The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the  We have identified a novel RNA element in RNA1 of the segmented positive-strand RNA genome of Red clover necrotic mosaic virus (RCNMV). Its similarity in structure to a known important RNA element in RNA2 suggests that the two RNA structures perform similar viral functions in their respective genome segments, and this was confirmed experimentally. Based on evolutionary considerations, the RNA element in RNA1 is proposed to have appeared earlier in RCNMV genome genesis, with the corresponding element in RNA2 arising subsequently as a functional mimic of the former. This information was integrated into a plausible pathway for the emergence and evolution of this segmented viral genome, thereby providing novel insights into the puzzling history of RCNMV. Dianthovirus is the only genus in the family Tombusvididae that possesses a segmented genome and, as such, its members face unique challenges [Fig 1A) [allenges . There aallenges –4. These[Fig 1A) , 3. The [Fig 1A) , 6, whil[Fig 1A) . An acce[Fig 1A) –12. The [Fig 1A) . RNA2 en[Fig 1A) .trans-activator (TA), and a sequence in RNA1, the TA binding site (TABS), located just upstream from the transcription start site [Fig 1A). This interaction forms a structure that blocks the RdRp’s progress during minus-strand synthesis of RNA1 [S1 Fig). Additionally, because the trans-interaction is concentration dependent, it coordinates CP production with high levels of both genome segments later in infections [Production of the CP-encoding sg mRNA requires an inter-genomic 8 bp-long interaction between the terminal loop of a stem-loop structure in RNA2, called the art site   of RNA1 , which gfections .in vitro assays showing that RNA fragments of the TA alone or a TA-TABS complex bind preferentially to recombinant CP, however the precise regions of CP contact in these RNAs were not defined [).The TA-TABS interaction between RNA1 and RNA2 is also proposed to facilitate copackaging of the two segments into the same particle , and the defined . In addi defined . ExactlyIn this study we have identified an RNA structure in RNA1 that has both structural and functional similarities to the TA, and, based on its likeness to the TA, the new element was termed the TA-like structure (TALS). Here we describe the discovery and analysis of this new RNA replication element, and compare and contrast its properties with those of the TA. The parallels between these RNA elements suggest an intriguing relationship, which we discuss with respect to the genesis of the RCNMV genome.). Regions with p-values corresponding to 0.001  or less are considered to be significant and represent prime candidates for harboring functionally relevant RNA elements. Results from this analysis of dianthovirus RNA1s revealed three regions meeting the significance threshold that corresponded to the frameshifting site, an internal segment in the p88 ORF, and the TABS region . The first and last regions were expected, as their positions overlapped with previously reported functional RNA structures [The ViRAD virus (re-)annotation database provides information on the likely presence of overlapping ORFs or functional RNA elements within the coding regions of viral RNA genomes . These dructures , 13. HowFig 1B) coincided with a section of the genome that exhibited well-defined secondary structure  . These results are consistent with the existence of a functional RNA structure located somewhere between nucleotides 1800 and 2000 in RNA1.The entire RCNMV RNA1 genome was also computationally-folded and analyzed for the conservation of RNA secondary structures in optimal and suboptimal folds . Well-maFig 2A). Selective 2ꞌ-hydroxyl acylation analyzed by primer extension (SHAPE) revealed high levels of reactivity in predicted single-stranded regions and minimal modifications for base paired residues. The varied reactivities observed in the region modeled to form a two-base-pair stem indicates that this interaction may be dynamic .Computational RNA folding of RCNMV RNA1 predicted an extended stem loop structure spanning coordinates 1841 to 1962, and corresponding solution structure mapping of this region within the context of a full-length RNA1 supported the proposed structure . While the general RNA conformation was maintained in all three viruses, most of the helical interruptions , were not conserved. One exception to this trend was a centrally-positioned asymmetrical internal loop (AIL) and its closing base pairs that was present in all species . In CRSV, the stem below its AIL was extended by two canonical base pairs, supporting the AIL’s existence and suggesting that pairing also occurs below the AILs in SCNMV and RCNMV.Similar extended stem loop structures were also predicted in other sequenced species of ), which is more apparent when the latter is rotated by 180° relative to its stem . Small differences in this region between the two genome segments included an adenylate in the TA’s AIL in RNA2 that was a uridylate in the RNA1 structures, and alternative closing pyrimidine-purine base pairs at one end of the AILs  . The RCNMV RNA1 structure and TA in RNA2 also possessed a second structural feature that was comparable; an eight-residue terminal loop, in which half of the nucleotides were identical . Based on both internal and terminal structural similarities to the TA, the novel RNA1 structure was named TA-like structure or TALS.Interestingly, the RNA1 AILs displayed a close resemblance to the AIL present in the TA in RNA2 of RCNMV . Transfection of R1-172 alone into plant protoplasts and subsequent northern blot analyses revealed that the introduced changes eliminated its accumulation . In cotransfections of R1-172 with wt RNA2, RNA2 replicated to notable levels, indicating that the mutations in R1-172 did not inhibit translation of replication proteins p27 and p88, and that, even though it was not amplifiable, the mutant was nonetheless stable . These results indicate that the inhibition observed is related to genome replication, and analysis of minus-strand RNA levels revealed a defect in minus-strand synthesis . These findings support an important role for the TALS (via its AIL) in mediating replication of RNA1, a property that is akin to the role of TA in facilitating replication of RNA2 [The conservation of the AIL in the TALS of different species and its similarity to the AIL in the TA prompted further investigation of its possible function. To address this, three silent nucleotide substitutions were introduced into the AIL in RCNMV RNA1, creating mutant R1-172 . This modification was made in an RNA2 mutant, R2-MPKO, in which the movement protein (MP) gene was inactivated by a premature stop codon, so that changes made to the TA would not affect corresponding coding of the MP. In contrast to the complete inhibition seen for the TALS modification in R1-172, the equivalent changes in the TA in R2-303 resulted in only a partial defect at both the positive- (~62%) and negative-strand (~72%) levels . Thus, although both AILs mediate replication of their respective genome segments, the same modification is less debilitating for RNA2.Next, the same three nucleotide substitutions made in R1-172 were introduced into the AIL of the TA in RNA2, generating R2-303 . The single nucleotide change in R2-244 resulted in a modest reduction in RNA2 levels to ~80%, whereas replacement of the wt loop with the tetraloop in R2-246 reduced accumulation to ~41% and, as anticipated, abolished activation of sg mRNA transcription . The observed level of R2-246 accumulation is consistent with another study which showed that substitutions within the TA terminal loop reduce RNA2 levels by ~50% [trans-activation of transcription. Finally, combining the loop and AIL modifications in R2-312 resulted in an additive negative effect (~3%) . Together, these data indicate that the AIL is the most important subcomponent of the TA for RNA2 replication, with the terminal loop contributing less substantially to the process.The functional resistance of the TA’s AIL to modification raised the question of its importance for RNA2 replication. Similarly, the potential contribution of the terminal loop of the TA to RNA2 levels was also of interest. The RNA2 mutants that were constructed to address these questions, as well as all other RNA2 mutants analyzed in this study, were made in an R2-MPKO background. Accordingly, R2-MPKO served as the “wt” 100% control for quantitative comparisons. In preparation for replacing the terminal loop of the TA with a cUUCGg super-stable tetraloop, the closing base pair in the wt 8-nt-loop was changed from UG to the required CG, creating R2-244 . A third silent substitution between these residues was not possible, because the corresponding amino acid was a singly-coded methionine. When R1-211 was transfected alone or in cotransfections with wt RNA2, both led to decreased levels of RNA1 to ~70% . Also notable was a twofold increase in RNA2 in the cotransfection, suggesting that the R1-211 defect rendered RNA2 more competitive for replication . Therefore, similar to the TA, both the AIL and terminal loop of the TALS contribute to its activity, but the former substructure appears to be more functionally relevant.The importance of the TA’s terminal loop for its activity suggested that the corresponding terminal loop of the TALS might also be functionally relevant. To test this possibility, two nucleotides, that could be exchanged silently, were substituted with alternative residues, creating R1-211 ; however, this amino acid position in the RdRps of tombusvirids is highly variable [Fig 6B). For RNA2, due to the absence of coding constraints in R2-MPKO, AIL substitutions that also included flanking regions were possible . In R2-265, only the AIL of the TALS was introduced, while in R2-313 and R2-314, the AIL and additional adjacent sequences were added. In all cases, the RNA2 mutants containing TALS-like AILs replicated to near-wt levels . Accordingly, the AILs of the TALS and the TA are functionally exchangeable.Having established a major role in replication for the