Influenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility. This tail contains the Nuclear Location Signal (NLS). The nuclear trafficking of the viral components mobilizes cellular import factors at different stages, making these host-pathogen interactions promising targets for new therapeutics. NP is imported into the nucleus by the importin-α/β pathway, through a direct interaction with importin-α isoforms. Here we provide a combined nuclear magnetic resonance and small-angle X-ray scattering (NMR/SAXS) analysis to describe the dynamics of the interaction between influenza B NP and the human importin-α. The NP of influenza B does not have a single NLS nor a bipartite NLS but our results suggest that the tail harbors several adjacent NLS sequences, located between residues 30 and 71. These are in particular the three subunits of the RNA-dependent RNA-polymerase of the virus and the nucleoprotein (NP), which is the major protein of the ribonucleoproteins (RNPs) that binds the viral RNA2 and has been shown to be essential for viral proliferation.Both transcription and replication of influenza virus take place in the nucleus of the infected cell. To ensure these two mechanisms, a number of viral proteins have to be transported from the cytoplasm to the nucleus6, which mediate the interaction with proteins of the karyopherin family, in particular different importin-α variants8. By specifically recognizing NLSs of cargo proteins, importin-alphas act as an adaptor protein for the nuclear transport, through a complex with an importin-β receptor9. Once inside the nucleus, the heterotrimeric importin-β:importin-α:cargo complex interacts with Ran:GTP, resulting in dissociation of the cargo from its carrier concomitant to the hydrolysis of GTP11.Transport of large proteins (>40 kDa) into the nucleus is generally mediated by the presence of a nuclear localization signal (NLS). NLSs are often made by short motifs with basic amino acids18. Recently, two crystal structures of the NLSs of influenza virus NP bound to importin-α have been published: the structure of NLS1 (residues 3–14) bound to the minor NLS-binding pocket of importin-α and the structure of NLS2 (residues 213–216) interacting with the major pocket of importin-α20. Both NLSs bind to importin-α with high dissociation constants (Kds between 2 and 5 µM for NLS1 and 70 µM for NLS2). Wu and coworkers suggest that importin-α can bind to both sites, either on the same monomer of NP or on two different protomers inside the NP-trimer, so that the synergy of the two sites is strong enough for the transport of NP into the nucleus20. Phosphorylation sites have been identified in the NLS1 of A/NP  suggesting that phosphorylation at these sites prevent the interaction of NLS1 with importin-α22.Two putative NLSs have been described in influenza A NP (A/NP): NLS1 is a non-classical (ncNLS) motif in the first 14 amino acids of the unfolded N-terminal region, while NLS2 consists of residues 198–216 located at the surface in the middle of the protein24. The main difference between the two proteins lies in their N-terminal extremities: A/NP has an N-terminal tail of about 20 residues with NLS1 whereas B/NP has a 70-residue N-terminal tail  in complex with importin-α was mapped using NMR spectroscopy, revealing the extent of the interacting region.With this paper we present biochemical and biophysical data that demonstrate that the 70 N-terminal amino acids of B/NP are intrinsically disordered. We show by size exclusion chromatography, NMR and SAXS, that this tail binds to human importin-α7, with a TAIL) using NMR spectroscopy and we therefore obtained the complete backbone resonance assignment of B/NPTAIL. We calculated secondary structure propensities (SSPs) based on experimental Cα and Cβ chemical shifts, which indicate that no strong propensities for either α-helical or extended structure exist  with this conformational ensemble , B/NP is mainly monomeric32. B/NP (residues 1 to 561) and B/NPCORE (residues 71 to 561) were injected separately or in complex with importin-α7, on a size exclusion chromatography column (SuperdexTM increase 200 10/300 GL column). B/NP and importin-α7 alone are both eluted as a single peak, with a respective elution volume of 13.8 mL and 13.7 mL . The elution peak at 10.5 mL corresponds to importin-α7 alone and the peak at 10.1 mL corresponds to the complex between B/NPTAIL and importin-α7. These results confirm that importin-α7 binds the full-length influenza B nucleoprotein and its N-terminal tail but not B/NPCORE.We have previously shown that, under the same experimental conditions as used here 33. A classical NLS is predicted, starting from residue 41. The analysis of the sequence by a prediction server that is specialized for the prediction of composite motifs, cNLS Mapper34 predicts indeed a bipartite NLS between residues 41 and 68. Interestingly the region of residues 41 to 68 also contains the region with increased helical propensity – a frequent observation in case small recognition motifs are encoded in an otherwise disordered chain. Residues 30–39, however, are additionally involved in the interaction and show characteristics of two conformational states in slow exchange on the NMR chemical shift time scale, visualized by peaks splitting in the course of the titration. While the ncNLS of influenza A has been shown to bind to the minor groove of importin-α120, bipartite NLS motifs are thought to bind both the major and the minor binding site35. The extended NLS region in B/NP may explain the convolved dynamic features observed in the interaction, switching between slow exchange from residues 30–39 and fast or intermediate exchange in the region of residues 41–50  were estimated (Table g) and Dmax values of each sample are listed in Table g) and the mass (through the extrapolated intensity at zero scattering angle (I(0))) of the analyzed sample. An absence of linearity in the Guinier plot reflects the presence of aggregates and/or attractive/repulsive interactions between the scattering particles. We have combined the processing of the dataset in order to further localize the B/NP NLS binding site on the importin-α7. The program MONSA builds ab initio shapes using the scattering curves for the complexes together with those of their individual components38. While the shapes obtained from a protein containing an intrinsically disordered tail will not be expected to provide realistic shapes . The experiment was done in triplicates. One of the titration curves is shown in Fig. TAIL binds human importin-α7 with a Kd of 844 nM, similar to the affinity measured between A/NP and the mouse importin-α1 (Kd between 2 and 5 µM)20. We tried to repeat the experiment with the full-length B/NP, but because of its tendency to adopt tetramers under these conditions, a meaningful interpretation of the ITC data could not be obtained.We have determined the binding affinity of B/NPTAIL samples a large space. The long N-terminal tail of influenza B NP can therefore change how the B/RNPs (RNPs from influenza B virus) pack together in comparison to what has been shown for influenza A39. CryoEM of paramyxoviruses like Sendai or measles viruses41, which have very long NTAILS43 shows that the nucleocapsids take a large volume inside the