cells. The top DE genes in CD11b+CD45hi cells were defined as those with expression levels above 100 cpm and at least 10-fold higher compared to CD11b+CD45lo cells. The top CD11b+CD45lo and CD11b+CD45hi cell-related genes were compared against mouse immune cell RNA-seq datasets at Immunological Genome Project (ImmGen) [R. The top CD11b+CD45hi cell-related genes were also uploaded to David: Functional Annotation tool [10(BJ) > 1.3]. The top CD11b+CD45hi cell-related genes were also imported into IPA for generation of gene interaction network.Following alignment and expression quantification, differential gene expression analyses were performed using edgeR 3.20.9 . We appl(ImmGen) , My Gene(ImmGen) . For plo(ImmGen)  and wereion tool , for genCcr2RFP/+Cx3cr1GFP/+ mouse strain  > 1.3 and number of genes in pathways ≥ 3. (B) Box plots showing the log2-transformed expression of Ctsd and genes involved in protein ubiquitination pathway in CD11b+C45lo and CD11b+CD45hi cells from CD or WD-fed mice. (C) Fold change (FC) of Ctsd and genes involved in protein ubiquitination pathway comparing CD11b+CD45hi cells from WD and CD-fed mice, colored by significance level using -log10(FDR). The positive FC value means the gene expression of CD11b+CD45hi from WD is larger in than that in CD-fed mice and vice versa. Figure S6. Shared transcriptomic features of brain myeloid cells in diet-fed, aged B6 and B6.APP/PS1 mice. (A) PCA plot showing the first and second component of transcriptional expression profiles between CD11b+C45lo and CD11b+CD45hi cells in aged WT (20 months) mice and APP/PS1 (6 months) mice. (B) Top 15 shared canonical pathways revealed by IPA based on DE genes between CD11b+C45lo and CD11b+CD45hi cells in mice fed a CD or WD (12 months), aged WT mice (20 months) and APP/PS1 (6 months). Figure S7. Top genes enriched in CD11b+CD45hi cells reflected peripheral myeloid cell profiles in WD-fed mice. Normalized gene expression plot (reproduced from ImmGen datasets) showing relative gene expression values for 34 CD11b+C45lo cell-enriched DE genes (A) or 73 CD11b+CD45hi cell-enriched DE genes (B) across all immune cell types available on ImmGen RNA-seq datasets. Figure S8. The number of OPN+IBA1+ cells per animal in the brain. (A) Box plot showing the number of OPN+IBA1+ cells per animal (the sum of cell numbers on seven images) in 12-month CD or WD-fed WT mice. . (B) Box plot showing the number of OPN+IBA1+ cells per animal (the sum of cell numbers on seven images) in 12-month CD or WD-fed Ccr2-KO mice. . (PDF 7836 kb)Additional file 2:lo with CD11b+CD45hi cells in WD-fed mice. Pairwise comparison of transcriptomes between CD11b+CD45lo and CD11b+CD45hi cells in WD-fed mice. The positive FC value means the gene expression is larger in CD11b+CD45hi than in CD11b+CD45lo and vice versa. DE genes were defined as FDR < 0.05. (XLSX 1720 kb) Gene list comparing the transcriptomes of CD11b+CD45Additional file 3:lo with CD11b+CD45hi cells in CD-fed mice. Pairwise comparison of transcriptomes between CD11b+CD45lo and CD11b+CD45hi cells in CD-fed mice. The positive FC value means the gene expression is larger in CD11b+CD45hi than in CD11b+CD45lo and vice versa. (XLSX 1700 kb) Gene list comparing the transcriptomes of CD11b+CD45Additional file 4:hi from CD-fed mice and WD-fed mice. The positive FC value means the gene expression CD11b+CD45hi is larger in WD-fed mice than that in CD-fed mice and vice versa. DE genes were defined as FDR < 0.05. (XLSX 1730 kb) Gene list comparing the transcriptomes of CD11b+CD45Additional file 5:lo cell-related genes in WD-fed mice. The top DE genes enriched in CD11b+CD45hi cells were defined as those with expression levels above 100 cpm and at least two-fold higher compared to CD11b+CD45lo cells. (XLSX 14 kb) The top CD11b+CD45Additional file 6:hi cell-related genes in WD-fed mice. The top DE genes enriched in CD11b+CD45hi cells were defined as those with expression levels above 100 cpm and at least 10-fold higher compared to CD11b+CD45lo cells. (XLSX 20 kb) The top CD11b+CD45


Endoscopic ultrasonography (EUS)-guided drainage has become the first-line therapy for late peri-pancreatic fluid collection (PFC). Double pigtail plastic stents (DPPS) and lumen-apposing metal stents (LAMS) are commonly used for PFC drainage. Recently, a multi-institutional consensus on PFC drainage has recommended that LAMS should be the standard care for patients with walled-off necrosis (WON). However, given the poor quality of evidence, we aim to perform a large-scale randomized controlled trial to determine whether LAMS is superior to DPPS for WON drainage.The study is an open-label, prospective, parallel-group, superiority, multicenter randomized controlled trial. Two hundred and fifty-six patients with WON who will attend 18 tertiary hospitals in China will be randomly