AILs in the TALS and TA in their respective genome segments, the next unknown to be addressed was the possibility of their functional equivalence. To this end, the AIL in the TALS was converted to the TA’s AIL with a single nucleotide substitution in R1-223, which concurrently resulted in a leucine to glutamine substitution in p88  that had its TALS disabled was utilized. Additionally, the CP coding ORF in this mutant was inactivated by a frameshift mutation, creating R1-172*, which allowed for segment insertions into this region free from CP coding-related complications . Initially, to test the feasibility of rescuing the TALS-defective mutant R1-172*, the wt TALS was inserted at five different positions in the non-coding CP region that were predicted to maintain proper TALS folding, as assessed by Mfold . Upon transfection, these RNA1 mutants (R1-268* through R1-272*) exhibited very low levels of accumulation, with the maximum recovery being ~13%, for R1-270* . These results indicate that the TALS can only function minimally when transposed into a new RNA1 context. Next, the TA was introduced at the same five positions in R1-172*, thereby generating R1-273* through R1-277* . In all cases, no recovery was observed , suggesting that the TA is not able to functionally substitute for the TALS. However, the low levels of recovery observed with the wt TALS insertions  indicate that this assay may not be sufficiently sensitive to detect possible low level TA-mediated replication of RNA1.To test if the entire TA could functionally substitute for the TALS, the R1-172 mutant . Deletion of the TA in R2-301 resulted in very low levels of RNA2 accumulation, while its replacement with the TALS in R2-287 led to a modest twofold increase over that for R2-301 . TA replacement with a defective TALS in R2-302 reduced accumulation down to that of the TA-deleted mutant R2-301, consistent with minimal TALS-mediated rescue for R2-287.Next the ability of the TALS to rescue a TA-less RNA2 was investigated. The R2-MPKO mutant context was used to create three different RNA2 mutants: (i) R2-301, with its TA deleted, (ii) R2-287, with its TA deleted and replaced by a wt TALS, and (iii) R2-302, with its TA deleted and replaced by a defective TALS (as in R1-172) , however complete replacement of the TA with the TALS resulted in very low levels of RNA2 accumulation . There are at least two possible explanations for this result. First, the AIL in the TALS is predicted to be less stable than that in the TA, and this could limit the ability of the former to fold properly. To test this idea, the stem below the AIL in the TALS that was inserted into RNA2 was strengthened by one or two canonical base pairs in R2-315, -316 and -317 . These targeted reinforcements resulted in notably improved levels of RNA2 accumulation up to ~32% , supporting improved functionality of the TALS’s AIL in RNA2 when present in a more stable context.The replacement of the TA’s AIL with the AIL from the TALS allowed for near wt replication of RNA2 , this modification modestly improved RNA2 accumulation levels up to ~43%, and led to activation of sg mRNA transcription . The limited enhancement observed suggests that specific spacing or orientation between its AIL and terminal loop may be required, which may not be possible in the TALS’s context.A second possible contributing factor to the minimal activity of the wt TALS in RNA2 could be that its terminal loop is not functionally comparable to that in the TA. To assess this option, the TALS terminal loop was replaced with that of the TA in R2-315, -316, -317, generating R2-319, -320, -321 . Each protoplast transfection was assessed for total viral RNA accumulation and corresponding packaging efficiency. For all of the mutants, near wt levels of viral RNAs were detected in transfections along with efficient packaging of both genome segments , indicating that the TALS’s AIL and its context are compatible with virion assembly.The TA has been implicated in nucleating particle assembly and facilitating copackaging , 17. To Fig 9C). The latter modification was necessary (i) to allow for a TA-TABS-like interaction to activate sg mRNA transcription, thereby providing coat protein, and (ii) for facilitating copackaging of the two viral genomic segments. Low levels of packaged RNA were observed in all cases , with efficiencies roughly correlating with corresponding overall accumulation levels of RNA2 . If the RNA2 level in these infections is considered as a limiting factor for packaging (e.g. 20% for R2-321), then packaging efficiency in the wt+R2-321 cotransfection (i.e. 20–24%) is comparable to that in the wt+MPKO control cotransfection . Thus, the results further support the AIL of the TALS as being compatible with assembly and copackaging functions.Next, RNA2 mutants with their TA replaced by the TALS were assessed. The TALS in these mutants contained either a wt (R2-298) or stabilized (R2-320 and R2-321) AIL, in addition to the TA’s terminal loop . This internal position, and the fact that it overlaps with conserved structural motif-D in the palm domain of the RdRp [de novo within this coding sequence. The post-frameshift portion of this ORF encounters lower levels of ribosome traffic, thus the TALS would have opportunities to refold between recoding events. This genomic position is also important for TALS function, because moving it to other locations in RNA1 greatly reduced its activity . It is unlikely that the TALS mediates either translation or ribosome frameshifting, since both processes were still operational when it was inactivated . Instead, the data are most consistent with it facilitating replication of RNA1.The TALS is located in the RdRp ORF, within the frameshifted portion of this coding region  that contain critical internal loops  have been identified in the readthrough regions of RdRp ORFs of other tombusvirids  and, like the TALS, these elements are implicated in assisting viral genome replication [Similar functionally-important extended stem loop RNA structures , with the TA being more sensitive to alteration. However, this difference could be related to the change made to the TA terminal loop being more substantial than that to the TALS. Nonetheless, the results suggest notable roles for both terminal loop sequences, which likely relates to their ability to interact with proteins or other viral RNA sequences. Regarding the latter possibility, a search of the RCNMV genome for complementary RNA sequences for the TALS and the TA (other than the TABS) did not yield any compelling candidates, thus, presently, protein partners for both seem more probable.The terminal loops and AILs are the structural features most pertinent to the functional correspondence of the TA and TALS. For terminal loop modifications, negative effects on RNA segment accumulation were observed in both structures , indicating fundamental mechanistic equivalence. Even so, the wt contexts of the two AILs are unique, as they are oppositely-oriented with respect to their terminal loops, and the AIL in the TALS is predicted to be less stable. The latter difference could, at least partially, explain why the TALS’s AIL was more susceptible to identical modifications. This disparity in local context could also represent a way for the virus to “fine tune” AIL activity and appropriately balance the replication and accumulation levels of its two genome segments. The critical requirement for the AILs implicates them as key ligands for a trans-acting protein(s). As mentioned, previous analysis indicated that the TA or TALS do not bind to viral replication proteins p27/88 [Exchanging the AILs between the two genome segments maintained near wt levels of replication for RNA1 and RNA2 . Interestingly, the wt TA-TABS interaction is predicted to downregulate the TA’s function in RNA2 replication, because base pairing of the TA’s terminal loop  to the TABS would make it unavailable for alternative interactions. It is also possible that the proximity of the rest of RNA1 to the TA-TABS interaction sterically hinders access of a candidate host factor to the TA’s AIL. Such inhibition of RNA2 replication by the TA-TABS interaction could be important for coordinating opposing processes; i.e. replication of RNA2 and sg mRNA transcription from RNA1. Indeed, down-regulating the synthesis of RNA2 minus-strands would prevent the RdRp from disrupting the TA-TABS interaction from the opposite direction, thus allowing for unimpeded production of the sg mRNA minus-strand from the RNA1 template.The TA-TABS interaction forms an RNA structure that stalls the RdRp, leading to the generation of a minus-strand RNA template that is utilized for sg mRNA synthesis . The TA’Fig 9A and 9B). Consequently, if the TA’s AIL does contribute to CP binding and/or nucleation of assembly [Fig 9C and 9D). This indicates that any AIL-mediated function in virus assembly does not require either precise spatial arrangement relative to the TA-TABS interface or a specific context for the AIL. If CP does bind to the AIL, it would compete with the proposed AIL-binding host protein(s) needed for genome replication. However, the late expression of CP during infections could function to temporally coordinate binding in a sequential manner.A key function proposed previously for the TA is its role as a packaging signal . Genome assembly , then thPrior to this study, the TA was viewed as a highly unique RNA element in the RCNMV genome. The discovery of a comparable structure in RNA1 prompts a reevaluation of this element and its origins. Here, we present a hypothetical evolutionary scenario for the emergence of RCNMV that is consistent with our current findings and existing information. Virus packaging is included in this scheme by considering the AIL as a determinant of particle assembly .de novo genesis of the TALS within the RdRp ORF, its physical and spatial coupling with the RdRp, and the absolute requirement that the RdRp-encoding RNA1 be replicable, all support the TALS arising first. At this point in its existence, RNA1 may have been the sole replicative substrate of its encoded RdRp, and