viral particle, unlike the nucleocapsids from rhabdovirus particles, because the nucleocapsids of rabies and VSV have no NTAILS45. In the influenza A viral particle the RNPs form the oval shape of the virus with a tight enwrapping of the envelope47 whereas the particles from Influenza virus B seem to be more loose and spherical50.This paper shows that the long N-terminal region of influenza B nucleoprotein is disordered. Figure TAIL carries the NLS of the protein. Whereas the non-classical NLS1 of A/NP is rather short with about 11 residues, the NLS of B/NP show three overlapping NLS sequences from residue 30 up to the end of the tail at residue 71. This result could explain why stepwise shortening of the tail gradually decreases the propensity of NP to go into the nucleus implicating a longer stretch in the interaction with importin-α rather than just a 4 residue linear motif between 44 and 4728. The complex between importin-α7 and B/NP was studied with SAXS. The analysis shows the volumes of the two proteins. Although the SAXS envelope of a disordered chain cannot give a single structure for B/NTAIL the complex shows that importin-α binds two thirds of the tail, concordant with the results from NMR, and no other parts of B/NP.Like the shorter N-terminal end of influenza A, the B/NPTAIL and importin-α we used importin-α7. For efficient virus replication in vivo, avian influenza viruses depend on importin-α3, whereas mammalian viruses depend on importin-α751. However, all importin-alphas are very similar in their sequence and their structures and a specific importin-α can bind a very large range of proteins. The affinity between B/NTAIL and importin-α is rather low (Kd around 900 nM) as measured with ITC and close to the affinity between the A/NTAIL and mouse importin-α1 (1.7 µM). This affinity is much lower than the affinity between the influenza polymerase protein PB2 and importin-α which has a Kd value of around 5 nM52. Maybe one of the reasons is that the infected cell makes a lot of NP and only very little PB2, at least in earlier hours of the infection54.For the complex between the B/N56, recent advances have demonstrated that these flexible domains can be targeted60 and biophysical descriptions of the behavior of linear motifs such as the NLS, and their complex interaction modes, will no doubt aid in the conception of rational peptide or small-molecule based strategies.Despite its intrinsic flexibility, the disordered N-terminal tail of NP may present a viable target for inhibition of viral infection, due to its essential role in nuclear import. Although the development of inhibitory molecules for intrinsically disordered targets represents a significant challenge52. The full length B/NP , its N-terminus  and its core , were cloned in the pETM11 vector (EMBL) to express N-terminal His-tag TEV protease-cleavable constructs. Escherichia coli BL21 (DE3) cells transformed with the corresponding plasmids were induced 12 hours by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C and collected by centrifugation. For B/NP constructs, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 M NDSB201 (Sigma), 5 mM β-mercaptoethanol (β-ME) and cOmplete EDTA-free protease inhibitor cocktail (Roche). For importin-α7, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM β-ME and cOmplete EDTA-free protease inhibitor cocktail. Purifications were performed at room temperature. Proteins were purified by Ni2+ affinity chromatography . For the B/NP and the B/NPCORE, this step was followed by a heparin column . Heparin elution fractions or nickel elution fractions were dialyzed overnight at 4 °C with TEV (1/100) against 20 mM Tris-HCl pH 7.5 at 150 mM, 5 mM β-ME and 20 mM imidazol, followed by Ni2+ affinity chromatography to remove the His-tag and the TEV protease. The proteins were then purified by size-exclusion chromatography using a SuperdexTM 200 increase 10/300 GL column . For ITC and NMR, the His-tag of B/NPTAIL was removed and purified by size-exclusion chromatography . The purities of the samples were confirmed by SDS-PAGE. Proteins were concentrated by centrifugation (Amicon concentrators with cutoff of 3 and/or 10 kDa). Protein concentrations were determined using the extinction molar coefficient at 280 nm ε = 46 785 M−1.cm−1 for importin-α7, ε = 24 995 M−1.cm−1 for B/NP and B/NPCORE and ε = 2980 M−1.cm−1 for B/NPTAIL. For B/NPTAIL without the his-tag, the concentration was determined using a bicinchoninic acid protein assay .The cloning of the human importin-α7  DNA coding sequence was described in13C, 15N proteins the medium was supplemented with 1.0 g.L−1 of 15NH4Cl  and 2.0 g.L−1 of D-glucose U-13C6 . Purification protocol was the same as above and all NMR experiments were performed in 20 mM Bis-Tris buffer pH 6.5, 50 mM NaCl.Samples for NMR spectroscopy were produced in M9 minimal medium containing MEM vitamins (Gibco). For producing 13C, 15N B/NPTAIL was obtained from a set of BEST-type triple resonance spectra: HNCO, intra-residue HN(CA)CO, HN(CO)CA, intra-residue HNCA, HN(COCA)CB, and intra-residue HN(CA)CB61. All assignment spectra were recorded at a 1H frequency of 700 MHz and at 5 °C. The spectra were processed with NMRPipe62 and automatic assignment was done with the program MARS63 and manually verified. Secondary chemical shifts were calculated using the random coil values from refDB64.Spectral assignment of TAIL was calculated based on the chemical shifts as obtained from the assignment using a combination of Flexible-Meccano and the genetic algorithm ASTEROIDS. Five times 200 conformers were selected from a statistical coil ensemble of 10,000 conformers31. A new ensemble of 8,500 conformers was generated based on the φ and ψ angles of the selected conformers, and supplemented with 1500 structures from the initial ensemble. This ensemble was subjected to another round of ASTEROIDS selection and this iteration was repeated four times30 until convergence was achieved with respect to the experimental chemical shifts.A multi-conformational model of B/NP15N labelled B/NPTAIL with importin-α, 1H-15N HSQC spectra of 71 μM B/NPTAIL were recorded at increasing concentrations of importin-α  at 25 °C and a 1H frequency of 850 MHz. 15N R2 (CPMG) were recorded at a 1H frequency of 950 MHz in the absence and presence of 55 μM importin-α at 25 °C using delay times of 4, 8, 20, 40, and 68 ms to measure the magnetization decay65. The time point at 8 ms was repeated for error estimation. Assignment experiments were performed with the purification tag present, whereas all interaction experiments with importin-α were performed with the purification tag cleaved off B/NPTAIL.For titration of TM 200 increase 10/300GL column for B/NP and B/NPCORE and a SuperdexTM 75 10/300GL column for B/NPTAIL. 