Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN)X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN)X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN)X motif containing sRNAs with target mRNAs on a “speed-dating” fashion, thereby supporting their regulatory function.The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq During stress and environmental changes, sRNAs orchestrate a complex and dynamic response, allowing the bacteria to rapidly adapt to new conditions. Thus, they play critical roles in the lifestyle switching of bacteria that are able to inhabit variable environments, as well as during infection and disease10.Non-coding RNAs play key roles in regulating gene expression in all domains of life. In bacteria, sRNAs control almost every aspect of bacterial physiology including metabolism, quorum sensing, and virulence13, regulating the translation of all of these with specificity and precision. Target recognition was shown to be generally initiated at short complementary ‘seed’ regions in the two RNAs15 and for most sRNA-mRNA pairs it is critically dependent on the RNA-chaperoning protein Hfq19. Consequently, bacteria with mutations in the hfq gene show reduced virulence and reduced adaptation potential18.Bacterial sRNAs are ~50–300 nucleotides long, and act by modulating the stability and translation of diverse mRNAs. They are expressed mostly independently from their mRNA targets and many of them can simultaneously act on several different mRNAs22. In sRNAs, Hfq was shown to preferentially bind 3′ to seed regions, whereas it interacts 5′ to sRNA-target regions in mRNAs23. Furthermore, Rho-independent terminators display a universal recognition motif for Hfq23. The Hfq hexamer (Hfq6) has three distinct RNA binding sites referred to as ‘proximal’, ‘distal’ and ‘lateral’  tracts present at the 3′ termini of most sRNAs33. At these poly(U) tails, Hfq also directly interacts with the free 3′-OH group, which helps trigger a constricted RNA conformation required for efficient sRNA binding and recognition32. The ‘distal’ site is located on the opposite face of the ring and has high affinity to A-rich sequences, which are commonly found in the 5′ untranslated regions (UTR) of mRNAs34. Crystal structures revealed that the distal site of each Hfq subunit can accommodate a triplet of RNA nucleotides (ARN or AAN) with differing specificities: the A-site binds specifically adenines, the R-site can accommodate both adenine and guanine with preference for A, while the third base points away from Hfq towards the solvent and can be any nucleotide (N)35. Six such sites come together in the hexamer to form a circular binding site accommodating an 18nt long A-rich RNA segment36. In agreement, genomic SELEX experiments revealed a specific enrichment of A-rich sequences among Hfq-bound RNAs and in vivo UV-crosslinking demonstrated that Hfq specifically binds to repeated ARN triplets (referred to as (ARN)X motifs) in the 5′-UTR of mRNAs37.The Hfq protein is a homo-hexameric ring-shaped RNA-binding protein of the Sm/LSm family that has several distinct ways of interacting with RNA39. A remarkable example is demonstrated in the crystal structure of the Hfq-RydC complex. Here, the 3′ U-rich tail of the sRNA binds to the proximal face of Hfq, while the 5′ end binds to the lateral surface. In addition, the external part of the rim as well as Hfq’s intrinsically disordered C-terminal tail are involved in contacts with RydC27. Furthermore, whereas most sRNAs primarily bind to Hfq’s proximal site, some also contain (ARN)X-like motifs, which contribute to their stability and can bind to the distal site of Hfq43. One prominent example is the oxidative stress response regulator, OxyS that is induced upon oxidative stress and acts on multiple mRNAs to fine-tune the expression of various stress response pathways45. In addition to proximal site binding regions43, OxyS contains an extended (ARN)X motif (positions 59–86) that is essential for its regulatory function, and biochemical studies and crystal structures have shown that it binds to Hfq’s distal site46.However, Hfq-RNA binding is not restricted to a single binding site. Recent reports indicate that Hfq-RNA interactions can simultaneously involve multiple sites on the RNA and/or the protein30. Moreover, the repetitive binding surfaces of the Hfq hexamer can accommodate multiple RNA molecules on the same surface, which was proposed to enable cycling of different RNA substrates on the ring and facilitate RNA release and turnover48. RNA turnover is further supported by Hfq’s C-terminal tail that helps displace RNA duplexes from the core binding sites49. Finally, Hfq can also interact with various proteins involved in RNA metabolism and translation, which help to mediate its function51. It appears that Hfq uses different mechanisms to catalyse annealing depending on the exact sRNA-mRNA pair, and the variety of the documented, partially complementary, mechanistic pathways enables this global ribo-regulator to act on many sRNA substrates and mRNA targets rapidly and accurately in the crowded milieu of the cell54. Nevertheless, the exact mechanisms of pairing remain incompletely understood for many sRNA-mRNA target pairs.To catalyse the annealing of diverse sets of RNA pairs, Hfq has been shown to employ a variety of mechanistic strategies X motif, that are flipped out by Hfq. Consistent with the structure, biochemical data with RNA probes that lack the base at the N-site and a systematic computational survey support the notion that base stacking of the N-site bases can help mediate RNA-RNA interaction between Hfq-bound (ARN)X motif-containing RNA molecules. We hypothesize that Hfq co-opts the N-site bases to initiate low-affinity interactions between RNA substrates so as to facilitate their partner search, adding yet another tool to the toolbox of this versatile RNA chaperone.Here, we present a crystal structure of 56. These data revealed how Hfq recognizes various RNA molecules and suggested mechanistic models for their annealing, however the structural basis of Hfq-mediated RNA-RNA interaction remains incompletely understood. Here, we present the crystal structure of an Hfq6-A18 RNA complex at 2.5 Å resolution  with A30 RNA and poly(A)-polymerase 1, but they contain only Hfq72 and an 18 nucleotide long poly(A) RNA segment. Hfq72, that lacks most of the intrinsically disordered C-terminal tail57, was used to facilitate crystallization. Recent data showed that deletion of the highly variable C-terminus58 has no effect on the affinity or annealing of A18-containing RNAs and indicate that its main function is to promote RNA turnover49. In the resulting crystal structure, the protein itself looks very similar to previously published structures55 and only small changes can be observed  with a neighbouring Hfq726:A18 complex resulting in a (Hfq726:A18)2 dimer  structures, the N-site adenines are tilted only slightly - approximately by 15 degrees - towards the surface of Hfq  in the sugar-phosphate backbone of the A18 chain of the partner Hfq726:A18 ring . This revealed a shift in the sedimentation coefficient (s = 5.1S) compared to the RNA-free Hfq72 control (s = 3.1S), indicating that larger molecular assemblies have formed. Notably, the shift was significantly larger than expected for a simple monomeric Hfq726:A20 complex  . These results indicate that Hfq726:A20 complexes can form dimeric assemblies. The reduced s-value probably indicates a dynamic equilibrium with the monomeric species. Such dynamic oligomerization equilibrium would result in average sedimentation coefficients that are between the predicted s-values of monomers and dimers and increase with the abundance of the larger assemblies as concentration increases60.To explore if the base stacking mediated dimerization observed in the crystal structure also occurs in solution, we analysed the oligomeric state of Hfq72-Are) Fig.  and its 20 RNA derivative, which contained an intact sugar-phosphate backbone, but every third nucleotide  was substituted with an abasic nucleotide (‘AA0’). This ‘AA0’ RNA was able to bind to Hfq72 equally well as A20 , and no shift to larger assemblies could be observed  are consistent with previously reported Hfq-poly(A) binding constants, as well as with the single binding constant measured for the ‘AA0’ RNA (KD-ITC = 40 nM)35. In contrast, the second binding has lower affinity (KD2-ITC = KD2-FA = 2.2 µM) and is only observed with A20 but not with ‘AA0’. This implies that the first high-affinity association event corresponds to primary Hfq-RNA binding, while the second A20-specific moderate-affinity event may represent Hfq-A20 dimerization. The moderate dimerization affinity observed with the Hfq726:A20 complex is also consistent with our size-exclusion data where the micro-molar affinity dimers cannot be observed  band was greatly reduced in the Hfq102R16A,R17A-‘AA0’ complexes  with full length Hfq (Hfq102). To avoid protein aggregation in EMSA, we used the Hfq102xes Fig. .20 variants, where every N-site base was replaced with G, C or U . Since base stacking can occur with any base, we predicted that dimers can form with diverse RNA sequences, but their affinity might differ depending on the base stacking efficiencies of different bases63. Consistently, we observed significant amount of supershift with ‘AAG’ that contains strongly stacking purine bases at the N-sites, but detected smaller amount of larger assemblies with pyrimidine bases as in ‘AAC’ and ‘AAU’ X motifs that are essential for Hfq-binding and RNA pairing in vivo65. Curiously, OxyS and fhlA share little sequence complementarity; two short (7–9nt long) complementary seed regions can be found at the tips of stable stem-loop structures in both RNAs that were proposed to interact via a “kissing complex”66, but the mechanism of OxyS-fhlA pairing remains incompletely understood.Our structural and biochemical data imply that Hfq can mediate RNA-RNA interactions via base stacking between A-rich RNA sequences. To test if this interaction can occur with physiological sRNAs and target mRNAs, we selected the prominent sRNA-mRNA pair, OxyS and X motifs from OxyS (positions 57–86) and fhlA . To test the importance of the flipped-out N-site bases, we also created an OxyS variant, Oxy0 where the predicted N-site nucleotides were replaced with abasic linkages (as for ‘AA0’ above). Since the sequence of the OxyS (ARN)X motif is complex and its exact binding mode on Hfq is difficult to predict from the available crystal structures with short OxyS fragments46, we manually inspected ARN triplets in the sequence to identify the N-site bases. We focused on the previously annotated ARN region42, searched for two purine bases followed by a variable nucleotide and removed the base at this putative N position. The oligonucleotides were differentially labelled with fluorescent probes , complexed with full length Hfq (Hfq102) alone or in combinations, and their oligomeric states were analysed by AUC  and did not pair. Remarkably, the Hfq102-fhlA and Hfq102-OxyS complexes also revealed simple monomeric Hfq1026:RNA complex species (s = 4.5S for both) and did not self-dimerize. This was surprising because