the expression of CP via sg mRNA transcription could have been directed by a cis-mediated mode. From a mechanistic perspective, it is reasonable to propose that sg mRNA transcription originally occurred by a simpler cis-mediated process and subsequently evolved to a more complex trans-based mechanism with its associated benefits. CP produced by a cis-mediated mechanism could be recruited to RNA1 for packaging via its TALS (possessing a more stable AIL), which we have shown is compatible with packaging . There are current examples of incomplete (i.e. non-self-sufficient) viral genomes that require coinfection with another virus to complement missing activities, and some are related to tombusvirids; e.g. umbraviruses and polerovirus-associated RNAs [A reasonable starting point is to ask the question: which appeared first, the TA or the TALS? The probable ted RNAs , 31. Accde novo formation of a precursor-TA (pre-TA) structure that structurally and functionally mimicked the TALS. The genesis of the pre-TA within the MP ORF would ensure their spatial coupling, and the AIL in the pre-TA would confer packaging competence to the precursor-RNA2, including the possibility of copackaging with RNA1. Additionally, the replication-related activity of the pre-TA would make the MP-coding RNA an attractive template for replication-mediated recombination with RNA1. Recombination, at sites flanking the MP ORF, could introduce terminal promoter elements from RNA1, thereby generating an RNA2-like genome segment; consistent with the presence of similar sequences and structures at the termini of current RNA1 and RNA2 segments [cis-activation to TA-dependent trans-activation via a TA-TABS interaction would enable timely CP production and facilitate copackaging of the genome segments [In this evolutionary scheme, the complementing viral RNA encoding the movement function would represent the precursor of RNA2. During coinfections with RNA1, the sequence corresponding to its MP-coding region would drift, allowing for the segments . Lastly,The discovery of the TALS and an unanticipated relationship with the TA has provided novel insights into the current status and conceivable origins of their corresponding genome segments. The hypothetical scenario presented above, which is in accordance with available related information, represents but one possible path to contemporary RCNMV. While it is understood that the proposed pathway cannot incorporate or explain all aspects of the present state of RCNMV, the scheme does provide a plausible evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.cDNA clones of wild-type RCNMV RNA1 and RNA2 genome segments (kindly provided by Tim Sit and Steven Lommel) were used to create all RCNMV clones for this study. Standard PCR-based site-directed mutagenesis was used to create RCNMV mutants, using the Q5 High-Fidelity DNA Polymerase kit (NEB). Sequencing was used to confirm that only the desired modifications were present in mutant constructs. Specific changes introduced into the original RCNMV genomes are shown in the accompanying figures.Uncapped in vitro transcripts of SmaI-linearized RCNMV constructs were produced individually using the T7-FlashScribe Transcription Kit (CELLSCRIPT) as previously described . RNA traRNA secondary structures and Pnum values were predicted at 37°C using Mfold version 3.6 using default settings , 33.Selective 2ꞌ-hydroxyl acylation analyzed by primer extension (SHAPE) was completed as described previously . Brieflyin vitro-transcribed viral genomic RNA. Specifically, 3x105 protoplasts were transfected with 3μg of RNA1 and/or 1μg of RNA2. The transfections were incubated under constant light for 22 hours at 17°C. Total nucleic acids were extracted as described previously [Cucumber cotyledon protoplasts were prepared and transfected with uncapped eviously .32P-radiolabeled probes complementary to the (+)3ꞌend of RNA1  or RNA2 . For minus(-) strand detection, total nucleic acid extracts of protoplast transfections were separated in denaturing 1% glyoxal gels as described previously [32P-UTP labeled riboprobes complementary to the 3ꞌ-ends of negative-sensed RNA1 (nt 3616–3890) or negative-sensed RNA2 (nt 1491–1449). Products of RNA1 and RNA2 were probed separately, as RNA2 and the subgenomic RNA are nearly identical in size. Viral RNA accumulation was monitored using a Typhoon FLA 9500 variable mode imager  and quantified by QuantityOne Software (Bio-Rad). All trials were repeated at least three times and averages were calculated with standard errors.For plus-strand RNA, total nucleic acid extracts from protoplast transfections were separated in non-denaturing 2% agarose gels. Viral RNAs were detected via northern blotting using two eviously . Minus-s5 protoplasts transfected with 12μg RNA1 and/or 4μg of RNA2. A tenth of the transfection was used to purify total nucleic isolation and perform northern blotting. The remaining 90% of the transfection was used to isolate virions. Briefly, protoplasts were suspended in 0.2M NaOAc pH 5.2. Sterile glass beads (212–300μm) were added, and protoplasts were homogenized with a Mini-Beadbeater (Biospec Products) using two 15 sec bursts. The homogenate was incubated on ice for 10 min prior to centrifugation at 11000 x g for 10 min. The supernatant was transferred to a new tube, and the precipitate was re-extracted as above. The combined supernatants were kept on ice for 1 hour, and then centrifuged for 10 minutes at 11000 x g. The supernatant was combined with an equal volume of 40%PEG/1M NaCl, and kept on ice overnight. Virions were collected by centrifugation and re-suspended in 10mM NaOAc pH 5.5. Virion RNAs were extracted by PCI, ethanol precipitated, and separated in non-denaturing 1% agarose gels. Viral RNAs were detected via northern blotting using the above described 32P-radiolabeled probes complementary to the 3ꞌ-ends of RNA1 and RNA2. Viral RNAs were quantified as described earlier.Packaging was assessed in protoplast transfections using 12x10S1 FigRNA1 and RNA2 genome segments are replicated by the viral p27/88 polymerase complex that first synthesizes full-length (-)strand RNAs complementary to the genome segments (grey dotted lines). These intermediate (-)strand RNAs are then used as templates for synthesis of progeny RNA genomes. Sg mRNA transcription occurs when the TA in RNA2 base pairs with the TABS in RNA1 at high concentrations of the two genome segments. The RNA structure formed by the intermolecular TA-TABS interaction causes the viral polymerase to terminate prematurely during (-)strand synthesis of RNA1, resulting in the generation of a sg mRNA-sized (-)strand RNA (green dotted line). This truncated (-)strand is then used as a template for transcription of sg mRNAs.(PDF)Click here for additional data file.


RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts. Parker Nichols et al. present an exact nuclear Overhauser enhancement (eNOE) protocol for defining small RNA structures at high resolution using only NOE distance data. They apply eNOE to a 14-mer UUCG tetraloop structure, obtaining a decrease in root-mean-square deviation from 1.52 Å to 0.44 Å, compared to conventional NOE. Second, NMR is one of the most powerful techniques for studying interactions between proteins, other nucleic acids, low molecular weight molecules, and solvent molecules. Finally, NMR is well-equipped to probe the inherent dynamics of RNA molecules, proven critical to their functions by amassing evidence5. In particular, residual dipolar couplings (RDCs) have been shown especially fruitful for investigating such dynamics, as they are capable of reporting on the orientation of bond vectors relative to a known molecular alignment frame6. Although X-ray crystallography and cryo-electron microscopy (cryo-EM) are still the techniques of choice for investigating RNAs and RNA–protein complexes larger than 50 kDa, which are difficult to study using NMR due to spectral overlap and fast T2 relaxation times, recent methodological advances have made NMR an alternative for studies of RNA of such sizes11.Although Nuclear Magnetic Resonance (NMR)-determined structures currently make up <10% of the total in the Protein Data Bank (PDB), they account for >40% of all RNA structures. This is mostly due to the inherent advantages of NMR over other structural techniques when it comes to RNA. First, solution-state NMR completely avoids the common difficulties involving crystallization of RNA common in X-ray12, which are employed this way due to various interfering mechanisms throughout the pulse sequence13, but mainly spin diffusion and dynamics14. The non-exact nature of these restraints means that important information about structure and dynamics is lost. Therefore, the only current NMR methods for probing dynamics are spin relaxation measurements, which are usually used for probing single-site flexibility and exchange, and RDCs, which require high technical sophistication when applied to RNA15.Despite these advantages, NMR has room for substantial improvement. One such area is the continued use of distance-dependent Nuclear Overhauser Enhancements (NOE) rate constants as semiquantitative upper limit distance restraints16. An additional difficulty with using NOEs in RNA structure determination is the low proton density of RNA compared with proteins, resulting in a sparser NOE network. The lack of sufficient NOE distance restraints means that traditional structure calculations have to rely on additional restraints such as dihedral angle restraints, RDCs, cross-correlated relaxation (CCR) rates19, electron paramagnetic resonance measurements20, as well as hydrogen bonding patterns, and rarely can these RNA structures achieve high resolution with NOEs alone21. Often, semi-empirical restraints such as base-pair planarity are added that cannot be used to characterize spatial sampling because they lack an accurate parameterization relating a specific conformation in a dynamic ensemble to an empirical observable. Therefore, a better use of the NOE would improve the quality of NMR structures and enable spatial sampling to be probed.RNA poses several