300 μL of each sample  were incubated 1 hour at room temperature before injection.All size exclusion chromatography (SEC) experiments were performed in 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME using a SuperdexTM increase 200 5/150 GL column  followed down-stream by SAXS sample capillary. SAXS measurements were performed every second with a Pilatus 1 M detector (distance of 2.87 m) allowing a q range of 0.03 to 4.5 nm with a wavelength of 0.01 nm.All datasets were collected on BM29 (ESRF). For in-line SEC-SAXS, the experimental setup consists of High Pressure Liquid Chromatography (HPLC) system connected to an analytical Superdex66. To verify the molecular mass, the Rambo and Tainer method was used67. Rg predictions using Guinier extrapolation were plotted against the elution volume to select the most monodisperse part of the protein elution peak. SAXS datasets within this zone were scaled and averaged to produce one unique I(q) curve. Distance distribution functions p(r) were calculated using the program GNOM68. The ab-initio models were generated by MONSA using when available, the individual data sets in order to fit them simultaneously37. Homologue PDB structure comparison was assessed using Crysol69. Homologue structure fitting within the DAMAVER envelope was performed with PyMOL70 and curve representations using Graphpad (Prism). SAXS curves with B/NP alone contained the poly-His tag, which is cleaved off for all samples probing interaction with importin-α.Experimental curves were subtracted and analyzed using Primus (ATSAS programs suite)23) and Flexible-Meccano30 to add the intrinsically disordered tail as a conformational ensemble. The backbone dihedral angle distribution identified by a Flexible-Meccano/ASTEROIDS31 combination to describe B/NPTAIL (see above) was assumed to be valid also within the full length protein construct. 200 conformers were generated and SAXS curves for each of the conformers were calculated using CRYSOL69 and averaged to obtain the expected SAXS curve for the full length B/NP ensemble.A description of the conformational ensemble of full length B/NP was obtained using the crystal structure of the folded domain  before the titration. The ITC titration experiments were done at 20 °C using a MicroCal ITC200  with 16 × 2.4 µL injections of 150 µM importin-α7 into a 20 µM B/NPTAIL solution. Integration of the titration curves was performed using the ORIGIN software  to extract thermodynamic parameters, stoichiometry N, equilibrium association constant Ka and the binding enthalpy ΔH. The Gibbs free energy of binding ΔG was calculated from the relation ΔG = −RT ln(Ka) and the binding entropy ΔS was deduced from the equation (ΔG = ΔH − TΔS). All titrations fit the single-binding site mechanism with 1:1 stoichiometry and binding parameters were calculated as the average of three independent experiments ± SD.B/NPSupplementary Information


Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5´-phosphate-dependent, 5´→3´ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5´-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell.Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5′-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior.We have found that in the pathogenic yeast Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry. Candida albicans the genes coding for rRNA (rDNA) are repeated multiple times in a tandem fashion [Ribosomes, the protein producing organelles of eukaryotic cells, are composed of four individual ribosomal RNAs (rRNA) and combined with 79 proteins . In yeas fashion . The ult fashion . RNA pol fashion . These t fashion . RNA pol fashion . Finally fashion .Candida albicans have been a focus in our laboratory [http://www.epibio.com/enzymes/nucleases-glycosylases-dna-binding-proteins/rna-exonucleases/terminator-5-phosphate-dependent-exonuclease) [Studies of ribosomal RNA (rRNA) in boratory –11. We rboratory . It doesuclease) . SurprisCandida albicans SC5314 grown overnight (16 h), therefore in the stationary phase, and we found that both 18S and 25S resisted the exonuclease digestion. Figure Our initial use of Terminator was with total RNA isolated from We did a more detailed timing experiment to see how the resistance development was related to growth phase Fig. a. As canhttps://www.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip) resulting in 5′-OH, again preventing exonuclease digestion.We carried out serial enzyme digestion experiments to examine whether the resistance to Terminator resided at the 5′-position of the rRNAs. In one experiment, we used tobacco acid pyrophosphatase (TAP) which cuts specifically between the α and β phosphate groups resulting in a monophosphate 5′ end. When TAP was followed by Terminator treatment, the previously preserved bands were eliminated Fig. d. AnotheTo see whether the transcription of these molecules was related to TOR activity, we exposed cells to rapamycin (1 µg/ml) for the same length of time as control cells (6 h), both in fresh YPD. Total RNA was extracted from the two conditions and submitted to Terminator. As can be seen on Fig. C. albicans for ribosome production, we isolated ribosomes by centrifugation through a sucrose gradient. Following RNA purification and precipitation, we performed a Terminator treatment  and our additional digestion experiments, point toward the 5′-end of these molecules, where the enzymes encounters resistance. Most unexpected was that TAP treatment allowed Terminator to fully digest both 18S and 25S. This enzyme cuts specifically between phosphate groups which can result in a monophosphate 5′end exactly what is required by Terminator. If correct, this would indicate that there is an additional phosphate at the 5′-end of these molecules. Diphosphorylated mRNA molecules have recently been described in Escherichia coli (E. coli) [E. coli. Another possibility it raises is new transcription of these molecules. This would be most remarkable, as transcription and processing of rRNAs has been extensively studied and transcription of individual 18S and 25S has not been seen. Clearly, any new transcription and the identification of the polymerase doing it would require much more direct evidence.Our data indicates that as ablished  but theyablished  where weE. coli) . These wC. albicans, a number of genes have been found to be expressed only in the stationary phase with some of them playing important roles in virulence [Inhibition of TOR function by rapamycin resulting in similar 18S and 25S molecules reinforces our observations with nutritional depletion. It might indicate that their production is independent of TOR regulation or that their production is suppressed by TOR during adequate nutritional availability. We also show that the development of these resistant 18S and 25S molecules is not just an incidental process, since the cells incorporate them into their ribosomes. As organisms have their nutrition depleted they do enter the stationary phase which allows them longer survival , 18. Theirulence . Thus, iFinally, survey of other yeasts finds that they also produce the same molecules while approaching the stationary phase, indicating a conserved role for some biological processes for them.Terminator, a 5´-phosphate-dependent processive 5´→3´ exonuclease, has revealed 18S and 25S rRNA molecules that differ from the currently recognized ones processed from full length transcripts. They were found in yeasts approaching decreasing nutritional states and those with TOR inhibition with rapamycin. What is known about the enzyme and our data point to a change at the 5′ side of these molecules but the basis of this resistance is unknown. The finding of the same results in three different yeast species suggests a preserved role for these changes in the