Hfq726:A20 complexes readily dimerized by themselves in our previous experiments. In contrast, an additional faster sedimenting peak appeared for the ternary Hfq102-fhlA-OxyS complex (s = 5.9S), indicating the formation of larger molecular assemblies. Importantly, the Hfq102-fhlA-Oxy0 complex did not dimerize (s = 4.4S), again highlighting the importance of the N-site bases.We synthesized oligonucleotides containing the (ARN)s Cy5, Cy, complexAUC Fig. . As expeX motifs seen in our crystal structure can also occur in OxyS and fhlA.These results indicate that the Hfq-mediated interactions between the N-site bases of (ARN)X motifs in Hfq-regulated sRNAs and mRNAs. An increasing body of evidence already recognizes the importance of these motifs for riboregulation in bacteria. For example, it was demonstrated that Hfq binding is specifically enriched at (ARN)X motifs in the 5′ UTR of mRNAs in vivo37 and these motifs are essential for their regulation68. Several sRNAs were also shown to bind Hfq at (ARN)X motifs that contribute to their stability43. To explore (ARN)X motifs in sRNAs more broadly, we screened 67 experimentally confirmed sRNAs from E. coli69. Since existing annotations of (ARN)X containing regions were incomplete and the constraints defining an (ARN)X motif were not clear, we constructed an iterative bioinformatics pipeline that consists of explorative pattern searches with various pattern definitions, secondary structure inspection, and conservation analysis s Figure . Based oX motif-containing sRNAs, such as OxyS42 and MicM 40, as well as several additional instances, where the role of (ARN)X motifs has not yet been implicated. In some sRNAs, we found multiple (two or four) non-adjacent (ARN)X motifs. Interestingly, most identified (ARN)X motifs contained at least 5 ARN triplets, even though our pattern searches required only 4 triplets. Analysing sequence conservation within the ARN triplets, also revealed a preference for A in the R position X motifs in 42 out of the 67 sRNAs tested, which include several well-studied sRNAs 55 that were shown to bind to the proximal and rim sites of Hfq and anneal with mRNAs bound to the distal site of the same Hfq hexamer21. The presence of conserved (ARN)X motifs in a distinct subset of sRNAs suggests that these sequence elements may have specific roles in the function of these sRNAs and would merit further investigation.The matching sRNAs include many known Hfq interactors and several previously documented examples of (ARN)X motifs in the above identified set of (ARN)X motif-containing sRNAs, we analysed their secondary structure and the arrangement of known functional modules (e.g. mRNA complementary seed regions) within their sequences. From the 25 sRNAs with predicted (ARN)X motifs, we selected 17 that - for simplicity - contain one single (ARN)X motif (Table X motif relative to secondary structure elements. This showed that the (ARN)X motifs are often flanked by predicted secondary structure elements such as stem loops on one side or both.To further explore the role of (ARN)X motif in a spatial arrangement that is so conserved that the different sRNA folds can be directly superimposed. To analyse these five examples (including OxyS) further, we prepared multiple sequence alignments for the sRNAs from related bacteria, which revealed high conservation of the (ARN)X motifs, further supporting their functional importance 42. Next, we mapped functionally relevant sequence regions on these five selected sRNAs. We found that the (ARN)X motif is positioned 20–40 nts away from the 3′ poly(U) tail in all cases , complementary regions localized to stem loops flanking the two sides of the (ARN)X motif  closely resemble OxyS in their overall structure Fig. . They alnce Figs –S9. In tes Table . This diAs Table . We seartif Figs –S9.X motif-containing sRNAs share a common functional architecture, with a conserved localization of (ARN)X motifs and seed regions within an overall similar structural arrangement.Taken together, these analyses reveal that unrelated (ARN)fhlA was previously shown to have a modular architecture, where several short seed regions flank a bipartite (ARN)X motif involved in Hfq binding66. Based on our observation that several (ARN)X motif-containing sRNAs share a common architecture, we wondered if the mRNA targets of these RNAs also share a similar architecture. To check this, we visually located (ARN)X motif containing regions in the respective target mRNAs . In contrast to the one complete (ARN)X motif in (ARN)X motif-containing sRNAs, generally two shorter (ARN)X regions were found in the 5′ UTRs of the target mRNAs 37. In several cases , the predicted (ARN)X regions also overlapped with experimentally identified Hfq-binding sites65. The spacing between the two (ARN)X regions was ~60 nts in all cases and often contained stem loops or other folded elements, suggesting that these regions may constitute two parts of a bipartite (ARN)X motif, which could come together in space upon folding of the mRNA (data not shown)65. In addition, common features extended to sRNA complementary regions: multiple short seed regions were found in the proximity of (ARN)X motifs, either upstream of the first (ARN)X region, between the two (ARN)X regions, or downstream of the second (ARN)X region at the beginning of the coding sequence . Of note, rpoS was a clear outlier in our analysis: it contains a long complementary region with OxyS, an (ARN)X region far upstream in the 5′UTR, and a quite different secondary structure (data not shown). However, we observed marked structural similarities in the other mRNA targets of our selected (ARN)X motif-containing sRNAs X motifs provide essential Hfq binding sites and interact with Hfq’s distal site71. Hfq binding involves up to six ARN triplets and occurs on a circular fashion36, as seen in the crystal structures . Interestingly, every third base at the N-site is excluded from Hfq binding and points towards the solvent. In this study, we present a crystal structure of an E. coli Hfq-A18 RNA complex, which reveals an additional structural feature of (ARN)X motifs. It shows that, when bound to Hfq, these motifs can create base-stacking interactions between two RNA molecules 6:RNA complexes that have been observed previously by others43 could not be excluded, our AUC experiments strongly suggest a 2:2 complex. 2:1 Hfq6:RNA complexes are also thought to have low abundance and little relevance at physiological Hfq-RNA ratios72. Furthermore, we show that the assemblies strongly depend on the presence of flipped-out N-site bases in the RNA and their stability scales with the base stacking affinity of these bases. This agrees well with the base-stacking mediated 2:2 assembly in our crystal structure, but is difficult to recapitulate with 2:1 Hfq6:RNA complexes as absence of the N-site bases would not be expected to influence tandem binding of two Hfq hexamers on one RNA , as neither of the two RNAs formed higher order complexes when binding to Hfq individually.Using abasic RNA probes that specifically lack the N-site bases, we provide several lines of biophysical evidence that support the occurrence of the structurally observed supramolecular interaction in solution and confirm its dependence on the N-site bases. Although in EMSA, ITC and FA experiments the exact composition of the higher order complexes could not be directly determined and the formation of e.g. 2:1 HfqE. coli sRNAs revealed that (ARN)X motifs are present in many sRNAs, where they are highly conserved and in some cases co-occur with a specific arrangement of characteristic sequence and secondary structure elements. These observations indicate that (ARN)X motifs can play a role not only in mRNAs, but also in some (ARN)X motif-containing sRNAs. Based on our structural data, we hypothesise that (ARN)X motif-containing sRNAs may bind to Hfq’s distal site and interact with mRNAs that are bound to a separate Hfq hexamer using interlocking base stacking of the flipped-out N-site bases as seen in our crystal structure  it has only micro-molar affinity enabling a rapid turnover73, (v) it requires additional sequence-specific interactions to create a stable pair for a proper gene regulatory response. This putative mechanism may act in concert with other known annealing pathways, supporting or specifying the function of specific (ARN)X motif containing sRNA. Due to its specific physicochemical properties, ARN base stacking can be particularly beneficial for sRNAs that act on multiple target mRNAs, with most of which they share only little sequence complementarity. Here, base stacking can enable interaction with many potential target RNAs and allow them to find even short complementary matches. In addition, the (ARN)X interactions can also help increase the affinity of these multifaceted sRNAs towards one or another of their targets and thus contribute to their specificity. Consistent with this idea, we find several multi-target sRNA in our list of (ARN)X motif-containing sRNAs  it occurs between appropriately pre-organized protein-RNA assemblies, (ii) it has low sequence specificity and can bring together a variety of RNAs, (iii) it positions the two RNA molecules in antiparallel orientation, as required for proper pairingX motif-containing sRNA-mRNA pair is the central oxidative stress response regulator OxyS and its prominent target fhlA. Various studies on OxyS-fhlA suggested a so-called ‘kissing loop’ annealing model, where short seed regions in stem loops flanking the Hfq binding sites interact with complementary segments in the partner RNA66. Now, our structural and biochemical results indicate that Hfq-bound OxyS and fhlA can interact via their (ARN)X motifs, perhaps initiating and/or facilitating the full RNA pairing. These results are consistent with previous studies showing that both OxyS and fhlA interact with the distal site of Hfq65 and can help elaborate their non-canonical mechanism of pairing.One example of a (ARN)X-containing sRNA-mRNA pairs contain similar architectural features including stem loops and seed sequences flanking the (ARN)X motifs. It will be interesting to test if these pairs can also associate via their (ARN)X motifs and follow annealing mechanisms that are similar to the OxyS-fhlA pair.Interestingly, our bioinformatics analysis also revealed that several other (ARN)X motifs contribute to Hfq binding and riboregulation in several sRNAs 75, whereas these motifs are absent and counterproductive to function if introduced in others 27. In fact, a recent study by Schu et al.21 showed that the stability and function of a number of sRNAs is compromised in Hfq distal face mutants and suggested that (ARN)X motifs define the stability, target choice and functional role of a specific class of sRNAs (defined as Class II). In agreement with these studies, we find (ARN)X motif matches in prominent Class II sRNAs (such as MicM and MgrR). Furthermore, Schu et al. also proposed that Class II sRNAs interact with mRNA targets bound to the rim of Hfq, in contrast to sRNAs without (ARN)X motifs (Class I) that bind to the proximal and rim sites and anneal with mRNA targets bound to Hfq’s distal site. As the rim site of Hfq