challenges over proteins regarding NOE spectroscopy (NOESY). As a biopolymer composed of only four chemically unique building blocks, as opposed to 20 for proteins, RNA results in a large amount of spectral overlap, causing resonance assignment to be more difficult. This is exemplified by the H2′-H5′′ ribose protons whose chemical shifts normally appear within the narrow range of 4–5 ppm where water signal predominates. The overlap problem is further increased by the predominately A-form helical structure of RNA, which results in a lack of chemical environment diversity, especially for larger RNAs. Chemical shift diversity is therefore often only seen in non-canonical RNA structures such as hairpin loops, bulges, or internal loops23. The r−6-averaged nature of eNOEs allows for the construction of multi-state ensembles that describe their conformational space  for the determination of distances up to 5 Å with less than a 0.1 Å error in proteins Fig. 22, 23. Tace Fig. 24, 25. A37. The aptly named four-nucleotide tetraloops are simple but important RNA structural motifs that stabilize the caps of RNA stem loops38 and have other functions including initiation of RNA folding40, participation in tertiary interactions in large RNAs such as ribosomal RNAs43 and self-splicing RNAs44, and as recognition sites for proteins in ribonucleoprotein complexes47. The secondary structure of the 14-mer, as well as the discussed defining features of the UUCG tetraloop are shown . The high thermodynamic stability of the UUCG tetraloop has been attributed to arise from a non-canonical base-pair between U6 and G7 of the loop, favorable base-stacking between U6 and U8, and stabilizing hydrogen bonds between the U6 and U7 hydroxyls and the purine of G948. In addition, the UUCG tetraloop adopts a Z-turn motif that is defined by favorable O4’-π stacking contacts between the ribose of C8 and the purine of G950. Here, we present evidence that eNOEs do not only provide interproton distances to high accuracy, but also contain enough information to define RNA structures to high resolution with no additional restraints.To determine the feasibility and accuracy of extracting eNOE distances from RNA, we have applied our eNOE protocol to the RNA 14-mer UUCG tetraloopown Fig. . The hig2O yielded 91 eNOEs involving exchangeable amino resonances, as well as the stable hydroxyl resonance of U6. The Watergate suppression of the water signal in the H2O NOESY was sub-par, therefore, a NOESY with water presaturation was acquired in D2O, which yielded 174 eNOEs between non-exchangeable base and ribose resonances. The eNOE coverage of the ribose sugars was poor due to their extreme overlap of the H2’ through H5′′ resonances in the 2D NOESY. However, this problem was alleviated by the use of 88 generic normalized eNOEs (gn-eNOEs)51, which are used as an upper distance limit, as described in Methods. The large number of eight mixing times allowed us to establish a general rule for the optimal NOESY mixing times for RNA (see details in the Methods section). Assuming an inverse relationship of the maximal mixing time with the overall tumbling time τc, we obtain a maximal mixing time of 4 × 10−10 s2τc−1. This is larger than our recommendation of 2.5 × 10−10 s2τc−1 for proteins26. The eNOE network spans most of the 14-mer , we excluded overlapped diagonal and cross peaks from analysis. This substantially reduced the number of distance restraints because of the large amount of spectral overlap in the 2D NOESY spectra. We could fit 265 eNOEs out of the totally available 677 semiquantitative NOEs. The NOESY acquired from the sample in Hmer Fig. . One notmer Fig. . These r21. 2KOC is a well-defined structure with input restraints from a large set of conventional NOEs, allowed dihedral angle ranges, and RDCs, making it an ideal reference structure. Effective distances were calculated from the 20-conformer 2KOC ensemble by taking the r−6 average ( < 1/r6 > ), which takes into account structural variation. This analysis showed that the determined eNOE distances correlate well with the back-calculated distances from 2KOC . Despite the good correlation, there were quite a few outliers suggesting that the distance-averaged nature of the eNOEs is sensitive enough to pick up dynamics within the 14-mer. Because 2KOC is an averaged representation of its input data, we also compared our eNOE data set with the 10-state bundle provided by Al-Hashimi (hereafter referred to as C1), which was calculated using RDCs from multiple alignment conditions and is thus more likely to represent the dynamic nature of the 14-mer15. However, in order to generate different alignment tensors, the stem of the tetraloop had to be extended and modulated through base mutations in a bulge between what would normally be the stem and UUCG tetraloop of the 14-mer15. To still enable a comparison, we discarded all atoms between the gUUCGc loop and the four stem base pairs in all 10 states and all distances between the stem and the loop. Again, the correlation between our eNOEs and the averaged distances was good , suggesting that the measured eNOE data set was in good agreement with both structures. We also compared our eNOE data set to the linearly averaged distances < r > from C1 . The correlation was of the same quality, however, there were several distances that agreed much better with the r−6 averaging than the averaged distance, indicative of the eNOEs sensitivity to motional effects. Overall, it is clear that the extracted eNOE distances are consistent with the previously determined high-resolution structures.To investigate the accuracy of the extracted eNOEs distances, we compared the eNOE distances from fits of 40–160 ms  alone, with no additional restraints such as, for example, base pairing and sugar pucker restraints. The increase in precision when using eNOEs as opposed to conventional NOEs is impressive, which results in an RMSD decrease from 1.52 Å for the conventional NOE structure  dihedral angles from both the conventional NOE and eNOE structures, and 2KOC ensembles  to represent the multi-state bundle. The TF for the 14-mer tetraloop RNA decreased with increasing number of states, indicating that multiple states were necessary to describe the input data Fig. . The lar57, we calculated order parameters S2 for all N-H and C-H covalent bonds and P-OP1 from the two-state ensemble. S2 is 1 if the bond orientation is completely restricted and 0 if the bond has complete angular freedom. S2 values for C5/H5, C6/H6, C8/H8 and C1’/H1’ are shown . The 2KOC bundle is extremely tight and thus showed almost no difference between the two methods. The difference between the two methods of distance calculation for C1 is due to a rather large amount of rotational dynamics of the bases G9 and G10 GUGCC-3′ were purchased from Dharmacon  with High-Performance Liquid Chromatography (HPLC) purification. Both samples were re-suspended and then dialyzed via centrifugation in their respective buffers to remove residual triethylammonium from the synthesis. The first sample was concentrated to a final concentration of 1 mM and a total volume of 500 µl in 20 mM potassium phosphate, pH 6.4, 0.4 mM EDTA and 5% D2O. The second sample was concentrated to a final concentration of 1.7 mM and a total volume of 500 µl in 20 mM potassium phosphate, pH 6.4, 0.4 mM EDTA and 100% D2O.Two 2.0 µmol scale synthesis reactions of unlabeled 14-mer-cUUCGg tetraloop RNA with the sequence 5′-PO1H/13C/15N cryo-probe with z axis gradient. For the sample in H2O, a 2D WaterGate [1H-1H] NOESY buildup series with eight mixing points  was acquired with 1470 × 200 complex points and a 1.3-s recycle delay. The number of scans was 64 and tmax was 73.5 ms in the direct and 10 ms in the indirect dimension. For the sample in 100% D2O, a 2D PreSat [1H-1H] NOESY buildup series with eight mixing points  was acquired with 1470 × 400 complex points and a 1.5-s recycle delay. The number of scans taken was 32, and the tmax was again 73.5 ms in the direct dimension, and 20 ms in the indirect dimension. All spectra were processed with the NmrPipe/NmrDraw/NlinLS package61. Each dimension was apodized using a 90o phase-shifted cosine-bell window function and zero-filled once. Assignment of peaks in the 320 ms NOESY spectra from both the H2O and D2O samples was done in ccpNMR62 based on the resonance assignments of the previously solved high-resolution structure21 . Cross and diagonal peak intensities at all eight mixing times were extracted using the NlinLS autofit script with the assignment from the longest mixing time (320 ms) as the reference spectrum.All spectra were acquired at 25 °C on a 900 MHz Direct Drive Varian spectrometer equipped with a 5-mm triple resonance σ) and auto-relaxation rate constants (ρ) were determined using the full-matrix approach63, which is a part of the eNORA2 package64 that has recently been implemented into the CYANA software package66. The protein-specific MATLAB code of eNORA2 has been transcribed into the Fortran programing language and generalized for use with DNA/RNA and any other molecules that can be handled by CYANA67. The implementation in CYANA also extends features such as the three-spin approximation of spin diffusion for partially deuterated molecules to deuteration of any configuration. The increase in computational efficiency in the CYANA framework furthermore allows for spin diffusion averaging over several conformers. Details of the CYANA implementation of eNORA2 will be described elsewhere. The full-matrix approach corrects for spin diffusion by simulating the active magnetization transfer pathways between all spins simultaneously. Spin diffusion corrections were calculated from the existing NMR structure (PDB entry 2KOC)21, as well as from relevant atoms in a 10-state bundle provided by Hashim Al-Hashimi15 (C1). The spin diffusion corrections and the extracted eNOE distances depend on the overall rotational correlation time τc via the spectral density function26. This requires an accurate overall τc value as input. For the sample in H2O, we used the previously determined τc of 2.23 ns determined