biology of these organisms.Candida albicans SC5314 (ATCC MYA 2876), Candida krusei (ATCC 14243), and Saccharomyces Cerevisiae AM109, (generous gift from Gera laboratory) were maintained in 50% glycerol in YPD broth  at − 80 °C. Cells were activated in YPD broth at 30 °C and maintained on Sabouraud dextrose agar at 4 °C, passaged every 4–6 weeks up to 4–5 times. Yeasts were lifted from agar surface and grown in YPD broth for variable length of times at 30 °C. Schizosaccharomyces Pombe  was obtained from Forsburg laboratory. For some experiments 1 μg/ml of rapamycin (EMD Millipore) was added to fresh YPD. Yeast cell concentrations were established using a hemocytometer.Cells were collected by centrifugation, washed with sterile phosphate buffered saline (PBS) and were put on ice pending total RNA extraction. Cells were disrupted with RNase-free zirconia beads and RNA was isolated using Ambion RiboPure RNA Purification kit for yeast (Ambion/ThermoFisher) according to the manufacturer’s instructions. RNA quantification was done using a Qubit 2.0 fluorometer.Total RNA was treated with Terminator (Epicentre) following the manufacturer’s protocol using Buffer B. The ratio of enzyme to substrate used was 1 U per 1 µg of RNA to ensure adequate cleavage.® pcr4 vector followed by transformation with TOP-10 chemically competent cells. Several colonies were screened for insert presence and sequenced (Laragen Inc). For probe preparation, bacteria were grown in Terrific Broth (Fisher) with ampicillin at 50 μg/ml, and plasmids were isolated with a QuickLyse Kit (Qiagen). Inserts were released with BamH I/EcoR I for 25S and 5S, and BamH I/NcoI for 18S, and were purified with a QIAquick Gel Extraction Kit (Qiagen). 50–100 ng of purified) inserts were biotinylated using the EZ-Link™ Psoralen-PEG3-Biotin (ThermoFisher Scientific). Membranes were pre-hybridized at 55 °C in North2South hybridization buffer (Pierce) for 30 min followed by overnight hybridization with denatured biotinylated probes at 5 ng/ml in the same buffer at 55 °C. Membranes were washed with low and high stringency buffers at room temperature and RNA was detected using Chemiluminescent Nucleic Acid Detection Kit (Pierce) according to the manufacturer’s protocol. Film was developed with the SRX-101A Konica film processor.The same amount of RNA was used for all conditions. RNA was separated on pre-fabricated formaldehyde agarose gels (Lonza) and stained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies) for 30 min. Gel images were captured with a digital camera (Canon Vixia HFS30). RNA was transferred by electro-blotting (Thermo Scientific Owl Hep-1) to a positively charged nylon membrane (Life Technologies) in 0.5 × TBE (standard Tris/Borate/EDTA buffer). The RNA was cross-linked to the membrane using UV (Stratagene UV Crosslinker). Probes specific for 25S, 18S and 5S components of the ribosomal RNA were prepared by PCR using specific 18S, 25S and 5S primers. PCR products were cloned into TOPO® measurement.RNA obtained from either stationary or mid-log organisms was treated with Alkaline phosphatase (Epicentre), Tobacco Acid pyrophosphatase (New England Bio) or 5′-Phosphate-Dependent Exonuclease (Terminator) (Epicentre) according to the manufacturer’s instructions. For experiments where sequential enzyme was required, RNA precipitation was performed immediately after first enzyme treatment using Acid-Phenol:Chloroform (Ambion) separation and ethanol precipitation. RNA quantity was established by Qubitalbicans were grown in YPD at 30 °C. Ribosome isolation was done by following a previously described protocol [® fluorometer (Life Technologies). Extracts (0.2 ml) containing 2–4 mg of total protein were loaded onto the top of 7–47% sucrose gradient. Fractions of 1 ml were collected by pipetting from top of the column and placed in 1.7 ml tubes containing 100 µl 10% SDS. Each sample was digested with 11 µl proteinase K (20 mg/ml) for 30 min at 37 °C. Fractions of stationary RNA were precipitated individually while mid-log fractions were pooled prior to precipitation. RNA was measured using a Qubit® fluorometer. RNA was digested with Teminator™ and electrophoresed through formaldehyde agarose gel along with undigested RNA as control.Stationary and mid-log C. protocol . Total p


Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disorder of unknown aetiology estimated to cause severe, indefinite fatigue in over 350,000 Canadians . It has Metagenomics provides a new avenue to investigate microbial causes for diseases of unknown aetiology. Combined with next-generation sequencing, a metagenomic approach enables researchers to sequence all of the nucleic acid in a sample in order to examine its microbial content. Compared to targeted discovery approaches looking for specific microbes, metagenomics offers the distinct advantage of discovering unexpected microbial associations .Despite its value as a holistic approach for investigating disease causation, metagenomics is not without its difficulties. Multiple studies have demonstrated contamination in metagenomic experiments from both environmental and reagent sources –9, meaniDespite these limitations, we chose to use a metagenomic approach to investigate potential microbial associations with ME/CFS, as we believe this to be the most sensitive and unbiased approach currently available. We also investigated a second disease of unknown aetiology, namely alternatively diagnosed chronic Lyme syndrome (ADCLS). ADCLS has a similar phenotype to ME/CFS, but is diagnosed on clinical grounds using alternative tests, the validity of which is questioned by major reference laboratories and the CDC , 11. We Recruitment was performed as detailed in Patrick et al. 2015 . In shorWhole blood was drawn from participants into EDTA Vacutainer tubes  and the plasma isolated and stored at -80°C until all collections were complete. Plasma specimen volumes ranging from 800 μL to 1 mL were then extracted using the NucliSens easyMAG system . A positive control consisting of a single patient respiratory specimen positive for human metapneumovirus (hMPV) by real time (RT)-PCR spiked into a pool of patient respiratory specimens negative for common respiratory viruses was extracted in parallel. Nuclease-free water  was included as a plasma-free negative control. All samples were eluted in 35 μL of elution buffer (bioMerieux). For each sample and control, 22.4 μl of the extracted nucleic acid was treated with Turbo DNase  for 30 minutes at 37°C. Each reaction was terminated using 0.1 X volume of inactivation reagent , followed by 5 min incubation at room temperature, centrifugation at 10,000 g x 1.5 min, and finally supernatant containing RNA was transferred to a new tube.An 8.5 μl aliquot of RNA was reverse transcribed with tagged random nonamers (5′-GTTTCCCACTGGAGGATA-N9-3′), and subsequently amplified as part of a modified two-step procedure, as previously described , 15. AmpSequencing was performed on an Illumina HiSeq 2000 sequencing system at McGill University and Genome Quebec Innovation Centre with 100 bp paired-end output . Nucleic acid concentrations were evaluated throughout the process using Qubit DNA and RNA High Sensitivity assay kits in a Qubit 2.0 fluorometer .2, 0.2-mM nucleotides, 0.4 μM of primers, 1.25 