is smaller and weaker than the other binding sites and many Class II mRNA targets also have (ARN)X motifs21, the ARN-ARN interactions observed here may help initiate or stabilize sRNA-mRNA contacts, thereby contributing to Class II sRNA function. In addition, several sRNAs, including OxyS, showed intermediate behaviours in the studies of Schu et al.21, suggesting that the mechanistic diversity of sRNA-mRNA pairing may be even greater. Our ARN pattern search protocol can help identify (ARN)X motifs in bacterial RNAs more broadly, thus helping to classify sRNAs and derive testable hypotheses for their functional and mechanistic features. In accord, our ARN-containing RNA set also contains sRNAs that have not yet been implicated to interact with Hfq, and it will be interesting to test if these RNAs may rely on Hfq under specific cellular conditions.Our results are consistent with the work of others showing that (ARN)X motifs in these species. Hfq proteins in Gram - positive bacteria bind RNA differently at their distal site, relying on a bipartite RNA-binding motif with no flipped-out bases; thus, our model is probably not applicable to these species. However, to investigate the exact impact of Hfq-mediated ARN base stacking in vivo and its species-specific features, further studies will be required.We speculate that the proposed RNA interaction model may be relevant in different Gram - negative bacteria, as the binding mode of poly(A) RNA is shared and our bioinformatics analysis revealed conservation of (ARN)78 and has accessory roles in organizing the tertiary fold. Now, we show that base stacking is not a sole property of complex folded RNAs, but it can also occur between two separate single-stranded RNAs if supported by the RNA-chaperone protein Hfq.One of our most surprising results is that Hfq can use base stacking to mediate RNA-RNA interactions. Base stacking is prominent in DNA, where it provides a major force stabilizing the structure of the double helix. In RNA, it was observed in structured tRNAs, rRNAs, ribozymes and in the ribosomefhlA complexes can self-pair, only their ternary complex forms base stacked Hfq6:RNA dimers. However, elucidating the exact role of base stacking in sRNA-mRNA pairing and the principles of their specificity will require substantial further analysis.The putative role of N-site base stacking in sRNA-mRNA interactions naturally raises the question if the identity of these bases matters for sRNA-mRNA pairing. In other words, are all bases and base combinations at the N-site able to interact equally well or does the identity of these bases convey a hidden code? If such a hidden code exists, this could contribute to specificity of sRNA-mRNA pairing and help ensure the selectivity of gene regulation. Our observations that poly(A) and ‘AAG’ containing RNAs interact more strongly than ‘AAC’ or ‘AAU’ sequences support this hidden code idea. Such preference for the pyrimidine bases can be explained by their advantageous base stacking and hydrogen bonding properties that help keep the RNA-mediated dimeric complex together. Consistently, we find that neither Hfq-OxyS nor Hfq-30 and A20 contain 30 and 20 consecutive adenine nucleotides, respectively. The ‘AA0’ RNA had the following sequence: (AA0)6AA, where 0 denotes an abasic nucleotide. Similarly, ‘AAC’, ‘AAU’ and ‘AAG’ sequences were (AAC)6AA, (AAU)6AA and (AAG)6AA, respectively. The oligonucleotide representing the ARN motifs of OxyS was derived from Gottesman et al.64 and comprises nucleotides 57 to 86 of full length OxyS, giving rise to the sequence: 5′-UCAACUCGAAUAACUAAAGCCAACGUGAAC-3′. In Oxy0, presumed N-site bases were exchanged for abasic nucleotides, denoted by 0: 5′-UCAA0UC0AA0AA0UAA0GCCAA0GU0AA-3′. The fhlA ARN segment oligonucleotide was constructed based on Salim et al.65 and comprises the two (ARN)X regions (nucleotides −78 to −65 and −14 to +5) directly fused to each other, giving rise to the sequence: 5′-CUAAUAAAAUUCUACCUAGAAGAACAAAAUGUC-3′. Residues −64 to −13 were replaced by a CC-dinucleotide, G at position −11 was replaced for A. For analytical ultracentrifugation, OxyS, Oxy0 and fhlA were synthesized with Cy5- and Cy3 fluorescence labels at their 3′-end, respectively. A modified A20 RNA, with an ATTO488-dye at the 3′-end was used for fluorescence anisotropy measurements.All RNA oligonucleotides were synthesized by Integrated DNA Technologies . AEscherichia coli Hfq72 (containing amino acids 1–72), Hfq102 , Hfq102R16A,R17A , and poly(A)-polymerase 1  were cloned into pETM28-SUMO vector and the 6xHis-SUMO-tagged proteins were expressed in E. coli BL21(DE3) cells in TB medium at 37 °C for 20 h  or 4 h (the PAP-1 construct). The cell lysate was applied to Ni-Sepharose column  in 0.1 M Hepes/NaOH pH 8.0, 0.5 M NaCl, 0.005 M TCEP. To remove nucleic acid contamination, the proteins were washed with 1 M LiCl on the column before eluting with imidazole. The eluate was then incubated with SenP2 protease (1:100) for 18 h at 4 °C and the cleaved SUMO-tag was removed via a second Ni purification. Proteins were further purified by size exclusion chromatography on a Superdex 200 column , concentrated to 10 mg/ml, and stored in 0.05 M Hepes/NaOH pH 8.0, 0.5 M NaCl at −80 °C until further use. For poly(A)-polymerase 1, a Heparin-Sepharose purification was included  after SenP2 cleavage to better remove the cleaved SUMO-tag and nucleic acid contaminations.DNA encoding 30 RNA and PAP-1 in a 1:1.2:1 molar ratio in HS-buffer  and dialyzing the solution against CX-buffer , and concentrated to 5 mg/ml. Crystals were grown at 20 °C in hanging drop vapor diffusion plates combining equal volumes of the complex solution with the well solution containing 0.1 M phosphate-citrate buffer pH 4.2, 27% PEG 1000, and 0.2 M LiSO4.For crystallization, complexes were formed by mixing Hfq72, A79. Even though the signal to noise ratio was still quite high at this resolution, the data was cut due to low completeness in the high resolution range (Table E. coli Hfq structure as a search model (PDB-ID:1HK9)81. The crystals belonged to space group R32 and the asymmetric unit contained two Hfq72 subunits bound to two adenine oligonucleotides, with crystallographic symmetry generating the biologically relevant homo-hexamers and the complete A18 chain. Model building in COOT82 was alternated with refinement in PHENIX83 until the R-values converged . Diffraction data was processed to 2.5 Å resolution with XDS2, 2% Glycerol) and run at 0.05 ml/min on a Superdex S200 10/300  pre-equilibrated in AUC buffer. UV-absorbance data were collected at 280 nm and 254 nm respectively.Hfq72-RNA complexes were prepared at 10 µM concentration in AUC-buffer  at a concentration of 33 µM. Sedimentation velocity experiments were performed at 20 °C in a Beckman OptimaXL-A centrifuge fitted with a four-hole AN-60 rotor and double-sector Epon centerpieces at 45 000 rpm. To unambiguously assign the composition of the complexes, absorbance data were collected at 280 nm for RNA-free Hfq, at 254 nm for samples containing unlabelled RNA or at 548 nm or 650 nm for fluorescent oligos. Data were analysed by the c(s) method using the Sedfit software package86. The observed s-values were compared with theoretical sedimentation coefficients calculated from our Hfq72-A20 crystal structure using HYDROPRO 5a88. The viscosity (1.087 mPa·s) and the density (1.015 g/ml) of the AUC-buffer were calculated using the program SEDNTERP V1.09 . The partial specific volumes were 0.530 ml/g for the RNA, 0.747 ml/g for the protein and 0.721 ml/g for the complex.Hfq72-RNA and Hfq102-RNA complexes were prepared in AUC-buffer . It was also previously shown that mutation of two arginine residues, R16 and R17 to alanine in E. coli Hfq reduces non-specific protein aggregation. Therefore, to ensure that potential higher order assemblies do not result from protein aggregation in electrophoretic mobility shift assays, we used a Hfq102R16A,R17A mutant62. These mutations at the Hfq’s rim site do not affect binding of A-rich RNA21. Hfq102R16A,R17A was expressed and purified as described above. ssRNA substrates were 5′-32P-labelled using T4 Polynucleotide Kinase (NEB) and [γ-32P]-ATP , and then purified on a Bio-Spin 6 column (Bio-Rad) following the manufacturers recommendations. Radiolabelled RNA was then incubated with varying amounts of Hfq102R16A,R17A in EMSA-buffer  Glycerol) for 30 min at 25 °C. Each 10 µl reaction contained 1 µl of 200 nM labelled ssRNA and an increasing excess of Hfq102R16A,R17A (1 μl of 20 nM–2000 nM). Complexes were separated via polyacrylamide gel electrophoresis using native 4–20% gradient gels in 1xTBE running buffer and results were imaged on a Typhoon FLA 9500 phosphoimager.Previous EMSA experiments indicated that full length Hfq102 migrates more effectively into native polyacrylamide gels than the truncated Hfq72 variant  at 25 °C in AUC buffer  after intensive dialysis of both Hfq72 and RNA overnight. The RNA was loaded in the sample cell at a concentration of 10 µM and was titrated with 150 µM protein solution from the injection syringe. The heat of dilution was measured in control titrations with buffer and subtracted from the binding data. Data were analysed using the Origin 7.0  software. After testing several binding models, the Hfq72-‘AA0’ binding data was best fit by a ‘one-set-of-binding-sites’ model, while the Hfq72-A20 ITC data corresponded best to a ‘two-set-of-binding-sites’ model.ITC experiments were carried out with an ITC20 (2 nM) in a final volume of 150 µl. Samples were prepared in triplicates in 96 - well plates. Anisotropy measurements were conducted in an Infinite M1000 plate reader (TECAN) at 25 °C. Excitation wavelength was 470 nm and the emitted light was recorded at 530 nm. Data were processed and fit according to a ‘two-set-of sites’-model in the GraphPad Prism software package.For fluorescence anisotropy, Hfq72 was dialyzed against AUC buffer. Starting from a concentration of 195 µM, Hfq72 was serially diluted by a factor of 0.66 in AUC buffer supplemented with BSA at a final concentration of 1 g/l. The resulting solutions were mixed with 3′-ATTO488-labelled AX motif were unknown, in order to survey the occurrence of (ARN)X motifs in sRNAs comprehensively we designed and implemented a custom algorithm that created distinct sets of ARN patterns with several different pattern definitions.Since the rules defining an (ARN)Our pattern definitions varied primarily in their degrees of ambiguity. The starting point was a conservative ARN pattern definition containing six consecutive ARN triplets directly following each other. Then, several properties of the pattern were defined in a more permissive manner, in a way that was consistent with known examples of ARN motif sequences. First, one or two non-functional (non-ARN) triplets were allowed within the pattern. Second, one or two single nucleotide gaps were introduced next to ARN triplets. Third, single gaps were allowed anywhere in the pattern. Forth, combinations of the above-described different ambiguity properties were also allowed.X motif and was used for pattern matching in sRNA sequences to produce distinct sets of results for each pattern definition.Using these pattern