by NMR and molecular dynamics simulations55. For the sample in D2O, we used τc = 2.74 ns, which was estimated from the 23% viscosity increase of D2O using the Stokes–Einstein equation68. An average auto-relaxation rate constant ρ of 2.9 s-1 was used for spins for which no value could be fitted. The spin diffusion corrections at each mixing time were derived from the simulation and applied to the extracted intensities. The diagonal peaks were fit to monoexponential decay curves to determine ρi and ρj and initial magnetization values ΔMii(0) and ΔMjj(0)63. The corrected cross-peak buildup curves were then fitted using ρi, ρj, ΔMii(0), and ΔMjj(0) as fixed input parameters, and the cross-relaxation rate constants σij and σji as free variables. Uni-directional buildups that were of sub-par quality when normalized to the spin of origin (i→j), but were of high quality when normalized to the destination spin (j→i), were normalized to the destination spin13. The quality of all fits was evaluated visually, and poor fits from both (i→j) and (j→i) were excluded. Then, σij and σji were converted into distance restraints r through the relationship σ ~ r−6. ΔMii(0), ΔMjj(0), ρi, ρj, σij, σji, and r’s were determined using the intensities from all eight mixing times (40–320 ms), as well as from the first four (40–160 ms). Further analysis described in the Supporting Information revealed that rates obtained from fits to 160 ms are more reliable and were used for structure calculation. For the extraction of the distances, we assumed isotropic tumbling of the molecule. Using the simulations , we estimate a maximal distance error of ca. 2% for a molecule with a ratio of 1.5 between the longitudinal and transverse axes of the diffusion tensor. In CYANA, this entire process is automated except a visual fit evaluation. Comparison of the extracted eNOE cross-relaxation rates σ from non-exchangeable resonances between the H2O and D2O NOESY buildup series showed a slope of 1.21, indicating that our τc approximation for the D2O sample was in good agreement with the data  (for amino protons only) values fitted from mixing times 40–320 ms were smaller than those fitted from 40–160 ms  values are underestimated by 12, 5, and 2% when fitting a monoexponential function to 40–160 ms. The effect is stronger for methylene or amino protons because their effective σ is dominated by the dipolar interaction with the geminal proton. These errors translate into relatively small distance errors of <2% even in the case of amino protons. However, fitting to 40–320 ms results in 45, 21, and 12% underestimation of ΔM(0) for the same extreme cases. The resulting distance errors of up to 6% (for amino protons) suggest that they can be reduced by restricting the fits of the diagonal peak decays to a maximal mixing time of 160 ms.The proton distribution and density in RNA is different from that of proteins. Thus, we expect an optimal maximum mixing time that is different from the previously established tumbling time-dependent value for proteinsown Fig. , there wσ from 40 to 320 ms and from 40 to 160 ms mixing time on the rates, as well as the extracted distances. Correlation plots between σ from fitting from 40 to 320 ms and 40 to 160 ms indicated that the most extreme difference occurred for interactions within methylene and amino spin pairs , where r is the distance between two atoms and < > denotes the ensemble average. eNOE distances were also compared with the average distances < r > calculated from just C1.The extracted eNOE distances (with spin diffusion correction from 2KOC) from buildups of exchangeable resonances in H13. Generic normalized eNOEs (gn-eNOEs)51 were converted into upper distance limit restraints and given a tolerance of ± 10%  and ρ. Amino, methylene, and all other hydrogen atoms had substantially different ΔM(0) and ρ values, and therefore the upper limit ΔM(0) and ρ values were based on the highest values in the corresponding atom groups. All structure calculations were performed in CYANA-3.9866, starting with 100 initial structures with random torsion angle values using the standard simulated annealing protocol with 10,000 torsion angle dynamics steps. The 20 structures with the lowest target function values were selected for the ensemble. For the structure calculation based on conventional NOEs, a total of 677 upper distance limit restraints were used as input. For the single-state structure calculated from eNOEs, a total of 75 bi-directional eNOEs, 190 uni-directional eNOEs, and 88 gn-eNOEs were used. The multi-state structures were calculated as previously described24 using the same input restraints as for the single-state structure. The symmetry restraint weight was 0.1 for all heavy atoms with a flat-bottom width of 1.2 Å in a harmonic potential.Distances extracted from bi-directional eNOEs  had no error tolerance applied and had the same values for the upper and lower limit distance restraints. For uni-directional eNOEs , a tolerance of ± 10% was applied for the conversion to upper and lower distance limitsThe final structure coordinates and processed spectra were deposited into the PDB/BMRB database .Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA  were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for  Thus, it is the most widely used supplement for culturing human and animal cells. One of the major concerns that has become evident recently is the presence of large amounts of extracellular vesicles (EVs) in FBS4. Secreted by most cell types, EVs are mediators of cell-to-cell communication and immune regulation6. They carry the genetic material (DNA and RNA) as well as proteins, lipids and other molecules, the transfer of which can alter the functions of the recipient cells. EVs present in FBS are co-isolated with cell-derived EVs and therefore act as contaminants that, affect the reliability of the read-out from cell culture experiments, as FBS-derived EVs are structurally and functionally similar to cell-derived EVs7. In addition, FBS-EVs taken up by cultured cells cause substantial physiological effects2. To overcome this problem, different methods are used to deplete EVs from FBS. Ultracentrifugation (UC) is commonly used, but this method removes EVs only partially8. Commercially available depleted FBS are produced using proprietary protocols to reach higher purity levels. Also, commercially available defined, serum-free and animal protein free (xeno-free) media developed for clinical cell therapy purposes are an alternative to the largely undefined FBS in culture media. However, we and others have shown that EV-depleted FBS produced by UC or even commercial EV-depleted FBS are not completely free of FBS-derived EVs4. Recently we have developed a simple, standardized, cost- and time-effective protocol to produce EV-depleted FBS by means of ultrafiltration (UF) for cell culture purposes that support cell viability and proliferation9.Fetal bovine serum (FBS) contains essential factors required for cell growth, metabolism, attachment, and stimulation of proliferationIn the EV research field, identifying the ‘RNA contaminants’ derived from media itself is of high importance when developing RNA biomarkers. To address this question, we undertook this study to characterize the extracellular small non-coding (nc)RNA contaminants present in FBS, EV-depleted FBS, commercially available EV-depleted FBS as well as serum- and xeno-free defined media to assess their small ncRNA content and diversity.11. WB results indicated a strong CD71 band in FBS and SBI-dFBS, whereas no bands were detected in UC-dFBS and UF-dFBS. Faint band of CD71 protein was detected in StemPRO , ultrafiltration EV depleted FBS (UF-dFBS), commercial depleted FBS (SBI-dFBS) and commercial serum- and xeno-free media (StemPRO) by ultracentrifugation Table . EVs werPRO Fig. . To addr11) and particle distribution  and serum- and xeno-free media Table . RNA seq4. The similarity between bovine and human miRNAs makes distinguishing bovine miRNAs from human miRNA difficult, which may be a confounding factor when assessing miRNA derived from cell culture experiments. In StemPRO medium the overlap was only 73%, which suggests that a much more significant proportion of these reads may not have originated from miRNA at all, but may rather be experimental noise, suggesting that the actual miRNA content of the defined media was very low. In contrast to miRNAs, other human small ncRNAs were also mapped to a high degree (90–100%) to the bovine genome. The reason for this is that the database for bovine small ncRNAs did not contain any tRNA fragments, which made up most of the human small ncRNA alignments. Therefore, almost all human small ncRNA reads were classified as mapping to the bovine genome instead of mapping to small ncRNAs.Small ncRNA sequencing reads were mapped to both human and bovine genome to detect the extent of species-specificity and overlap between the species. Our analysis showed Fig.  that humWe further characterized the small ncRNA composition of FBS media preparations. As a quality control, serum and xeno-free media (StemPRO) was included in the study and evaluated for RNA contamination, as this medium should not contain any animal derived components. To facilitate comparison between different media types, total miRNA and other small ncRNA levels were normalized according to the external spike-in levels. As shown in the Fig. For depicting the overlap of small ncRNAs in the different media, Euler diagrams were created Fig. . Our datSubsequently, we evaluated the RNA depletion efficiency of different EV depletion methods. Unsupervised clustering analysis Fig.  showed tThe sequencing data clearly indicated ineffective depletion of RNA in dFBS, commercially available EV-depleted FBS and defined media, and the efficiency of miRNA depletion in these depleted media is unclear. To address this, we focused on specific miRNAs and traced their levels in the different media preparations. Based on the clustering analysis, two groups of miRNAs were selected for further analysis; miRNAs strongly affected by depletion methods and miRNAs weakly