U of Hot Start Polymerase , 1 μl of template total nucleic acids, and water in a 25 μl volume. Thermocycler conditions were as follows: 94°C × 5 min, 35 cycles of 45 s at 94°C, 45 s at 50°C, and 60 s at 72°C, and a final cycle of 10 min at 72°C. Nuclease-free water  and E. coli genomic DNA were used as a negative and positive control, respectively. Aliquots of 5 μl for each PCR reaction were analyzed on a 1.5% agarose gel.To confirm the presence of bacteria identified by metagenomics, we performed conventional PCR amplification of the V3-V4 regions from the 16S rRNA-genes on the nucleic acid extracts prior to the DNase treatment and random amplification step. Each PCR reaction consisted of 1.5-mM MgClhttp://picard.sourceforge.net). Illumina adapters and remaining Sol-B primer sequences from library preparation were removed using cutadapt v1.3 in paired end mode, with minimum length 70 bp [Sequence files were downloaded as de-multiplexed BAM files and converted to fastq format using picard-tools-1.74  and human rRNA (ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens/RNA/). After extracting the unmapped reads using samtools v0.1.19 [http://www.arb-silva.de/). Finally, prinseq-lite was used in single end mode to remove any duplicate single reads, not accounting for read pairs. Bacterial taxonomies were identified from the filtered non-host reads using Kraken [Host subtraction was performed using bowtie2 v2.1.0 –very-sensitive  against  v0.1.19  and bamt v0.1.19 , further v0.1.19  to the Lg Kraken  v0.10.6 g Kraken  for taxoStatistical analysis was performed using R version 3.2.4. Clustering and heat maps were drawn using heatmap.3, multivariable regression performed using generalised linear models, and plots drawn using ggplot2.Twenty-five ME/CFS cases, 25 healthy controls, 13 ADCLS cases and 11 SLE controls were included in the final study. Demographic details of the participants recruited are given in P<0.0001, Kruskal-Wallis), therefore, read counts were normalised by the number of filtered reads prior to host subtraction. A summary of read statistics for each sample can be found in Sequencing was performed in three batches, with RNA extracted and libraries prepared on three separate occasions. Across all batches of sequencing results, there was a mean of 16,855,866 paired end reads sequenced per sample, of which the majority were removed during quality filtering and host subtraction, leaving a mean of 282,525 reads for analysis. We identified a significant difference in number of reads produced between sequencing batches  phyla and 254 (49.6%) genera were present in one or more of the plasma-free negative control samples. RNA from Proteobacteria, Firmicutes, Actinobacteria and Bacteriodes were all detected at high abundance in both study samples and plasma-free negative controls .In order to determine whether there were differences in RNA levels between samples, hierarchical clustering was performed. Hierarchical clustering provided no evidence of samples clustering according to study group, with the exception of the plasma-free negative control samples, which clustered together. However, clustering by sequencing batch was observed, with sequencing batch 3 largely separating out from the other two batches.To further investigate the relationship between bacterial RNA content and study group or sequencing batch, Principal Component Analysis (PCA) was performed at the phylum and genus levels using the normalised read abundances. Neither analysis showed samples from the same study group clustering together; however, samples did cluster according to the batch in which nucleic acid extraction and sequencing were performed .Propionibacterium, Pandoraea, Acidovorax, Rubrivivax, Cupriavidus, and Alteromonas (P<0.005), however, RNA corresponding to these six genera also differed significantly between sequencing batch. There were also 36 genera whose RNA differed significantly between sequencing batch only (P<0.005), indicating that sequencing batch had an effect on the RNA levels of more genera than study group  also had high levels of RNA in the plasma-free negative control samples and are known common contaminants [Pandoraea and Rubrivivax) did not differ significantly in RNA abundance between any single study groups using a Wald test to determine the effects of the individual groups [Of the six genera whose RNA differed significantly between study group and sequencing batch, four  matching to RNA from bacterial phyla. This suggests that the level of bacteria is very low in our samples, and it is even possible that the observed bacteria came from contamination during sample processing. As the levels of bacteria were so low based on the PCR, results are highly subject to contamination and minor variances from extraction and other laboratory processes, highlighting the need for controls and tracking of reagents lots, as well as the batching of extraction, library preparation and sequencing runs.We also investigated the study samples for viral RNA content. Due to the comparatively small genome size of viruses compared to bacteria, we expected viral RNA to be present at lower abundance in our samples. Therefore, we used the more sensitive BLASTn megablast to map all filtered reads to the NCBI RefSeq viral database. RNA from six and 47 viral phyla and genera were identified respectively, of which RNA from three (50%) and 15 (31.9%) were in at least one plasma-free negative control sample . In the Betabaculovirus, significantly differed between study groups after Bonferroni correction. However, despite reaching significance at the group level, no single study group differed from any other in its level of Betabaculovirus RNA using a Wald test. This virus also differed significantly between sequencing batch, with sequencing batch 3 having significantly higher abundance of Betabaculovirus RNA using a Wald test (P<0.0005). Such a significant difference between batches suggests that the batch effect was far larger than the study group effect. Furthermore, RNA from Betabaculovirus was present in the plasma-free negative control samples, particularly the one from batch 3, and since it is a dsDNA virus, we are measuring transcription rather than DNA copy number. Also, its natural hosts are arthropods, supporting our conclusion that it is a contaminant rather than a true component in human plasma. See We again used multivariable regression to investigate the effect of study group and sequencing batch on viral RNA content, revealing that RNA from only one virus, Our metagenomic investigation of plasma RNA content revealed significant variation between sequencing batches, and potential sample contamination, but did not reveal differences in the plasma RNA content between individuals with ME/CFS or ACDLS and healthy controls or controls with SLE. Our findings highlight the necessary care that must be taken when performing metagenomic experiments both to control for batch-to-batch effects and the need to include negative and positive controls in the experimental design to identify contaminants.We chose to analyse the RNA content of our samples, meaning that our results represent a composite picture combining the actual cellular abundance with metabolic and replicative activity of the microbes present. This