definitions, we then algorithmically created comprehensive pattern sets by permuting the combinations and positions of non-ARN elements (triplets or gaps) within the patterns. The resulting ensemble of pattern sets allowed us to cover the entire possible diversity that may occur in a potential (ARN)E. coli sRNA sequences were extracted from the Storz lab resource69 and homologous sRNA sequences in other bacteria were identified using BLASTN in NCBI and KEGG91. We did not attempt to sort the sRNA dataset into positive and negative interactors, as negatives under one condition may interact under different cell or experimental conditions, as was recently seen with McaS92. Sequences were aligned with Clustal W 2.093 and displayed in Jalview94. E. coli mRNA sequence data was retrieved from Genolist95 and analysed within a sequence window containing the 5′ UTR and the first 80 nts (+1–+80) of the coding sequence. In cases where 5′ UTR annotation was not available (e.g. for downstream genes in multigene operons), position −80 was used as a default starting point. These coordinate ranges were chosen to include all known functional regions of well-annotated mRNAs (such as fhlA) and exceed them by a safety margin.X regions as well as the requirements of strong Hfq binding at this region, and algorithmically generated a number of ARN pattern definitions containing different numbers of ARN triplets in combination with non-ARN triplets and distinct single gaps. With these, we performed pattern matching in sRNAs to get an overview of the distribution of different kinds of ARN motifs in these sequences. This survey identified commonly observed patterns, which were then further refined iteratively by analysing similarities in the identified candidate (ARN)X regions, conservation of their sequence, and their structural features. The resulting refined ARN pattern was then used to annotate (ARN)X motifs in 67 sRNAs. Overlapping matches were merged into a single (ARN)X region X motifs were protected from base-pairing. Plots were generated with Mfold and colour-coding was added manually. For identifying complementary regions between sRNAs and their target mRNAs, we defined the seed region in our search algorithm to minimally contain either seven consecutive base pairs or six consecutive base pairs flanked by a single gap followed by two base pairs.Secondary structure predictions were performed using KineFold, Mfold, and RNAfoldCoordinates and structure factors have been deposited in the Protein Data Bank under accession code 5NEW.Supplementary Information


Head and neck cancer (HNC) ranks as the 6th most common malignancy across the world. Metastasis is a hallmark of cancer, primarily contributing to the relapse and poor prognosis of HNC. Recently, long noncoding RNAs (lncRNAs), previously considered as non-functional, are increasingly appreciated by scholars to play crucial roles in mediating HNC metastasis. LncRNAs, which are located in the nucleus and cytoplasm, mainly exert their function via epigenetic modification, transcriptional control and translational regulation. As several lncRNAs are presently demonstrated to participate in HNC metastasis, we make a summary of the functions and mechanisms regarding these lncRNAs. As shown in the literature, most lncRNAs appear to promote the metastasis of HNC. Hence, we primarily discuss the lncRNAs involved in enhancing metastasis. Additionally, more studies are needed to understand those lncRNAs without clear mechanisms. Furthermore, we introduced the upstream regulator for the aberrant expression of lncRNAs in HNC. Finally, we concisely addressed future research prospects of lncRNAs, particularly the interplay between lncRNAs and tumor immunity as well as lncRNA-targeted therapeutic techniques, and we introduced clustered regularly interspaced short palindromic repeats (CRISPR)-Display as a possibly transformative tool to study lncRNAs. Although lncRNA research is still in the initial stage, it holds great promise to be applied as a prognosticator of HNC and a therapeutic target to inhibit HNC metastasis, which could significantly enhance the outcome of HNC patients. Head and neck cancer (HNC), which ranks sixth among the frequent malignant neoplasms worldwide, has an estimate of over 500,000 new cases detected annually , 2. The Metastasis is a common characteristic of cancer progression with multiple sequential steps, presenting as enhancement of the invasive ability of tumor cells and its spread to secondary sites of the body . EpithelLong noncoding RNAs (lncRNAs) are one subtype of RNA transcripts which contain more than 200 nucleotides, lacking in capability of encoding protein or exhibiting limited potential . LncRNA In the present paper, we review these aberrantly expressing lncRNAs in the mediation of EMT, migration, invasion and metastasis of HNC Table , and proIn general, lncRNAs perform their function based on their functional domains in the secondary or tertiary structure, and these mature structures originate from alternative splicing. Specifically, the domains facilitate the interaction of lncRNAs with chromatin, RNA and proteins; thus lncRNAs could function in HNC metastasis via chromatin remodeling, transcriptional control and post-transcriptional regulation , 22. In Several studies have suggested that most lncRNAs in the nucleus could function as a guide to lead chromatin modulating complexes into certain genomic loci and then initiate chromatin modification to activate or silence gene expression . AdditioRegarding the great number of cytoplasmic lncRNAs, they typically modulate gene expression by base pairing with specific genes, improving or attenuating the mRNA stability and acting as miRNA sponges; in addition, they could influence mRNA translation by binding to different elements. Moreover, cytoplasmic lncRNAs are also responsible for protein stability control , 27.Presently, various lncRNAs have been demonstrated to function positively in metastasis, as well as in the EMT, migration and invasion of HNC. Here, we discuss their possible roles and attempt to elucidate the mechanisms.vivo through the phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) pathway [MALAT1 was first uncovered as a poor prognosticator in NSCLC patients, whose overexpression could predict a higher risk of distant metastasis . MALAT1  pathway . Additio pathway . However pathway . In addi pathway , 34–39.Zhou and the colleagues, by employing siRNA to inhibit MALAT1 in HNC, indicated that the EMT, invasion and migration of HNC cells were attenuated possibly by inactivation of the β-catenin and nuclear factor-κB (NF-κB) pathways, which are potent regulators of EMT; furthermore, the in vivo study demonstrated the suppression of EMT markers, N-cadherin and Vimentin, in MALAT1 knockdown HNC tumors . MoreoveApart from HNSCC, MALAT1 also presents as a modulator of metastatic capability for other types of HNC in some in vitro studies. NPC is a disease more prevalent in southeastern Asia; despite the improvement in treatment modalities, distant metastasis remains a main culprit for its poor prognosis . Xie andHOTAIR is located antisense to HOXC mRNA . MechaniRecently, HOTAIR was implicated to undertake a crucial role in HNC metastasis. Wu et al., after initially validating the positive association between HOTAIR expression and the LNM of oral squamous cell carcinoma (OSCC) within clinical samples, conducted an in vitro study and determined the oncogenic function of HOTAIR in driving the invasion and migration of OSCC. Their further data suggested that HOTAIR contributed to EMT by decreasing E-cadherin, the speculated mechanism of which is that HOTAIR could regulate the binding of the enhancer of zeste homolog 2 (EZH2), as well as trimethylation of lysine 27 in histone 3 (H3K27me3) to the promoter region of E-cadherin, thus enhancing the metastatic ability . MoreoveUrothelial carcinoma-associated 1 (UCA1) comprises of three exons and encodes two transcripts . lncRNA Recently, a few studies have unveiled the function of UCA1 in HNC metastasis. As a lncRNA dysregulated in TSCC, UCA1 is closely related to its LNM, and its silencing markedly dampens cancer invasiveness. Further study demonstrated that UCA1 triggers Wnt/ β-catenin pathway activation and contributes to cancer metastasis . SimilarTaurine upregulated gene 1 (TUG1) is another frequently reported lncRNA . PreviouTo date, only one study has discussed TUG1 in HNC metastasis. Liang and colleagues, after confirming the positive correlation between TUG1 and LNM, showed that TUG1 might inhibit the invasion of OSCC along with the downregulation of β-catenin, while a stimulator of the Wnt/β-catenin pathway could reverse the repression effect of TUG1 . Thus, tActin filament associated protein 1 antisense RNA 1 (AFAP1-AS1), which is derived from the complementary chain of the Actin Filament Associated Protein 1 (AFAP1) gene, is presently implicated in modulating the metastasis of lung cancer, hepatocellular carcinoma, and notably, NPC –69. InitAfter investigating the lncRNAs profiles between PTC tissue and normal thyroid tissue, Li et al. identified an obviously elevated lncRNA in PTC, termed as HIT000218960, which is related to LNM of PTC. In addition, inhibition of HIT000218960 repressed migration and invasion of PTC possibly by downregulating high mobility group AT-hook 2 (HMGA2) mRNA level . HoweverENST00000470135 is a lncRNA identified to be markedly elevated in highly metastatic NPC cells, and its positive role in the LNM of NPC was further confirmed. Additionally, knocking down ENST00000470135 could remarkably repress the migration and invasion of NPC cells . As anotHOXA transcript at the distal tip (HOTTIP), as a lncRNA highly expressed in HNC, is associated with its distant metastasis and is responsible for its dismal prognosis . RelyingTo date, only a few lncRNAs have been highlighted to suppress HNC metastasis. Herein, we generally introduced them and investigated NKILA as a representative.Nuclear factor-κB interacting lncRNA (NKILA) is major inhibiting checkpoint for NF-κB activation in breast cancer . EncodedAccording to the previous finding about NKILA, the same group also reported its similar influence on TSCC metastasis. To begin with, NKILA was pervasively reduced in TSCC samples by contrast with that in normal tongue tissue; notably, it was negatively correlated with the LNM of TSCC. Cell invasion and migration studies further verified its negative role in TSCC. In theory, the mechanism of NKILA in downregulating the TSCC metastatic capacity is identical as to that in breast cancer. NKILA could initially bind to IκB, suppressing NF-κB activation and thereby blocking its downstream EMT phenotype. In agreement, animal investigations also showed that NKILA knockdown contributed to more lung metastasis of TSCC . OverallBased on microarray analysis, NONHSAT037832 was detected as a novel lncRNA remarkably downregulated in PTC, next implied to function as an inhibitor in the LNM of PTC . BesidesPresently, diverse lncRNAs are solely involved in driving the EMT, migration and invasion of HNC, whereas the in vivo functional roles await deep discovery.FOXCUT is a lncRNA encoded from the