affected by depletion methods  in the 14. EV isolation and RNA sequencing was done according to the stringent workflow of the Qiagen services. Furthermore, qPCR was performed on our media samples with endogenous miRNAs . mir-451a, used as an EV marker15, was clearly expressed in our samples, indicating that miRNAs analyzed are EV related. Despite using a stringent protocol for isolating EV-related RNA for RNA sequencing, we still could not rule out the possibility of presence of non-vesicular RNA in our data. As the genuine association of sequenced RNA with EVs was not demonstrated  in our study due to technical difficulties, it is more likely that our samples contained both “cell-free RNA” (non-vesicular RNA) and “EV-bound RNA” (vesicular RNA). Therefore we prefer to use the term “vesicular or non-vesicular fractions” in this study.It is well known that the specificity of isolating EVs is low for both ultracentrifugation and other EV isolation methods. Various studies have demonstrated that EV preparations derived using these methods contain small amounts of non-EV soluble components, which might carry RNA as well17. Based on information retrieved from the literature, similar trend was observed for our miRNA data  in our samples. As expected, all the samples except UF-dFBS showed positive expression of HDL, indicating that none of the EV-depleted media are EV or lipoprotein free. These HDL-associated miRNA, could be considered as one of the source of miRNA contamination.A vast majority of miRNAs (90–95%) are estimated to be non-vesicularet al.4, where 100% overlap of RNA sequencing between bovine and human genome was observed. These results indicate, that media selected for cell culture experiments should be chosen carefully, prioritizing EV/RNA-free media. Screening of contaminants from the culture media should be a routine procedure in order to establish the baseline level of RNAs as a control to minimize the confounding effect of media. To highlight the importance of these findings, we collected the most commonly reported miRNAs in cell-culture-based EV studies  contaminants are clearly more difficult to eliminate, even with our UF-dFBS Figs  and 6. D26.Regardless of its source, presence of RNA (vesicular/non-vesicular) in all the tested media should still be considered contamination, which needs careful consideration for further applications. While there is no data readily available, we speculate that possible source of the RNA in these media could be from the recombinant proteins such as human serum albumin, essential amino acids, or vitamin constituentsIt is likely that RNA detected in our study could have originated from the different components of media supplements. In the future additional studies are needed to formulate an RNA free culture media, where all media components should be precisely screened. This will provide the details about the function and origin of the different RNA components in media.in vitro studies in the EV field, specifically related to the investigation of RNA biomarkers. Furthermore, functional impact of these contaminant RNA in recipient cells need to be addressed to understand their biological roles.In conclusion, our results highlight the need and importance of screening of EV- and RNA-free media for the FBS was obtained from two different suppliers, product details are described in Table et al.9. Briefly, UC-dFBS was prepared by 19 hours UC of regular FBS at 26,000 rpm (121 896 gmax) with SW28 rotor . Light-colored top layer of supernatant was filtered with a 0.22 µm filter . UF-dFBS was prepared by centrifuging regular FBS for 55 min at 3,000 g in Amicon ultra-15 centrifugal filters  and collecting of flow-through. FBS-1, UC-dFBS-1 and UF-dFBS-1 refer to FBS obtained from Sigma and FBS-2, UC-dFBS-2 and UF-dFBS-2 refer to FBS obtained from Gibco  to collect an EV pellet, which was washed with filtered DPBS (0.1 μm filter) and stored in Protein LoBind microcentrifuge tubes (Eppendorf) at −80 °C. For small ncRNA sequencing, EVs and RNA were isolated from 3.5 ml of FBS, UC-dFBS, UF-dFBS, SBI-dFBS, and StemPRO by exoRNeasy Serum/Plasma Maxi Kit (Qiagen).EV characterization was performed on EVs isolated by UC due to technical issues related to using exoRNeasy Serum/Plasma Maxi Kit (Qiagen) used for the sequencing study. This issue has been described in our previous articlehttp://www.malvern.com) as described previously9. Briefly, isolated EVs were diluted in filtered (0.1 µm) DPBS to obtain the optimal detection concentration of 106–109 particles/ml, and triplicate videos were recorded. The data was analysed using NTA software28.The number and size distribution of particles in EV samples were analysed using NTA  DPBS, loaded into 200 mesh copper grids and negatively stained with neutral uranyl acetate and embedded in methyl cellulose uranyl acetate mixture.Particle morphology was examined using TEM as described previously9 using primary antibodies against anti-transferrin receptor/CD71-H68.4  at 1:1000 dilution and ApoA130 (kind gift from Matti Jauhiainen) at 1:2000 EVs isolated by ultracentrifugation from equal volumes (20 ml) of each sample were loaded to gels. As a control, protein from adipose tissue derived mesenchymal stem cell lysates were used. Samples were denatured at 95 °C for 5 min in reducing Laemmli sample buffer, separated using Mini-PROTEAN® TGX™ 12% gradient SDS-PAGE gel  with BlueSTAR Prestained Protein Marker  as a standard. Running conditions were 150 V for 60 minutes. Blotting involved semi-dry transfer of proteins on nitrocellulose membranes, 0.2 µm , using 40 mA per gel for 60 minutes. Blocking and antibody incubations were performed in Odyssey blocking buffer (PBS)  with and without 0.1% Tween-20, respectively. After primary antibody overnight incubation at +4 °C, membranes were washed 4 × 5 minutes in TBS-T and probed with secondary IRDye® 800CW Goat  at 1: 15,000 and goat anti-rabbit  at 1:10000 for 1 hour at room temperature (RT). After incubation, membranes were washed 4 × 5 minutes in TBS-T at RT and briefly rinsed with PBS 1X, then imaged on an Odyssey FC Imager (LI-COR).WB was performed as described previouslySmall ncRNA sequencing was performed by Qiagen . EVs and RNA were isolated using exoRNeasy Serum/Plasma Maxi Kit. EVs were isolated from 3.5 ml of media followed by RNA isolation protocol optimized for serum/plasma (no carrier RNA was added). Library preparation was done using the QIAseq miRNA Library Kit. Out of 12 µl of isolated RNA, 5 µl was converted into microRNA NGS libraries. Adapters containing 12nt-long unique molecular indices (UMIs) were ligated to the RNA. Then RNA was converted to cDNA. The cDNA was amplified using PCR (22 cycles) and during the PCR indices were added. After PCR the samples were purified. Library preparation quality control was performed using either Bioanalyzer 2100 (Agilent) or TapeStation 4200 (Agilent). Based on the quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pools were quantified using the qPCR ExiSEQ LNA™ Quant kit (Exiqon). The library pools were then sequenced on a NextSeq500 sequencing instrument according to the manufacturer instructions using 1 × 75 bp reads. Raw data was demultiplexed and FASTQ files for each sample were generated using the bcl2fastq software (Illumina Inc.).31 was used on each raw read with the adapter sequence to acquire information about the presence of adapter. Only reads that fulfilled the following criteria were kept: (1) read contained adapters, (2) read had at least 16nt insert sequence length, and (3) read had at least 10nt-long UMI sequence. Second, insert sequences with incomplete UMI sequence were extracted as partial-UMI reads. Third, all unique insert + UMI combinations were identified from reads with full-length UMI and collapsed into a single read. Fourth, collapsed full-length UMI reads and partial-UMI reads were combined. After UMI-correction, reads were trimmed using cutadapt output and an in-house script.To correct PCR bias with UMI information, raw reads were processed as follows: First, cutadapt 1.1132 as follows: First, reads mapped to spike-ins or outmapped  were filtered out. Perfect match to the reference sequence was required. Second, reads were aligned to mature sequences of miRBase 20, requiring perfect matches. Third, unmapped reads were mapped to either to human GRCh37 or bovine UMD3 reference genome, allowing one mismatch in the first 32 bases of the read. Reads aligned to known miRNA loci in the genome were combined with the miRBase-mapped reads and reads aligned to small ncRNA reqions in the Exiqon small ncRNA database were classified as small ncRNA. Reads mapping to the reference genome outside the miRNA or small ncRNA regions were classified as genome-mapped. No indels were allowed in any of the mapping steps.Reads were mapped using Bowtie 2 (2.2.2)Read counts were normalized using the external spike-ins (UniSp100–UniSp151) that were present in three relative concentrations of 0.1 (low), 1 (moderate) and 5 (high), reflecting the expected range of miRNAs in the samples. Linear functions with intercept fixed at zero were fitted to the spike-in concentration versus observed counts in each sample. The linear fit was used calculate an estimated relative concentration for each RNA. For the clustering analysis, only RNAs with moderate (>1) spike-in normalized counts in at least one sample were included. Clustering analysis was performed on lists of miRNAs and small ncRNAs, which included the top 60 most abundant miRNAs and 40 most abundant small ncRNAs in all samples. Euclidean distance metric and average linkage were used in the unsupervised clustering analysis. Euler diagrams were created using a threshold of 1 spike-in normalized counts (moderate to high abundance) using the eulerr-package in R.33 and miRandola34 databases. Studies using human cultured cells were included. In miRandola, only studies with exosome or microvesicle material were included. This resulted in 19 included studies in EVpedia and 10 studies in miRandola. Three studies were reported in both databases, resulting in a total of 26 studies .To identify most commonly reported EV miRNAs in cell culture studies, we used EVpediaSupplemental figure 1Supplementary 1Supplementary 2


Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of  Histoplasma capsulatum, an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism.Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA. Histoplasma capsulatum is a major human fungal pathogen on the global stage that causes disease in both immunocompetent and immunocompromised individuals, albeit the risk for severe disease increases with compromised immunity . In the United States, it is the most common cause of fungal pneumonia (H. capsulatum is of particular concern in certain developing regions (H. capsulatum. An interesting facet of the biology of H. capsulatum is its ability to release extracellular vesicles (EVs)  , 8.EVs are bilayered lipid structures released by remarkably diverse cells across all kingdoms . We have10–Cryptococcus neoformans, Paracoccidioides brasiliensis, Candida albicans, Saccharomyces cerevisiae, and Malassezia sympodialis . In addition, proteomic data allowed the identification of 139 RNA-binding proteins (RBPs) in the EVs, suggesting that proteins involved in RNA metabolism might play an important role in cell communication through the EVs. Our results add this important pathogen to the list of fungal species with the ability to use EVs for the extracellular release of RNA.EV-mediated transport of fungal RNA was recently shown in both commensal and opportunistic fungi. EV RNA molecules, mostly smaller than 250 nucleotides (nt), were identified in podialis , 18. SinH. capsulatum strains G186AR and G217B. These strains belong to distinct clades, and G217B has been shown to be more virulent than G186AR in experimental models  were aligned with each strain-specific genome available at the NCBI (G186AR ABBS02 and G217B ABBT01). For data validation, we considered only sequences with expression values of transcripts per million (TPM) of ≥100 in all biological replicates and transcripts with reads covering at least 50% of the coding DNA sequence (CDS). The small RNA (sRNA) fraction was analyzed for the presence of different species of noncoding RNAs (ncRNAs) by aligning the sRNA fraction (reads of <200 nt) with the H. capsulatum.We identified a total of 124 mRNA sequences in EV samples from the two strains and carried out paired comparisons between the G186AR and G217B samples. We applied the statistical negative binomial test with filters corresponding to TPM values of ≥100, log2 values of ≥2, and false-discovery-rate (FDR) values of ≤0.05. We observed 93 transcripts enriched in EVs derived from the G217B strain, while 31 transcripts were enriched in the G186AR strain see . In the 10.1128/mSphere.00176-19.1TABLE S1H. capsulatum G217B and G186AR strains. Download Table S1, XLSX file, 1.4 MB.List of transcripts differentially enriched in Copyright © 2019 Alves et al.2019Alves et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. capsulatum genome code hypothetical proteins and/or do not present a conserved domain, which impedes our current ability to determine specific biological activities. Those associated with DNA metabolism/biogenesis were the second most abundant for both EV samples (22 for G217B versus 16 for G186AR), followed by transport for G217B and by protein modification for both strain EVs. Other processes related to short RNAs identified in both strain EVs were oxidation-reduction, signaling, and carbohydrate and lipid metabolism (n = 11) but not in G186AR (n = 2) EVs, while those related to response to stress were found exclusively in the G217B sample. The corresponding proteins are stress response protein whi2, DNA repair protein rad5, and a thermotolerance protein ; about 50% of these short fragments aligned to the reverse strand, including 172 (G217B) and 80 (G186AR) sequences of this type . A totaltabolism . RNA fra protein . Analysi protein .l-isoaspartate O-methyltransferase, serine/threonine-protein kinase, proteasome component C5, pre-rRNA processing protein Utp22, C-x8-C-x5-C-x3-H zinc finger protein, fungus-specific transcription factor domain-containing protein, and DNA damage-responsive transcriptional repressor RPH1 , two Dicer-like proteins, the QIP (quelling interaction protein), and the RNA-dependent RNA polymerase (QDE1)  complexes. The proteins associated with RNAs are named RNA-binding proteins (RBPs). These proteins participate in several biological processes, ranging from transcription to RNA decay . In thisB strain , and we B strain   .10.1128/mSphere.00176-19.3TABLE S3H. capsulatum G217B strain    with theCopyright © 2019 Alves et al.2019Alves et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans, C. neoformans, P. brasiliensis, and S. cerevisiae  resulted in the production of both sense and antisense sRNAs .In our study, we identified short reads that aligned specifically to exons; however, these sequences did not correspond to complete mRNAs in the EVs. They instead corresponded to 25-nt-long fragments that were enriched in specific exons of the transcript. These fragments of mRNAs were previously described in human cells , where mse sRNAs –33. Seque sRNAs –. This reH. capsulatum EVs contain different RNA molecules, it is reasonable to hypothesize that proteins that regulate RNA metabolism are also present in the EVs, probably associated with RNA. If validated, this hypothesis could indicate how the RNAs in a specific subset are directed to the vesicles and exported. RNA-binding proteins (RBPs) participate in several biological processes, from RNA transcription to decay -induced silencing (N. crassa led to the identification of miRNA-like RNAs (milRNAs) in this organism  and various small interfering RNAs (siRNAs), including exo-siRNAs, endogenous siRNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs) . Previouus fungi , 27, 40.. crassa –45. The  crassa –. A simile growth . In N. corganism . The ideCryptococcus gattii, pathogen-to-pathogen communication via EVs resulted in reversion of an avirulent phenotype through mechanisms that required vesicular RNA  supplemented with glucose (18.2 g/liter), l-cysteine (8.4 mg/liter), HEPES (6 g/liter), and glutamic acid (1 g/liter) and cultivated at 37°C with constant shaking at 150 rpm. Viability assessments were performed using Janus green 0.02%, and all aliquots used had >99% live yeast cells. EVs were then isolated from fungal culture supernatants as previously described  and were then treated with an RNeasy MinElute cleanup kit (Qiagen), according to the manufacturer’s protocol, to obtain small RNA-enriched fractions. The sRNA profile was assessed in an Agilent 2100 Bioanalyzer (Agilent Technologies).Purified sRNA (100 ng) was used for RNA-seq analysis with two independent biological replicates. The RNA-seq analysis was performed using a SOLiD 3 Plus platform and an RNA-Seq kit (Life Sciences) according to the manufacturer's recommendations.H. capsulatum G186AR strain ABBS02 and G217B strain ABBT01). The alignment was performed using the following parameters: additional number of bases of upstream and downstream sequences, 100; minimum number of reads, 10; maximum number of mismatches, 2; nonspecific match limit, −2, minimum fraction length, 0.7 for the genome mapping or 0.8 for the RNA mapping. The minimum proportion of read similarity mapped on the reference genome was 80%. Only uniquely mapped reads were considered in the analysis. The libraries were normalized per million, and the expression values for the transcripts were recorded in RPKM (reads per kilobase per million). We also analyzed other expression values, including TPM (transcripts per million) and CPM (counts per million). The statistical test applied was the DGE  test. For the ncRNA analysis, the database used was the ncRNA database from Histoplasma capsulatum (EnsemblFungi G186AR GCA_000150115 assembly ASM15011v1). The secondary structure analysis was performed using the PPFold plugin in CLC Genomics Workbench v. 10.1 and the default parameters. The entire RNA-seq database was subjected to PPFold analysis, and the putative structures were determined. Analysis of the relationship between the profile of RNA sequences detected in this study and the protein composition of H. capsulatum EVs was based on results recently obtained with strain G217B using a proteomic approach (SRR2015219 and SRR2015223)  .PRJNA514312.The data were deposited into the SRA database under study accession number


Journal of Molecular Cell Biology on RNA biology, combining a series of reviews and research articles that reflect most of the current trends and topics emerging from recent Chinese–German interactions in RNA research.NA biology has developed in recent years to a new emerging field, with new surprises and insights ranging from basic mechanisms to topics of clinical interest and opening up biotechnological applications in molecular medicine and novel therapeutic options. Here, we edit this Special Issue of The Sino-German Symposium on RNA Biology has been held since 2016, each year concentrating around a special theme of RNA research. The symposium is funded by the Sino-German Center for Research Promotion, a joint venture between the Deutsche Forschungsgemeinschaft (DFG) and the National Natural Science Foundation of China (NSFC), which supports scientific collaborations between China and Germany, in particular workshop-type meetings with ~40 scientists from both countries, all presenting their latest work, discussing ongoing projects, and initiating new co-operations. Renowned scientists from other countries were also invited, such as Prof. Xiangdong Fu from UCSD, USA. Albrecht Bindereif and Zefeng Wang co-organized the first two meetings, which were held in the castle of Rauischholzhausen  focusing on the role of RNA in human disease and at the Chinese Academy of Sciences (CAS) campus in Shanghai  on RNA-related complex structures and novel mechanisms, respectively. The 3rd meeting co-organized by Markus Landthaler from the Max-Delbrück-Center for Molecular Medicine (MDC) in Berlin and Wei Chen from the Southern University of Science and Technology (SUSTech) in Shenzhen was held last year in Berlin  and emphasized emerging technologies and new trends in RNA research. The 4th meeting will soon take place in Shenzhen in November 2019, continuing this short but successful history.These Sino-German RNA meetings also rely on a core of long-standing interactions, collaborations, and personal friendships, some extending over the last two decades. For instance, Wei Chen spent many years in Berlin , with a long history of successful German–Chinese collaborations; Jingyi Hui once worked in Albrecht Bindereif’s group in Berlin and Giessen before moving back to Shanghai; and Zefeng Wang co-organized the Otto-Warburg Summer Schools alternating between Berlin and Shanghai and recently became the Mercator Fellow of a DFG-funded Research Training Group coordinated by Albrecht Bindereif. In addition, new collaborations were initiated during these meetings, reflecting the need for an interaction forum for German and Chinese scientists in RNA research. We are very grateful to the Sino-German Center for Research Promotion (Beijing) for continuous support and funding to make this possible.6A RNA modification in plants , with the following topics: RNA structural biology , aging an plants , new insn plants , regulatn plants , generalmechanisms of auto-regulation by RNA-binding proteins , as well


Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three‐reaction high‐resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High‐resolution melting assay 1 is described in a previously published report. High‐resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High‐resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three‐reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples.The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of  M. tuberculosis, M. africanum, M. microti, M. bovis, M. caprae, and M. bovis BCG from directly extracted samples and cultured material. The use of this powerful assay may save several months of cultivation time and supports improvement of efficient tuberculosis combating strategies worldwide.The HRM assay enables rapid identification and differentiation of the clinically most relevant species of MTBC namely  TubercuMycobacterium tuberculosis complex (MTBC) comprises the closely related species Mycobacterium (M.) tuberculosis, M. bovis, M. bovis Bacillus Calmette and Guérin (BCG), M. caprae, M. africanum, M. microti, M. pinnipedii, M. orygis, and four further species known as animal‐adapted Clade A1   resistance, in contrast to the common M. bovis strains, detected in samples from Malawi  of dissociating PCR amplicons  approaches are cheap and rapid assays, which are able to detect single nucleotide polymorphism (SNP) according to altered melting temperatures , M. africanum/M. orygis/M. pinnipedii/Clade A1, M. bovis/M. bovis BCG, M. caprae/rare M. caprae/M. bovis ecotypes, and M. microti in cultured isolates and clinical samples. If requested, it is possible to further differentiate M. bovis, M. bovis BCG, and one of the two rare M. caprae/M. bovis ecotypes (ecotype I) by conducting another HRM assay (HRM assay 3) based on two SNPs within gyrA and thus completing the three‐step paradigm. For most diagnostic applications, however, a combination of HRM assays 1 and 2 will be sufficient.We have previously reported the design and evaluation of a HRM assay (HRM assay 1) for the identification and differentiation of MTBC into three groups most relevant for veterinarians  were used in each qPCR run. Clinical samples were received in the Laboratory of Veterinary Bacteriology, University of Zurich, between 2013 and 2016.Sixty‐one samples positive for MTBC were collected from 39 different animals Table . One wil2.2Sample preparation, culture, and DNA extraction were conducted as described in a former study . In order to extend the obtained SNP differentiation scheme with the recently described two rare ecotypes of M. caprae/M. bovis (ecotypes I and II), a rare M. canettii subtype , and one SNP on gyrA  were included in the assay design . The designed primers amplify a conserved 107‐base pair (bp) fragment within gyrB comprising two SNPs and gyrA comprising one SNP, respectively  was used for data analysis to generate normalized and difference plots as described in a previous study  with the Type‐it HRM PCR Kit (Qiagen). The qPCRs were performed as described previously . The analytical sensitivity of HRM assay 3 was tested for M. bovis BCG Pasteur ATCC 35734 and the clinical isolate M. bovis ZH20665 in an analogous manner by analyzing 10‐fold DNA dilution series. Based on an estimated genome size of 4.4 Mb for members of MTBC, 1 GE correlates with a DNA quantity of 4.8 fg. The slope of the resulting standard curve corresponded to the amplification efficiency of each tested strain. The limit of detection (LOD) was defined as lowest dilution for samples with successful PCR amplification of all triplicates having a Ct < 38 and a standard deviation of ≤ 0.5.The analytical sensitivity of HRM assay 2 was measured by triplicate testing of a 10‐fold dilution series of DNA isolated from strains 33.1mT  and the interassay CVs were ranging between 0.01%–0.04% and 0.11%–0.17%, respectively . One sample revealed significantly lower mT values compared with the other results. This clinical sample , however, has not yet been successfully cultured and is in the progress of further investigations. High‐resolution melting assay 3 showed a clear distinction of all cultured isolates as well as directly isolated clinical samples of M. bovis from M. bovis BCG in 100% concordance with the GenoType MTBC test (Hain Lifescience) results.For 22/23 (96%) clinical samples tested in HRM assay 2, obtained normalized and difference plots Figure b,c showeIn HRM assay 2, intra‐ and interassay CVs were between 0.02%–0.04% and between 0.12%–0.19%, respectively Table .High‐resolution melting assay 3 showed intra‐assay CVs between 0.01% and 0.02% and an interassay CV of 0.04% Table .3.3N. paucivorans, E. coli, and S. suis were tested for specificity of HRM assay 2 resulting in no melting curves or melting curves with entirely different mT in respect to mT deriving from samples of MTBC. In HRM assay 3, some NTM showed similar curves and mT values. Therefore, HRM assay 3 is recommended to be applied only after identification of M. bovis or M. bovis BCG.Forty‐one NTM and 3.4M. microti, 97% for M. bovis, 112% for M. caprae, and 91% for H37Rv  were complied with 10 GE, corresponding to 50 fg of template DNA in the qPCR, for is Table . The LOD4M. microti, M. tuberculosis, M. caprae, M. bovis, and M. bovis BCG were demonstrated to undoubtedly and consistently be differentiated from each other by distinctive difference plots  from M. caprae. To resolve this problem, it would be possible to expand the assay by designing a novel primer pair covering a SNP at base pair position 1,310 of gyrB and thus reliably identifying M. caprae/M. bovis ecotype II. Since M. caprae/M. bovis ecotype II is a rare type of strains, it can be neglected in routine diagnostic laboratories, where it is essential to rely on simply performable assays. Moreover, an additional differentiation of M. caprae/M. bovis ecotype II from M. caprae has no advantage in respect to the choice of antibiotic treatment, because both strain types are not intrinsically resistant to PZA.The only drawback of the described HRM approach compared to the Hain test is the inability to differentiate three strains originating from China , M. tuberculosis/M. africanum/M. orygis/M. pinnipedii/Clade A1, and M. caprae/M. bovis/M. bovis BCG/rare M. caprae/M. bovis ecotypes), secondly into six groups , M. africanum/M. orygis/M. pinnipedi/Clade A1, M. caprae/rare M. caprae/M. bovis ecotypes, and M. bovis/M. bovis BCG), and finally on gyrA, M. bovis BCG, M. bovis, and rare M. caprae/M. bovis ecotype I can further be separated leading to a clear differentiation into the main human‐ and veterinary‐associated MTBC species , the members of MTBC can be separated into distinct groups  display a weak fluorescence signal  deriving from an alpaca, however, showed a nonspecific melting curve preventing a correct assessment of the sample to MTBC applying the developed HRM assays. A possible explanation of this interference of the melting curve could be correlated to the inhibitory substances of the complex sample mixture originating from different tissues including lymph node, lung, heart, liver, and cervical vertebra. Investigations to unravel this finding were not successful yet. Although all remaining 22 clinical samples deriving from lymph nodes, lung, or liver tissues showed an unambiguous and correct result Table , it is i% derivinmT, which is demonstrated in very low values of intra‐ and interassay CVs. Since HRM assays 1 and 2 are proven to be 100% specific, they can be used unambiguously for the identification and differentiation of MTBC. Moreover, the assays yielded PCR efficiencies of more than 91% and 89%, respectively. The sensitivity of HRM assay 2 showing a LOD of 10 GE is adequate. The LOD of HRM assay 3 is slightly higher with a measured LOD of 100 GE. However, since the tested collective of samples did not cover species of all eight genotype groups, it is suggested to further evaluate the assay by testing a more extensive collection of isolates.Summarizing the obtained data, both HRM assays 2 and 3 showed a very good reproducibility with small variations of 5M. tuberculosis/M. canettii, M. canettii (rare subtype), M. africanum/M. orygis/M. pinnipedii/Clade A1, M. microti, M. caprae/rare M. caprae/M. bovis ecotype II, M. bovis, M. bovis BCG, and rare M. caprae/M. bovis ecotype I extracted from clinical samples and from isolates. Several months of cultivation time may be saved by using these potent HRM assays. Since most species within MTBC are implicated in human infections  were followed. The animal samples were analyzed in the context of a monitoring program of lymph nodes aiming at an early recognition of bovine tuberculosis and NTM infections. No animals were killed for the purposes of this research project, and no ethical approval was required.