therefore may not entirely reflect the bacterial abundance of each sample. Despite this limitation, batch-to-batch effects and sample contamination were both clearly identified, highlighting their strong impact. It should be noted that it is possible that differences between study groups would have been identified if we had performed DNA analysis, which is as a more precise measure of cellular abundance, however, the large batch and contamination effects, as well as the low levels of microbial RNA present, make this highly unlikely.There has been little research performed on metagenomics of plasma. One study investigated the plasma microbiome in patients with HIV and found it was dominated by the orders Pseudomonadales, Lactobacillus, Burkholderiales, Bacillales and Enterobacteriales . HoweverIn fact, it could be argued that a plasma microbiome does not exist at all in healthy subjects. Such an assertion is supported by the fact that even in the sample with the highest number of RNA reads mapping to bacteria in this study, we were unable to amplify any microbial ribosomal RNA from the plasma extracts. However, we believe it likely that the plasma has some microbial content, even if it is a low concentration of exogenous elements circulating in the blood that act as an indicator of microbial presence elsewhere in the body. As nucleic acid extraction techniques, purity of molecular biology reagents, and availability of methods that require less sample manipulation and amplification continue to improve, it will become possible to study nucleic acids from increasingly small volumes , 32. ThuSince all plasma-free negative controls used in this study were put through every step of our protocol, we are unable to determine where contamination arose. To determine the source of contamination, further experiments would be required, in which a new negative control was introduced at each step. Since our initial aim was not to measure contamination, our study did not use this approach. However, other studies have shown contamination arises from multiple sources, including ultrapure water  and nuclBorrelia, which was proven to consist of largely incorrect alignments when validated using BLASTn. Similarly, for viral alignments, Orthobunyavirus RNA was found in 84/102 (82%) of samples (data not shown), however, Orthobunyavirus is often identified due to non-specific alignments of bacterial sequences to viral databases, thus may not truly be present in our samples. It is likely that aligning all reads to the complete NCBI database using BLASTn would reduce false positive taxonomic assignments, however, such an analysis would be highly time consuming, hence alternative faster alignment methods were chosen.It is also possible that some taxonomies identified could be false positives occurring due to incorrect alignments made by Kraken or BLASTn. Using a bacterial mock community, we calculated that our bioinformatics pipeline has sensitivity and specificity of 98%, suggesting that 2% of reads may be false positive or false negative alignments. This is particularly likely for bacterial genera seen at low levels, for example Lactonifactor and decrease in Holdemania in ME/CFS patients compared to controls [Actinobacteria and one other phylum in ME/CFS patients compared to controls, but no significant differences in bacterial phyla from blood samples. However, both ME/CFS studies were small, with sample sizes of 43 and 10 ME/CFS patients respectively. Furthermore, neither study used a negative control, so they were unable to rule out contamination as a source of their differences. Thus, confirmation of these results requires replication in other populations and laboratories, with careful use of control samples.The lack of a positive association between ME/CFS and ADCLS and plasma microbial content in this study does not mean that these syndromes do not have an infectious cause, and future experiments should examine the microbiome of other body tissues, including the cellular blood microbiome, which may harbour higher concentrations of microbes. The gut microbiome is of particular interest, as it has been associated with other chronic diseases, including for example, inflammatory bowel disease , 34, astcontrols . A seconDue to its exploratory nature, our study had a relatively small sample size (25 ME/CFS participants), which reduced our power to detect differences in the metagenome between study groups. A large sample size is particularly important for investigating ME/CFS, since it is a complex disease defined by a group of symptoms, which are not all required to be present. To reduce within-case heterogeneity, we chose to use the Canadian Case Definition for ME/CFS  as it waIt is also important to perform case-control studies using carefully matched controls. In our study we matched for sex and age within five years. However, it may also be beneficial to match on other characteristics, such as ethnic background or level of activity, as ME/CFS sufferers often tend to be quite sedentary. Additionally, nested case-control studies may increase a study’s power to find significant associations without requiring a large number of ME/CFS participants.Another possible method of matching is to follow the same ME/CFS patient over time. One of the hallmarks of ME/CFS is post-exertional malaise (PEM), which involves symptom flare-up after exertion and which can be simulated using an exercise test. Shukla et al. 2015 measured the blood and stool microbiome of the same patients before and after PEM and were able to find differences in the abundance of seven of the nine bacterial phyla they investigated .Our study was unable to find a positive association between the plasma RNA content of individuals with ME/CFS or ACDLS and healthy controls or controls with SLE. Plasma represents a difficult medium for metagenomic analysis, since very low levels of microbes mean it is very difficult to distinguish the microbiome from background contamination. This is not to say that associations with ME/CFS or other syndromes might not be identified in the microbiome of other body sites, or perhaps in the investigation of host factors such as host gene expression or immunological profiles. However, future exploration must be performed rigorously with large sample sizes, clear definitions and careful use of positive and negative controls.S1 Info(DOCX)Click here for additional data file.S2 Info(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableNumbers are raw read counts.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