upstream area of the FOXC1 promoter. As indicated, FOXCUT functions together with FOXC1 as a lncRNA-mRNA pair in enhancing the migration of OSCC and NPC cells , 84. PreColon cancer-associated transcript1 (CCAT1) serves as a lncRNA involved in regulating the metastatic ability of various cancers via distinct or similar mechanisms, including colon cancer , cervicalncRNA H19 is the transcript product of the H19 gene . EmerginNuclear enriched abundant transcript 1 (NEAT1), as its name implies, is a lncRNA localized exclusively to paraspeckles, a sub-nuclear structure . PreviouLncRNA-Regulator of Reprogramming (LncRNA-ROR) is overexpressed in solid tumors including NPC. LncRNA-ROR deficiency contributes to the suppressed tumor invasion with reduced expression levels of the EMT marker . SimilarEmerging lncRNAs are also implicated in facilitating HNC metastasis. For example, long intergenic non-coding RNA 673 (LINC00673) was identified to be the most highly expressed lncRNA in TSCC based on the microarray analysis of two TSCC cohorts in the Gene Expression Omnibus dataset. Thereafter, it was validated in TSCC samples . AdditioApart from these lncRNAs that are positively related to the metastatic ability of HNC in vitro, a few lncRNAs are inversely correlated with its metastasis in vitro. Zou et al. carried out transcriptome sequencing to investigate dysregulated lncRNAs in HNC; as a result, they discovered two dramatically reduced lncRNAs, namely, KCTD6–3 and LCE5A-1. Moreover, transfection of both lncRNAs into HNC cells reduced its migration significantly because these two lncRNAs might lead to EMT alterations. Specifically, KCTD6–3 could decrease vimentin and twist, and LCE5A-1 might increase E-cadherin, while reducing vimentin . In anotThe tumor microenvironment (TME) of HNC is characterized by immunosuppression . CD8+ T Based on previous findings, there are three main mechanisms that are responsible for the aberrant expression of lncRNA in HNC, which are miRNAs, functional proteins such as RNA binding proteins (RBPs), NF-κB and TP53, and genetic changes such as genomic mutation and epigenetic alteration , 118–125In the past decade, lncRNAs, previously regarded as non-functional , have atIn this review, we introduced the lncRNAs implicated in modulating the metastatic potential of HNC and attempted to illuminate the mechanisms; meanwhile, the involvement of these lncRNAs in the metastasis of other common malignancies are also briefly summarized to facilitate their study in HNC. Additionally, the upstream regulation of lncRNA that underlies its abnormal expression in HNC was discussed. Based on the screening result of lncRNAs, most of these are suggested to promote the EMT, migration, invasion or metastasis of HNC, while a small proportion of these have opposite effects of inhibiting HNC metastasis in vitro and/or in vivo. In theory, these lncRNAs highly expressed in HNC tissue or cells, driving its metastasis, have the potential to be utilized as therapeutic targets and predictors of poor outcome. However, the prerequisite of its application is the illumination of mechanisms validated in vitro and in vivo. Moreover, those lncRNAs downregulated in HNC and exhibiting the capability of suppressing its metastasis also hold the promise to serve as prognostic predictors.In terms of clinical translation of lncRNAs, it still has a long way to go since there exist multiple unmet challenges for us to overcome. In order to realize the goal, we need to seek solutions for the listed issues: 1. exploring more lncRNAs affecting HNC metastasis; 2. investigating the detailed mechanisms of these lncRNAs; 3. figuring out the key lncRNAs pathways; 4. constructing a lncRNA interacting and regulating network since TME is complex and not dependent on a single lncRNA; 5. since HNC includes a range of cancers with various genetic background, individualized key lncRNAs and its involved pathways in a specific cancer might be studied; 6. intervening tumor metastasis-promoting lncRNAs to inhibit metastasis by targeting lncRNAs.To target lncRNAs in HNC, diverse techniques have been developed, namely, RNA interference (RNAi) , antisenRecently, a novel approach, namely, clustered regularly interspaced short palindromic repeats (CRISPR)-Display, has emerged as a potentially transformative tool to probe into the function and mechanisms of lncRNAs . SpecifiIn summary, lncRNAs contribute to HNC invasion and metastasis by distinct mechanisms based on their subcellular localization, as well as modulating or regulated by the cancer immune microenvironment. Emerging evidence has identified lncRNAs as a novel set of potential prognosticators and therapeutic targets. The characterization of an expanded repertoire of lncRNAs and their interaction with the cancer immune microenvironment are promising to further improve combinatorial treatment protocols for HNC.


It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection. To date, 32 species of viroid have been described, and broadly classified into two families: the Avsunviroidae and the Pospiviroidae2. The members of the family Avsunviroidae, whose type species is the Avocado sunblotch viroid (ASBVd), replicate in chloroplasts through a rolling-circle mechanism and exhibit self-cleavage activity. In contrast, members of the family Pospiviroidae, whose type species is the Potato spindle tuber viroid (PSTVd), replicate in the nucleus and contain five structural/functional domains: the Terminal Left (TL), Pathogenicity (P), Central (C), Variable (V) and Terminal Right (TR) domains4.Viroids are non-coding, highly structured, circular, single-stranded RNA (ssRNA) molecules of 246–401 nucleotides (nts) in length5. Either the double-stranded RNA (dsRNA) or the self-complementary RNA triggers RNA silencing through the activity of the DICER-like (DCL) RNase III-type ribonucleases, and results in the production of small interfering RNAs (siRNA). These small RNAs (sRNA) comprise the sequence-specific effectors that regulate gene expression6. Due to their high internal base pairing and RNA-RNA mode of replication, viroids are the elicitors of the host defense system via RNA silencing. As a result, viroid derived sRNA (vd-sRNA), which are 21- to 24-nts long, are often recovered from viroid infected host plants9. Previously, it has been shown that the (−) sRNAs derived from Peach latent mosaic viroid (PLMVd) can target the coding region of the heat shock protein 90 (cHS90) in peach plants (Prunus persica), and the (+) sRNA derived from the virulence modulating region (VMR) of PSTVd can down-regulate soluble inorganic pyrophosphatase gene in Nicotiana species and callose synthase gene in tomato13. However, to date there is no explanation for the higher level of down-regulation of endogenous mRNAs in the presence of fewer amounts of recovered target binding vd-sRNAs.RNA interference (RNAi) regulates gene expression both transcriptionally and post-transcriptionally14. For example, previously it has been used to demonstrate the viroid’s (i) structure in solution by high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)3, (ii) cell-to-cell trafficking through plasmodesmata and its systemic movement through phloem15, (iii) role of vd-sRNA in different host species such as tomato and Nicotiana species13, and, (iv) to develop viroid resistant plants17. It has also been used to monitor tomato gene regulation by microarray analysis and its effect on tomato miRNA expression18. In addition, the availability of the tomato genome allows for prediction of sRNA target genes19. In this study, the least expressed (+) and (−) PSTVd-sRNA were selected as a starting point in order to analyze the fate of their target mRNA during PSTVd infection in tomato plants using both computational and molecular biology approaches.PSTVd is a good model for studying viroid-host interactions, as it is a widely-studied viroidSolanum lycopersicum cv. Rutgers) infected with PSTVd variants  were analyzed so as to identify the regions producing the sRNA of 21 to 24-nt in length  strand of PSTVd was predicted to target the 3′ UTR of the chloride channel protein CLC-b-like mRNA  and 196 (0.26%) vd-sRNAs targeting the chloride channel protein CLC-b-like mRNA in PSTVd-I and PSTVd-RG1 infected plants were obtained, respectively. None of the sRNA derived from healthy plants targeted chloride channel protein CLC-b-like mRNA  strand of PSTVd as described above was predicted to bind the coding region of the 40S ribosomal protein S3a-like mRNA . This protein has also been shown to interact with the DNA damage-inducible transcript 327. Analysis of sRNA data obtained for tomato plants infected with PSTVd variants revealed only 4 (0.005%) and 8 (0.01%) vd-sRNAs, targeting the RPS3a-like mRNA from the (−) strands of PSTVd-I and PSTVd-RG1 in infected plants, respectively, but none from healthy plants.In order to elucidate the most susceptible regions of both the PSTVd (+) and (−) strands for RNA silencing, the vd-sRNA profiles obtained at 21 days post inoculation (dpi) from tomato plants  assay was performed on tomato plant. Approximately 400-nt long chloride channel protein CLC-b-like and RPS3a-like genes were expressed in the pTRV2 vector under the control of the 35S promoter. The resulting binary vectors were named pTRV2:CLC-b-like (targets the chloride channel protein CLC-b-like mRNA) and pTRV2:RPS3a (targets the RPS3a-like mRNA). pTRV1 and pTRV2, or their derivatives, were then transformed into an Agrobacterium tumefaciens, and were then used for agroinfiltration into tomato seedlings as previously described28. pTRV2:PDS, which is capable of targeting Phytoene desaturase mRNA (PDS), was used as a positive control, while a negative control was maintained by agro-infiltrating the plants with empty pTRV2 vector. At approximately 18-dpi, all the virus induced gene silenced (VIGSed) plants exhibited phenotypic alterations when they were compared to control plants  using gene specific primers. As presented in Fig. chloride channel protein CLC-b-like mRNA level at 14-dpi, PSTVd-I down-regulated it at 21-dpi. On the other hand, both PSTVd variants down-regulated the RPS3a-like mRNA level at 14 and 21-dpi  system was developed for the transient expression of the vd-sRNA derived from positions 191 to 211 (5′-UUCUUCGGGUGUCCUUCCUCG-3′) of the (+) strand and positions 69 to 49 (5′-GCCGCCUUCUUUUUUCUUUUC-3′) of the (−) strand of PSTVd in Nicotiana benthamiana (N. benthamiana) leaves. All of the amiRNA constructs and the green fluorescent protein (GFP) tagged target constructs were created on pBIN61 vector as previously12. If the amiRNA produced vd-sRNA binds to the target sequence, the fluorescence level of the GFP will decrease as this binding blocks the translational process and/or affects the stability of the target mRNA29. The amiRNA construct which targets the chloride channel protein CLC-b-like mRNA was named amiR:(+)191; that targeting the RPS3a-like mRNA was named amiR:(−)69. The GFP constructs with the chloride channel protein CLC-b-like mRNA target sites were named GFP:CLCb, which is targeted by amiR:(+)191, and GFP:RPS3a, which is targeted by amiR:(−)69. The GFP constructs were co-agroinfiltrated in N. benthamiana leaves along