allocated to the LAMS or DPPS group before the procedure. The primary endpoint is the clinical success at one month after drainage (reduction in the size of WON to < 2 cm). Secondary endpoints include technical success, operation time, recurrence, adverse events, and secondary interventions.The LVPWON trial is designed to determine whether LAMS is effective, safe, and superior to DPPS for WON drainage.NCT03027895. Registered on 14 January 2017.ClinicalTrials.gov, The online version of this article (10.1186/s13063-018-2901-3) contains supplementary material, which is available to authorized users. Walled-off necrosis (WON) is a type of pancreatic fluid collection (PFC) that develops in the setting of acute or chronic pancreatitis, trauma, or pancreatic duct obstruction . WON, a In the past decade, the treatment of symptomatic WON has evolved from surgical to endoscopic necrosectomy. Endoscopic treatment has a reduced proinflammatory response compared to surgery for the treatment of WONs . It is aDouble pigtail plastic stents (DPPS) is the standard choice for pancreatic pseudocyst drainage with > 90% technical and clinical success rates . DPPS haHowever, the safety of LAMS is still controversial. Some studies have reported that LAMS is superior to DPPS in terms of overall treatment efficacy (90% vs 81%) and a significantly lower number of procedures (2.2 vs 3.6) . LAMS inThe LVPWON trial is an open-label, prospective, parallel-group, superiority, multi-center RCT designed to determine whether LAMS is effective and safe in the EUS-guided drainage of WON and superior to DPPS. Patients with WON admitted at 18 tertiary hospitals in China will be randomly allocated to the LAMS or DPPS group before the procedure. The study protocol has been approved by the Institutional Review Board and Ethics Committee of Shanghai Changhai Hospital. Recommendations for Interventional Trial (SPIRIT) Checklist have been provided  to the LAMS or DPPS group after obtaining signed informed consent  and the participant will be randomized to receive either LAMS Fig.  or DPPS The operation will be performed under mild sedation, monitored under anesthesia care or under general anesthesia, and prophylactic antibiotics will be used when appropriate. EUS-guided drainage will be performed by expert endosonographers after unified standard operation training. First, the position and size of the WON will be determined by endoscopic ultrasonography and the appropriate puncture point will be selected. Next, a 19G puncture needle will be inserted into the WON via the endoscope. Under fluoroscopy guidance, a 0.035 in. (1 in. = 0.025 m) yellow zebra guide wire will be inserted and coiled into the WON cavity. A Wilson COOK cystotome electric knife will be placed along the guide wire to cut the wall of stomach and the wall of the WON. Balloon expansion will be used if necessary. Subsequently, after drainage of the liquid content of the WON in to the stomach, LAMS or DPPSs would be placed to maintain the opening.Investigators will save a recording of the operation and note the procedure time. After EUS-guided drainage, patients will be sent back to the ward. Patients will be monitored for 3 h and 24 h serum amylase after the operation and prophylactic antibiotics will be administered postoperatively. Detailed data information can be found in the SPIRIT figure , it will be recorded as drainage failure. The secondary outcome is technical success, operation time, recurrence, AEs, and secondary interventions. The definitions of the primary and secondary outcomes are presented in Table The sample size calculation is based on a literature review and conference discussion regarding the clinical success rate of LAMS or DPPS drainage for WON. The LVPWON trial is a superiority trial in which the sample size calculation was based on the assumption that the incidence of the primary endpoint with LAMS and DPPS is 90% and 75%, respectively. Assuming a one-side alpha (type I error) of 0.025 and a power of 85% , we used the following formula to calculate sample size and found that 256 patients (128 per condition) would be necessary, including a possible dropout rate of 10%.pT represents expected success rate of the LAMS group, pC indicates expected success rate of the DPPS group, μ represents the quantile of standard normal distribution.In this formula, Patient characteristics will be recorded in a CRF. An electronic data collection system (electronic data capture [EDC]) will be used to complete the trial data collection. The EDC system has been rigorously tested to fully meet the requirements of the “Quality Management Practice for Medical Device Clinical Trials” and “Technical Guidelines for Clinical Trial Data Management Work.” Before the system is officially launched, training tests will be conducted on relevant users to ensure