So from Shewanella oneidensis and LacY, GalP, GlpT and XylE from Escherichia coli, as well as a known stable mutant of LacY, LacY-C154G. CD stability measurements revealed that these transporters fall into two broad categories. The ‘urea-sensitive’ category includes LacY-WT, GalP and GlpT, which each lose around a third of their secondary structure in 8 M urea and two-thirds in the harsher denaturant guanidine hydrochloride (GuHCl). The ‘urea-resistant’ category includes LacY-C154G, XylE and PepTSo. These resistant transporters lose very little secondary structure in 8 M urea, and LacY-C154G and PepTSo resist denaturation by GuHCl up to a concentration of 4 M. The stabilities of LacY, GlpT, XylE and PepTSo correlated with their crystal structure conformations, implying that a similar conformation is adopted in vitro. The ‘urea-sensitive’ transporters LacY and GlpT were crystallised inward-open states, while XylE and PepTSo were crystallised in occluded states. This study highlights the importance of studying a wide range of similar proteins, as a similar secondary structure and overall function does not necessarily confer the same stability in vitro.Membrane transporters are a vital class of proteins for which there is little available structural and thermodynamic information. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates in eukaryotes and prokaryotes. We have used far-UV circular dichroism (CD) to assess whether transporters from this superfamily have the same chemical and thermal stability. We have compared the stability of five different MFS transporters; PepTThe online version of this article (doi:10.1007/s00249-017-1197-7) contains supplementary material, which is available to authorized users. The Major Facilitator Superfamily is a large group of secondary transporters found in all organisms, responsible for transporting a wide range of substrates Yan . WhetherThe current model for MFS substrate transport is the alternating access mechanism. Although the majority of the MFS high resolution structures are in an inward-facing conformation Yan , there aMembrane proteins, and transporters in particular, are very difficult to study in vitro due to their hydrophobic nature rendering them highly unstable once removed from their native membrane environment. In the case of transporters, the flexibility that is essential for their function further increases their instability in vitro. There is therefore very little information available on the stability of transporters. High resolution structures of mammalian MFS transporters have only recently been solved, after considerable effort  is a commonly used spectroscopic technique, which can provide information on the secondary structure content of proteins in the 170–240 nm wavelength range. The use of Synchrotron Radiation CD (SRCD) is particularly useful, as the high light flux of the synchrotron source enables measurement to lower wavelengths than an in-house machine and gives better quality data with a better signal to noise ratio. SRCD therefore enables measurements of membrane proteins at the lower protein concentrations at which they can typically be worked with, and also minimises the artefacts which arise from absorbance of the sample. The more comprehensive wavelength range also allows for more accurate deconvolution of secondary structure. There have been many recent advances in the use of CD on membrane proteins, including the development of a reference dataset, SMP180, which contains membrane protein structural information to aid deconvolution of CD spectra for more accurate structure determination . In this paper, we have expanded these stability studies by comparing LacY and GalP with the E. coli glycerol-3-phosphate transporter GlpT and the E. coli xylose transporter XylE, a homologue to the GLUT transporters  was purchased from Generon. PepTSo was cloned from pWaldo into pET-28a modified with a 10-His tag for protein overexpression. The pWaldo-GFPe_PepTSo was a gift from So Iwata and Simon Newstead (Addgene plasmid # 58334).All the standard reagents used were purchased from Sigma–Aldrich or Fisher Scientific UK Ltd. All reagents used were of the highest available grade. The >99.5% E. coli BL21-AI, using the vector pET28a modified with a 10-His tag. The purification procedures for LacY-WT and GalP have been published previously  to unfold. Each MFS transporter was measured at a protein concentration from 0.1 to 3.5 mg.ml−1, in 0.01–0.2 mm pathlength cells. Synchrotron Radiation CD (SRCD) was measured using the CD12 beamline at ANKA .CD chemical denaturation was performed and analysed following the method described  were measured at a protein concentration of 0.01 mg mlAll MFS transporters have a very similar secondary structure; two bundles of six α-helices connected by a cytoplasmic loop, with the substrate binding site in between the two domains. The two domains are commonly referred to as the ‘N’ and ‘C’ domains. The CD spectra of all the MFS transporters studied are therefore very similar, and are 70–90% α-helical when the secondary structure content is calculated using the Dichroweb server and the SMP180 reference dataset Below is the link to the electronic supplementary material.


P53, KRAS, or BRCA2 are the most useful biomarkers in clinical practice. In addition to these, recent translational research has provided evidence of new biomarkers based on different molecules involved in endoplasmic reticulum stress, epithelial-to-mesenchymal transition, and noncoding RNA panels, especially microRNAs and long noncoding RNAs. These new prospects open new paths to tumour detection using minimally or noninvasive techniques such as liquid biopsies. To find sensitive and specific biomarkers to manage these patients constitutes a challenge for the research community and for public health policies.Pancreatic ductal adenocarcinoma is one of the most lethal types of tumour, and its incidence is rising worldwide. Although survival can be improved when these tumours are detected at an early stage, this cancer is usually asymptomatic, and the disease only becomes apparent after metastasis. The only prognostic biomarker approved by the FDA to date is carbohydrate antigen 19-9 (CA19-9); however, the specificity of this biomarker has been called into question, and diagnosis is usually based on clinical parameters. Tumour size, degree of differentiation, lymph node status, presence of distant metastasis at diagnosis, protein levels of KI-67 or C-reactive protein, and mutational status of Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in both sexes in the USA. In 2014, the number of deaths from PDAC in the USA was 39,590, and PDAC is the cause of 227,000 deaths per year worldwide . Helicobacter pylori: a meta-analysis comprising seven studies found that presence of Helicobacter pylori was correlated with as much as a 65% increased risk of developing PDAC . Se. Se61]. < 0.001) . Recentlreatment .In addition to these, new molecular biomarkers have appeared which can dissect disease information. Deletions, mutations, translocations, amplifications, overexpression or downregulation of DNA, RNA, protein, or noncoding RNA factors are the most commonly described in scientific reports. A selection of these molecular biomarkers is summarised below to broaden the understanding of their functions and potential clinical uses.P = 0.0049) ..122]. in situ hybridisation, microarray, and deep-sequencing will be considered the best tools for pathological diagnosis in the future (Protein detection has been the gold-standard methodology for pathological diagnosis. Nowadays, immunohistochemistry is losing favour relative to qRT-PCR, and it shows thate future .On the other hand, biomarkers studies sometimes lead to controversial results. Therefore, new biomarkers and larger validation cohorts are required. In addition, only biomarkers that combine high-sensitivity and specificity and being highly cost-effective will be incorporated in healthcare systems.