with either their respective targeting amiRNAs or with empty vector. All of the vd-sRNA:target complexes predicted to be formed by the interaction between the amiRNAs and the putative mRNA target sequences are illustrated in Fig. 7S signal recognition particle RNA (7SL) mRNA level for normalization. In order to verify the amount of GFP produced in the various vd-sRNA:amiRNA plus GFP:target combinations, an immunoblot assay was performed using anti-GFP antibody  mediated cleavage of both the 30. The same technique has been applied in order to validate the cleavage of predicted target mRNAs in viroid infected plants12. The RISC-mediated cleavage of predicted target sequence of RPS3a-like mRNA was verified by performing a 5′ RLM RACE experiment. However, vd-sRNA which was predicted to bind 3′ UTR of chloride channel protein CLC-b-like mRNA, leaves little space to design primers for 5′ RLM RACE. Hence, the RISC-mediated cleavage of predicted target sequence of chloride channel protein CLC-b-like mRNA was not evaluated by 5′ RLM RACE experiment. The cDNAs were produced in 5′ RLM RACE by ligating an RNA linker to the free 5′-phosphate end of an uncapped mRNA followed by reverse transcription using oligo(dT) primer. The ligation products were subsequently amplified by nested PCR, cloned and sequenced. To verify RISC-mediated cleavage at the predicted site, RLM-RACE experiment was performed on the 7-dpi RNA preparations using a gene-specific nested PCR. The resulting amplicons of expected size were cloned and sequenced. The cDNA clones obtained from the RPS3a-like transcripts at 7-dpi were aligned to detect the 5′ termini. Of the twenty obtained clones seventeen had a single cleavage site at 5′ termini that was identical to the predicted cleavage site  is the widely accepted technique to confirm the RISC-mediated cleavage of a target RNAite Fig. . No PCR 31. The PARE data obtained were then aligned against the whole lengths of both the chloride channel protein CLC-b-like and the RPS3a-like mRNAs, and the sequences that showed homologies were retrieved. Plotting of these retrieved sequences against the respective mRNAs after normalization revealed widespread cleavage throughout the lengths of the mRNAs  and (−) strands of PSTVd. If there was any sequence homology, the mRNA wide cleavage might have occurred due to vd-sRNA:mRNA interaction. None of the sequences showed any sequence similarities with PSTVd other than the predicted ones. Taken together, data presented here clearly shows that viroid infection indirectly induces the degradation of endogenous mRNA.In order to verify whether viroid infection induces widespread degradation of putative target mRNA, the same RNA samples (21-dpi) that were used for the analysis of the viroid accumulation levels were used for a Parallel analysis of RNA ends (PARE) experiment, as described previously with modificationsNAs Fig. . Visual 32. Due to this RNA-RNA base pairing, viroids produce both (+) and (−) strand specific sRNAs. Previously, we have shown that sRNA derived from virulence modulating region (VMR) of PSTVd induces can down-regulate callose synthase genes CalS11-like and CalS12-like by RNA silencing. The products of CalS11-like and CalS12-like are essential for the formation of callose during pathogen infection, hence plays important role in plant’s defense by preventing systemic spreading of the pathogen. By performing mutatgenic studies on PSTVd, we verified the role of VMR derived vd-sRNA on the viroid accumulation. The polymorphism in the vd-sRNA binding region of CalS11-like mRNA greatly affected viroid accumulation in different tomato cultivars12. In the present study, to understand the mechanism behind the observed higher level of down-regulation of endogenous mRNAs in the presence of fewer amounts of recovered target binding vd-sRNAs, the two least expressed vd-sRNA of both the PSTVd (+) and (−) strands were selected for an in silico analysis that searched for possible RNA:RNA interactions with the tomato transcriptome19. This search revealed the possibility of vd-sRNA:mRNA duplex formation with both the 3′ UTR of the chloride channel protein CLC-b-like mRNA and the coding region of the RPS3a mRNA , has gained importance as these techniques permit the evaluation of a the large number of samples in a single experiment and have diverse applications for the study of the RNA degradome38. All of these methods comprise a modified 5′ rapid amplification of cDNA ends (5′ RACE) followed by deep sequencing and bioinformatics analysis in order to determine the 3′ cleaved ends of the mRNAs. As these methods use global sampling, they allow comparison of the relative abundances of the mRNAs cleaved by vd-sRNA interaction, as well as of the non-specific degradation of mRNA caused by altered state of the diseased plant. In order to verify the vd-sRNA independent degradation of endogenous mRNAs, the RNA samples extracted at 21-dpi were subjected for PARE. Analysis of PARE data revealed very few cleavages at the predicted target site of RPS3a-like mRNA in comparison to the data obtained by 5′ RLM RACE. This inconsistency observed between the two different experiments might be due to: (i) samples from different time of infection were used (7-dpi samples were used for 5′ RLM RACE and 21-dpi samples were used for PARE); and, (ii) viroid infection induces non-specific degradation of host mRNA in the later stages of the infection. Plotting of the PARE data against the respective target mRNA revealed the presence of cleavage sites throughout the mRNA, indicating the possibility of the induction of the degradation of host mRNA by viroid infection  mRNA in viroid infected plants have been demonstrated previously39. More recently, transcriptome analyses of PSTVd infected tomato plants have revealed genome-wide alterations40. Our data is consistent with those previous observations, further supporting the viroid infection induced vd-sRNA independent degradation of host mRNAs.5′ RLM RACE is a widely accepted technique used in order to confirm the RISC-mediated cleavage of a target mRNAion Fig. . Instancchloride channel protein CLC-b-like and their RPS3a-like genes were suspected. A BLAST search of the vd-sRNA binding sites of the tomato chloride channel protein CLC-b-like mRNA against the potato genome revealed 95% sequence identity (GenBank Acc. No. XM_006357128). The sequence alignment of the vd-sRNA binding site of potato against positions 191 to 211 of the vd-sRNA revealed the presence of a mismatch located 9 nt from the 5′ end of the vd-sRNA . As a consequence, it forms an RNA:RNA duplex similar to that of the RPS3a-like mRNA of tomato . A BLAST analysis of the vd-sRNA binding site of the cyc07-like protein mRNA against the tomato genome did not reveal any homology. Additionally, the search for possible 5′ RACE ends in the tomato PARE library did not reveal any sequence identity, indicating the possibility of sequence diversity in the cyc07-like protein gene among tomato and potato cultivars. Nicotiana species are the other widely used laboratory plant for viroid research. A BLAST analysis of the vd-sRNA binding site of the chloride channel protein CLC-b-like mRNA against the Nicotiana genome did not reveal the presence of any sequence similarities, whereas the vd-sRNA binding site of the RPS3a-like mRNA of tomato possessed a 100% sequence similarity with Nicotiana sylvestris (N. sylvestris), suggesting a possible vd-sRNA:target RNA duplex formation in this plant  and the (−) strands of PSTVd have a potential to impact transcriptome, even though these vd-sRNAs are present in low numbers in infected plants. Additionally, the PARE data presented using different PSTVd variants, showed that vd-sRNAs are not the sole culprit in viroid pathogenicity. These findings increase our comprehension of the molecular basis of viroid etiology, a concept which to date has not been understood to its depth.12. Recombinant plasmids were linearized with the appropriate restriction enzymes, and were then transcribed using T3 RNA polymerase. The resulting products were analyzed by electrophoresis in 1.0% agarose gels containing 1X TAE buffer  and 1 mM EDTA).The head-to-tail dimeric constructs of both PSTVd-I (GenBank Acc. No. AY937179) and PSTVd-RG1 (GenBank Acc. No. U23058) were created in the pBluseScript KS (+) vector (Stratagene) using monomeric PSTVd-I, PSTVd-RG1 and PSTVd-M obtained from gBlock Gene Fragments  as described previouslyS. lycopersicum cv Rutgers; Livingston Seed Co, Ohio, USA) used for the viroid inoculation assays were grown in a growth chamber at 25 °C with 16 h of light and 8 h of darkness. After the development of the primary leaves, the plants were inoculated with the 1 µg of RNA transcripts of respective viroid variants. The tomato plants used for the VIGS assay were grown at 22 °C with 16 h of light and 8 h of darkness. After the development of the primary leaves, the cotyledons were agroinfiltrated with either pTRV1 and pTRV2, or with their derivatives as described previously28. The N. benthamiana plants used for the amiRNA experiments were grown at 23 °C with 16 h of light and 8 h of darkness and the agroinforltration was performed as described previously12.The tomato plants , and resulting plasmids were then digested with the restriction endonucleases XbaI and BamHI. The digestion products were ligated into the same restriction sites of the binary vector pTRV2. The resulting binary vectors were then transformed into the Agrobacterium tumefaciens strain GV3101, and were used for agro-infiltration as previously described28. Details of oligonucleotides used are available upon request.The pTRV1 and pTRV2 vectors for the VIGS assay were procured from Arabidopsis Biological Resource Centre . In order to produce the pTRV2 derivative targeting either the mirVanaTM miRNA isolation kit  and complementary DNA (cDNA) was synthesized as described previously12. For the evaluation of both the PSTVd titer and the gene expression levels, 10 ng of this cDNA was used in the qPCR experiments along with appropriate primer combinations. mRNAs are amplified using gene specific primers. Ubiquitin-conjugating enzyme (UBC), Transducing/WD40 repeat family protein and the ARF-like GTPase family protein (ASAR1) were used as house-keeping genes to normalize of the RT-qPCR results42. Every qPCR run included no-template primer-pair control samples, and these were consistently negative. All the oligonucleotides used are available upon request. The relative expression levels were calculated using the qBASE framework43. All of the RT-qPCR analyses were performed commercially at the Laboratoire de Génomique Fonctionnelle de l’Université de Sherbrooke .Total RNA from infected leaf samples was extracted using the 29. The target construct was prepared by ligating 21-nt long predicted target sequences to the 3′ UTR of the GFP gene in the pBIN61 vector. The resulting binary vectors were then transformed into the Agrobacterium tumefaciens strain C58C1 carrying the virulence plasmid pCH32, and were used for agro-infiltration as previously described12.The amiRNA for different vd-sRNA sequences were synthesized on an osa-MIR528 backbone using appropriate oligonucleotide pairs in