that the system meets the trial requirements. After the official launch, related personnel will receive the account number and password. The account is bound to the user’s role and permissions: the user must properly maintain the account information, not inform others about the account information, and exercise appropriate rights for others. Clinical auditors will monitor the clinical trial center’s work at least once a month. This trial has an interim analysis once half of the required number of the patients have been enrolled, during which the data will be analyzed. If a serious AE is identified, the study will be terminated immediately.t-test. For non-normally distributed continuous data, Wilcoxon rank sum test will be used for comparison between groups. This is a multi-center study. The test conditions of each center are not identical  and the outcomes may be different. For the primary outcome, comparison between groups will be performed using the CMH (Cochran–Mantel–Haensel) χ2 analysis to adjust for the central effect. The differences between the success rates of the groups and their 95% confidence intervals (CI) will also be estimated. AEs will be described by the number and incidence of AEs; the likelihood ratios between groups will be compared using the χ2 test or Fisher’s exact probability test. Major investigators will have access to the final trial dataset and the statistician involved will be blinded to the treatment assignments. Statistical analysis will be performed with two-sided tests at a level of significance of 0.05 (except where specified). Data analysis will be conducted using SAS®9.4 statistical software.Categorical data will be described using frequency and composition ratios. Continuous data will be described using mean, standard deviation, maximum, minimum, and median, as well as 25th and 75th quantiles. Baseline demographic analysis based on the descriptive analysis, the likelihood ratio χ2 test will be used for the comparison between the categorical data groups. Fisher’s exact probability method will be used when theoretical frequency of cells exceeding 25% is < 5. Normally distributed continuous datasets will be compared using a group The LVPWON trial has been designed to answer the question of whether LAMS is superior to DPPS for EUS-guided drainage of WON with regards to the clinical success rate and the incidence of AEs. We also want to prospectively investigate the risk factors for success of treatment and complications of EUS-guided drainage, which will provide a reference for the clinical treatment of WON.EUS-guided drainage has been maturely applied in late PFC with well-defined inflammatory walls , 16–18. Due to their small diameter, several DPPSs are needed to achieve adequate drainage of a WON. Metal stents that have a larger diameter are theoretically considered superior to DPPS as they allow for the possibility of WON debridement. A previous study directly comparing DPPS and LAMS demonstrated that the latter had a success rate of 90%, which was higher than the former, and a significantly fewer number of procedures were required with LAMS for WON resolution . In termHowever, with the increasing use of LAMS for WON drainage, LAMS-related bleeding has been more frequently reported –15. In cRecently, a consensus guideline formulated by the Asian EUS group RAND/UCLA expert panel raised the issue of stent selection . There aThe LVPWON trial was initiated by Changhai Hospital, which is a National Clinical Research Center for Digestive Diseases. This is the largest prospective, open-label, parallel-group, superiority, multi-center RCT to address the appropriate selection of the stent for WON drainage. The trial includes 18 Chinese tertiary hospitals. Despite discrepancies in diagnostic and operational skill levels at different centers, as the research initiator, Changhai Hospital will provide technical training. Only experienced endosonographers will perform drainage procedures. The trial will also adopt “centralized readings” to reduce the heterogeneity between centers. Given the differences in the type of stent that will be placed, endoscopists and patients will not be blinded to the treatment allocation. However, the outcomes of the trial are unlikely to be affected by the patient’s psychological factors.In conclusion, the major focus of the LVPWON trial is to prospectively compare the efficacy and safety of LAMS and DPPS in EUS-guided drainage of WON and to identify the risk factors associated with LAMS-related complications, thus further benefiting WON patients treated with LAMS.This multicenter RCT is expected to begin enrolling patients on 30 September 2018. Protocol version number: V 2.0, 2017-10-10. The approximate date when recruitment will be completed is 30 September 2019.Additional file 1:SPIRIT checklist. (DOC 118 kb)