the PCR reaction and were ligated to the binary vector pBIN61 as described previouslyKpnI restriction endonuclease followed by transcription using the T7 MAXIscript kit (Ambion) according to the manufacturer’s protocol. In order to detect the chloride channel protein CLC-b-like, the RPS3a-like and the 5S rRNA, mRNAs, 5′ ends of the DNA oligo nucleotides were radiolabeled with [γ-32P]ATP in the presence of T4 polynucleotide kinase (PNK).The riboprobes for the detection of both the GFP and the 7SL mRNAs were prepared using the GFP and the 7SL genes previously cloned in the pBlueScript KS (+) vector. The riboprobes were prepared by linearizing the pBlueScript KS (+) vectors with For the GFP-mRNA expression analysis, a northern blot was performed using radiolabelled GFP probes. Briefly, two leaf discs were homogenized in TRIzol® buffer and the RNA was extracted as per the manufacturer’s instructions . A total of 250 ng of the RNA sample was dissolved in 5 µl of a loading buffer containing 10% (v/v) glycerol and 0.01% each of bromo-phenol blue and xylene cyanol FF. The samples were heat-denatured at 65 °C for 15 min in the presence of 3 vol of sample buffer , and were then were separated by electrophoresis in 1.0% agarose-formamide gels containing 1x MOPS buffer. The RNAs were then transferred to a Hybond-XL nylon membrane  and hybridized with radiolabelled riboprobe at 55 °C for 16 h in order to detect the GFP. The membranes were washed twice for 20 min at 62 °C with washing buffer , and the hybridization signals were then visualized using a Typhoon imaging system .chloride channel protein CLC-b-like, the RPS3a-like and the 5S rRNA mRNAs. The chloride channel protein CLC-b-like and the RPS3a-like probes were hybridized overnight at 45 °C, while the 5S rRNA probe was hybridized at 55 °C. The membranes were washed twice with washing buffer, and the RNA gel blot hybridization signals were visualized using a Typhoon imaging system.For the RNA gel blot hybridizations of the VIGSed plants, 2.0 μg of the total RNA extracted from 18-dpi plants were transferred to a Hybond-XL nylon membrane as described above. The transferred RNAs were hybridized with radiolabeled probes in order to detect the 12. Anti-GFP-HRP antibodies were used to detect GFP according to the manufacturer’s instructions (Santa Cruz Biotechnology). Anti-PEPC rabbit antibodies  were used to detect PEPC, followed by a subsequent incubation with donkey anti-IgG rabbit-HRP polyclonal antibodies (BioLegend). The proteins were subsequently visualized using ECL substrate (Bio-Rad) as per the manufacturer’s instructions. The amount of GFP produced was quantified using ImageJ software (http://imagej.nih.gov/ij). The data obtained for the housekeeping gene (PEPC) was used for the normalization.For GFP expression analysis, total protein was extracted from two leaf discs using 1.5X of Laemmli sample buffer as described previously44, and only 21 to 24-nt long vd-sRNAs were filtered using a Python program45. The filtered 21 to 24-nt vd-sRNAs were plotted against both the (+) and the (−) strands of the respective PSTVd variants after normalizing to the Reads Per Million (RPM) scale using a standard pattern-matching algorithm developed in the Python programming language46.For the high-throughput sequencing of the small RNAs (sRNAs), the small RNA of 15 to 50-nt were column purified from the total RNA extracted from 21-dpi plants, and were then subjected to sRNA library preparation using the NEBNext Multiplex Small RNA Library Prep Set for Illumina according to the manufacturer’s instructions (New England Biolabs). The products obtained were then processed simultaneously in the Illumina Mi-Seq platform using a multiplex strategy. The adapters were trimmedRPS3a mRNA, a 5′ RLM RACE was performed by ligating an RNA adaptor to the 5′-phosphate of the total RNA extracted as described previously (12). The resulting product was reverse transcribed using Oligo (dT) primer. The products were subjected to nested PCR using RPS3a-like gene specific primers. The nested PCR products were separated, eluted, cloned, sequenced and analyzed for the 5′ termini as described before.In order to detect the cleavage sites of the 31. Briefly, 120 µg of total RNA obtained from 21-dpi plants were used to prepare mRNA using the GenElute mRNA Mini Prep kit according to the manufacturer’s instructions (Sigma-Aldrich). A 5′-PARE RNA adapter was ligated to the 5′-uncapped ends of the purified mRNA by incubation at 37 °C for 1 h in the presence of T4 RNA ligase I . The resulting RNA products were reverse-transcribed to cDNA using the Target RT-primer in the presence of SuperScript® III Reverse transcriptase according to the manufacturer’s instructions . This was followed by 7 cycles of PCR amplification using both the 5′- and 3′-cDNA PCR primers. The amplicons were then subjected to Mme I restriction endonuclease digestion for 1 h according to the manufacturer’s instructions . The digestion products were separated on 5% polyacrylamide gels, and the gel slices containing a band of approximately 70-bp were excised and the DNA was eluted overnight using an elution buffer . The eluted products were subjected to 15 cycles of PCR amplification with the Illumina universal primer using the multiplex strategy. The obtained products were purified and sequenced in the Illumina Mi-Seq platform at the Laboratoire de Génomique Fonctionnelle de l’Université de Sherbrooke . The adapter sequences were trimmed, and only 20-nt long sequences were filtered as before. The filtered sequences were mapped against mRNA sequences using a standard pattern-matching algorithm developed in Python46. The data was normalized to TPM and mapped on the respective mRNA sequences as described previously47.For the PARE analysis, the protocol designed for the Illumina Hi-Seq platforms was modified for the Illumina Mi-Seq platformThe deep sequence data used in this study is deposited in GEO with the Accession No. GSE70256 (sRNA derived from control plant); Accession No. GSE70096 (sRNA derived from PSTVd-I inoculated plant); GSE70166 (sRNA derived from PSTVd-RG1 inoculated plant); GSE88707 (PARE data obtained from mock inoculated plants); GSE70037 (PARE data obtained from PSTVd-I inoculated plants) and GSE70062 (PARE data obtained from PSTVd-RG1 inoculated plants).Supplemental data


ATXN2 encodes Ataxin-2, a protein involved in RNA metabolism and metabolic homeostasis [ATXN2 locus was found for familial PD [eostasis . CAG repilial PD .ATXN2 mutations and controls [ATXN2 triplet expansions. Notably, we observed strong similarities between sporadic PD and the SCA2p patients with involvement of molecular pathways already described in PD. Of interest, we also observed gene expression perturbations of the ALS signaling in SCA2p and sporadic PD compared to controls. Disturbances of this pathway also showed up after our analysis of Yokoshi and collaborator's data from high-throughput sequencing of Ataxin-2-bound RNAs [To seek the complex role of CAG repeats in familial PD (herein referred to as SCA2p), we performed a without a priori transcriptome analysis of mononuclear blood cells from SCA2c, SCA2p as well as sporadic PD without controls . We founund RNAs . They reund RNAs .SNCA overexpression and several proteins regulating translation including ATXN2 as well as a decrease in translation initiation factors has recently been identified [ATXN2 [ATXN2 resulting in the synthesis of a toxic anti-sense ATXN2 transcript with CUG repeats in SCA2c and ALS patient samples. It is thus possible that such mechanisms also contribute to the physiopathology of PD all the more so as we observed a significant decrease of Ataxin-2 expression in Epstein–Barr virus immortalized from SCA2c and SCA2p patients with more than 35 CAG compared to controls.The possible mechanisms contributing to neurodegeneration are multiple, with neuronal toxicity being one of the first associated with CAG repeat expansions. RNA metabolism dysfunctions including the sequestration of RNA binding proteins, have emerged as a deleterious feature of several neurodegenerative disorders. Given that Ataxin-2 is involved in RNA metabolism, we could expect deregulations of this fundamental mechanism induced by CAG expansions. Indeed, in SCA2p, sporadic PD but also SCA2c group, our transcriptome analysis revealed a high disturbance of molecular functions linked to RNA binding, polyA RNA binding, mRNA binding as well as structural constituent of ribosome. We found numerous functions linked to translation in patients with a PD phenotype (SCA2p and sporadic PD) compared to SCA2c. RNA transport and mRNA surveillance pathways were perturbed in SCA2c, while splicing deregulations were found in sporadic PD and SCA2c. These data show that RNA metabolism disturbances are a common feature of these different forms of degenerative diseases suggesting the involvement of potential similar biological processes but also specificities for each type of patients. Thus, some of the deleterious mechanism might be similar between these 2 disorders. Of interest a link between entified , comfortentified . Moreoved [ATXN2  was reveTaken together, these recent data support our conclusion underlying the perturbation of RNA metabolisms as a common feature in PD and SCA2. These data not only contribute to a better understanding of the mechanisms leading to PD with CAG expansions, but also conceptually to understanding alternative mechanisms leading to the vast majority of PD cases.- Our study was performed on a small number of patients, will the future transcriptome studies confirm or refute our results?- Will extended PD patient studies confirm or refute the decreased expression of Ataxin-2 associated of SCA2p with CAG repeats >35 in familial PD?ATXN2 of the SCA2p group with CAG repeats >35?- Will we find a mutated non coding RNA in - Do PABP proteins play a role in physiopathology of PD?- Since hnRNPs family is involved in TDP-43 aggregation, is it also linked to alpha-synuclein aggregation?- Since PABPC1 is sequestrated by CAG42 expansions, is it still sequestrated by Ataxin-2 with a lower number of repeats? And/or by aggregated alpha-synuclein?- Will new PD genes be involved in RNA metabolism, splicing, translation or non-coding RNA?Several questions need to be answered:


The genetic basis of metastasis is still unclear because metastases carry individual karyotypes and phenotypes, rather than consistent mutations, and are rare compared to conventional mutation. There is however correlative evidence that metastasis depends on cancer-specific aneuploidy, and that metastases are karyotypically related to parental cancers. Accordingly we propose that metastasis is a speciation event. This theory holds that cancer-specific aneuploidy varies the clonal karyotypes of cancers automatically by unbalancing thousands of genes, and that rare variants form new autonomous subspecies with metastatic or other non-parental phenotypes like drug-resistance – similar to conventional subspeciation.To test this theory, we analyzed the karyotypic and morphological relationships between seven cancers and corresponding metastases. We found (1) that the cellular phenotypes of metastases were closely related to those of parental cancers, (2) that metastases shared 29 to 96% of their clonal karyotypic elements or aneusomies with the clonal karyotypes of parental cancers and (3) that, unexpectedly, the karyotypic complexity of metastases was very similar to that of the parental cancer. This suggests that metastases derive cancer-specific autonomy by conserving the overall complexity of the parental karyotype. We deduced from these results that cancers cause metastases by karyotypic variations and selection for rare metastatic subspecies. Further we asked whether metastases with multiple metastasis-specific aneusomies are assembled in one or multiple, sequential steps. Since (1) no stable karyotypic intermediates of metastases were observed in cancers here and previously by others, and (2) the karyotypic complexities of cancers are conserved in metastases, we concluded that metastases are generated from cancers in one step – like subspecies in conventional speciation.We conclude that the risk of cancers to metastasize is proportional to the degree of cancer-specific aneuploidy, because aneuploidy catalyzes the generation of subspecies, including metastases, at aneuploidy-dependent rates. Since speciation by random chromosomal rearrangements and selection is unpredictable, the theory that metastases are karyotypic subspecies of cancers also explains Foulds’ rules, which hold that the origins of metastases are “abrupt” and that their phenotypes are “unpredictable.” The Molecular Biology of the Cell states that metastasis “is the most deadly – and least understood – aspect of cancer” [Metastasis is defined as the development of secondary malignant growths at a distance from a primary site of cancer . The rel cancer” .The Biology of Cancer states in 2014–60 years after Foulds first questioned mutations - that “it is clear that the identities of many of the genes that are specifically involved in programming metastasis have been elusive.” [In 1954 Foulds showed that cancers progress to metastases stochastically or “abruptly” and with diverse “unpredictable” phenotypes and concluded that “it is inadvisable to attribute to mutation” the great diversity of the individual phenotypes of metastases –19, aneulusive.” . In searlusive.”  or “predlusive.” . Considelusive.” , 75–77 wIn an effort to find a mutation-independent mechanism of metastasis, we reviewed previously unexplained findings for clues. As a result we found two conspicuous links: (1) the risk of metastasis correlates with the degrees of cancer-specific aneuploidy, and (2) the aneuploid karyotypes of metastases are related to those of primary cancers. In the following we first summarize the evidence for these two links between aneuploidy and metastasis, and then show that a karyotypic, rather than a mutational theory can explain the origins of metastasis.P < 0.02) higher in the aneuploid cases (8/15) than in the diploid cases (3/21)” [Studying cancer cytogenetics Atkin found in 1972 that “Carcinomas of the breast fell into two discrete groups. The lower near-diploid group showed a significantly better eight year survival rate than the higher triploid-tetraploid group.” . Likewis (3/21)” . In 1997 (3/21)” . And Tor (3/21)” . Concurr (3/21)” , which w (3/21)” .Despite these consistent correlations between degrees of aneuploidy and metastasis, the functional role of aneuploidy in metastasis remained unexplained , 86, 87.Numerous independent studies found that the individual aneuploid karyotypes of metastases are related to those of individual primary cancers, but not to those of metastases of other cancers , 75, 88.But in view of the individuality of the karyotypes and transcriptomes of metastases, compared to the expected common metastasis-specific mutations, a coherent theory of the role of the individual karyotypes and transcriptomes of metastases has not emerged. Therefore, we test here the theory that metastasis is a form of speciation, which predicts individual metastasis-specific karyotypes and phenotypes, rather than common mutations. This theory extends and confirms preliminary karyotypic evidence for metastasis from two labs including ours , 75, 88.The theory that metastases are karyotypic subspecies of cancers is a logical extension of the theory that cancers are karyotypic species of their own , 90, 91.One may argue, however, that cancers are not species, because they lack a natural habitat beyond their original host. The American evolutionary biologist Lee Van Valen acknowledged this reservation, because “cancer cells depend for their existence on the continuation of the practice of tissue culture”. But Van Valen maintained that this “wholely artificial” habitat is not an argument against the species definition of cancers, “the expected persistence of its habitat is never used as a criterion for judging the reality of a species, but rather for judging its susceptibility to extinction” . MoreoveIn the following we have tested the theory that metastases are subspecies of cancers by comparing the karyotypes of seven independent cancers with corresponding metastases.The breast cancer, HIM-2 and a brain metastasis, HIM-5 .The melanoma, WM-115 and a metastasis of undefined origin, WM-266-4 –23. It iThe liver cancer, H2P and a metastasis in the portal vein to the liver, H2M .The medulloblastoma, M-458 and a metastasis from a cerebrospinal fluid relapse, M-425 , 96. A cThe colon cancer, SW-480 and a lymph node metastasis, SW-620 –101 withThe melanoma, IGR-39 from a leg and a metastasis from the groin, IGR-37 .The pancreatic cancer, A13-B and two independent metastases, a pancreatic metastasis A13-A and a liver metastasis A13-D .As a first test of our theory that metastases are subspecies of cancers, we analyzed the karyotypic and cellular phenotypic relationships between seven published pairs of cancers and metastases:The provenance of these cancers and corresponding metastases, and the conditions under which they were propagated are described in Methods.The theory that metastases are subspecies of cancers makes two testable predictions: (1) Each pair of a cancer and corresponding metastasis is karyotypically related. (2) Each pair is also distinct in cancer- and metastasis-specific karyotypic and phenotypic markers. To test these predictions we compared the karyotypes and morphological cellular phenotypes of each of these seven cancer-metastasis pairs.As can be seen in Figs. The microscopic cellular phenotypes of all seven cancers were similar to, but also distinct from those of the corresponding metastases. In addition we found non-microscopic distinctions that set apart cancers from metastases. For example, the cellular growth rate of the primary melanoma WM-115, shown in Fig. The numbers of chromosomes of all seven cancers were very similar to, but not identical to those of the corresponding metastases.The karyotypes of all seven cancers shared a majority of structurally and numerically-defined aneusomies with corresponding metastases. But they also all differed from each other in distinct cancer- and metastasis-specific aneusomies.The phenotypic and karyotypic differences between cancers and metastases were roughly proportional to the degrees of cancer-specific of aneuploidy: The more aneuploid the cancer, the more different are the karyotypes of cancers from those of corresponding metastases. For example, it is shown Fig. We deduce from these comparative analyses that the karyotypes and phenotypes of metastases are exclusively related to, but also distinct from parental cancers – exactly as predicted by the theory that metastases are subspecies of cancers. Our observations that metastases appear to conserve the average numbers of parental chromosomes and conserve many parental aneusomies further supports the subspeciation theory – as conventional species typically conserve the genetic complexities and most of the karyotypes of their precursors .It could be argued, however, that the differences setting apart the karyotypes of metastasis from parental cancers shown in Figs. By contrast, if metastases were karyotypic subspecies of cancers, their karyotypes would be clonally related to parental cancers and would be stabilized by selection for autonomy - like those of parental cancers and of conventional species.To distinguish between these possibilities we next have investigated, whether the karyotypes of metastases are clonal and are clonally related to parental cancers.To prove that cancers cause metastases by karyotypic variation rather than by gene mutation, we need evidence that the karyotypes of metastases are clonal and clonally related to parental karyotypes.To determine karyotypic clonality, the karyotypes of multiple cells of a metastasis and of a primary cancer must be compared and shown to be identical. But, owing to the inherent variability or flexibility of cancer karyotypes and the resulting clonal heterogeneity of cancers , 105  there would be no stable precursors of metastases with less than the full set of authentic metastasis-specific aneusomies, and that (2) all metastases retain the karyotypic complexity of parental cancer as a whole, despite metastasis-specific aneusomic variations (see previous section). At the same time the single-step model also predicts there would be no precursors of the many non-clonal, non-metastatic variants of cancer-karyotypes with multiple non-parental aneusomies  A near-diploid breast cancer, termed HIM-2 and a brain metastasis, termed HIM-5 were a generous gift of Elaine Mardis. They were isolated and sequenced as described by her and her collaborators in 2010 . (2) A hOne to two days before karyotyping, cells were seeded at about 50% confluence in a 5-cm culture dish with 3 ml of the medium described above. After reaching ~75% confluence, 250–300 ng colcemid in 25–30 μl solution  was added to 3 ml medium. The culture was then incubated at 37 °C for 4–8 h. Subsequently cells were washed once with 3 ml of physiological saline, dissociated with trypsin, pelleted and then incubated in 0.075 molar KCl at 37 °C for 15 min. The cell suspension was then cooled in ice-water, mixed (‘prefixed’) with 0.1 volume of the freshly mixed glacial acetic acid-methanol  and centrifuged at 800 g for 6 min at room temperature. The cell pellet was then suspended in about 100 μl supernatant and mixed drop-wise with 5 ml of the ice-cold acetic acid-methanol solution and then incubated at room temperature for 15–30 min or overnight at -20C. This cell suspension was then pelleted and was then either once more re-suspended in fixative and pelleted, or was directly re-suspended in a small volume of the acetic acid-methanol solution for microscopic examination. For this purpose an aliquot of a visually turbid suspension was transferred with a micropipette tip to a glass microscope slide, allowed to evaporate at room temperature and inspected under the microscope at x 200 for an adequate, non-overlapping density of metaphase chromosomes. Metaphase chromosomes attached to glass slides were then hybridized to color-coded, chromosome-specific DNA probes as described by the manufacturer, MetaSystems . Karyotypes were analyzed under a fluorescence microscope, as described by us previously , 49.