X chromosome inactivation (XCI) is a dosage compensation process that was adopted by female mammals to balance gene dosage between XX females and XY males. XCI starts with the upregulation of the non-coding RNA Xist, after which most X-linked genes are silenced and acquire a repressive chromatin state. Even though the chromatin marks of the inactive X have been fairly well described, the mechanisms responsible for the initiation of XCI remain largely unknown. In this review, we discuss recent developments that revealed unexpected factors playing a role in XCI and that might be of crucial importance to understand the mechanisms responsible for the very first steps of this chromosome-wide gene-silencing event. This generates a genetic imbalance for ancestral genes between males and females, for which various dosage compensation strategies have evolved across different species. In mammals, females transcriptionally inactivate one of their two X chromosomes in a process called X chromosome inactivation (XCI)4.In many animal species, males and females have unbalanced genetic content regarding the sex chromosomes 7. The Xp is then reactivated at the blastocyst stage and random XCI occurs, implying that either the maternal X chromosome (Xm) or the Xp is silenced8. After this choice is made, the inactive state is inherited throughout cell division. XCI begins with expression of the long non-coding RNA (lncRNA) Xist from the chromosome that will be silenced9. Xist RNA coats the X chromosome incis, resulting first in rapid depletion of RNA polymerase II (RNA Pol II) and euchromatic histone marks (such as histone acetylation)10. Xist RNA-coated chromatin then becomes enriched in repressive histone ubiquitination and methylation marks (H2AK119ub and H3K27me3)12. In addition, Xist RNA coating triggers spatial reorganization of the X chromosome14.In mice, XCI occurs in two waves during early development. A first wave of XCI starts between the 4–8-cell stagecis-limited epigenetic memory of the X chromosome during XCI. The chromatin modifications that decorate the inactive X (Xi) and the kinetics of their deposition have been well defined using immunofluorescence during embryonic stem cell (ESC) differentiation or early embryogenesis11 or chromatin immunoprecipitation for candidate histone modifications followed by qPCR for target genomic regions15. Despite several decades of research, the transcription- or chromatin-associated mechanisms responsible for the initiation ofXist-mediated gene silencing have remained unclear.Chromatin seems an obvious candidate as the template targeted by Xist RNA to carry the18. These approaches identified factors that might have important roles during the initiation of XCI. Highlighting their roles, two genetic screens for factors involved in the onset of Xist-mediated gene silencing (as opposed to its maintenance19) identified the same proteins21.Recently, three different groups developed methods that allow the pull down of endogenous Xist and its associated proteinsIn this review, we will discuss these exciting new discoveries as well as some of the open questions regarding Xist-mediated gene repression during XCI.Xist expression, genes along the Xi start to be progressively silenced, with groups of genes that are silenced at early, mid, or late stages during XCI23. The reasons why the kinetics of gene silencing vary so dramatically from one gene to another are not clear. It has been suggested that silencing might progress as a linear gradient from theXist gene within the X-inactivation center (Xic)24, with the genes closest to the Xic being silenced first. However, although some genes close to the Xic are silenced early, others are not23. In fact, the spread of XCI should be considered in the context of the 3D chromosome organization of the X, whereby distant genomic regions can come in close contact because of chromosome folding25. This was investigated using RNA-antisense purification (RAP) that allows the purification ofXist RNA–DNA contacts26. By sequencing the RNA-bound DNA, the chromosome contact regions of Xist RNA were mapped during XCI, revealing that the earliest regions associated with Xist were those that came into closest proximity to the Xic region based on chromosome conformation capture (HiC) data27. Similar results were found using a capture hybridization analysis of RNA targets with deep sequencing, or CHART-seq28. Both studies further revealed that Xist RNA tends to initially bind in close proximity to active gene-rich regions, correlating with sites of H3K27me3. How do these findings relate to gene silencing kinetics? Although the ESC systems involving doxycycline induction ofXist used in these studies undergo more synchronized XCI, this might not entirely reflect the normal kinetics of XCI. To address this questionin vivo, a recent study examined the X-chromosome-wide gene-silencing kinetics during imprinted XCI, a system with endogenous initiation of XCI that is triggered by paternal Xist expression from the 2–4-cell stage. Single-cell RNA-seq of single blastomeres from F1 hybrid mouse embryos at every stage of pre-implantation development revealed that many of the genes silenced earliest in embryos lie within the firstXist-bound regions as defined in ESCs. However, some late-silenced genes, or even escapees, also lie in their vicinity29. Similar findings were reported in hybrid mouse ESCs during differentiation towards embryoid bodies and neural progenitor cells30. This implies that although 3D proximity toXist indeed seems to promote XCI of early silenced genes, this is not the only factor predicting the silencing kinetics of any particular region. Whether there is local spread incis from Xist-coated regions and whether this relates to the local chromatin context or proximity of certain regions to the nuclear envelope remains to be found.XCI is a dynamic process operating at different levels, including gene silencing, chromatin modifications, and chromosome reorganization  or else in its chromatin structure that prevent Xist RNA from spreading into this region. The sequences responsible for escape from gene silencing remain to be identified. Transgenesis studies have shown thatJarid1c has an intrinsic capacity to escape, withJarid1c transgenes being capable of escape from XCI at different locations on the X chromosome33. More detailed BAC integration studies mapped elements required for escape to the region upstream or within theJarid1c gene34. A candidate sequence-specific escape-promoting factor that has been proposed is CTCF (CCCTC-binding factor)35. CTCF is a zinc-finger DNA-binding protein that has been identified as an insulator between different genomic regions36, maintaining a gene in a spatial functional domain with its own regulatory elements37. CTCF is widely bound across the mouse and human genomes38. It is also bound to the X chromosome, including transcription start sites (TSS) of escapees39, suggesting a possible role for the transcriptional state of these genes. In fact, escape from Xist-mediated gene silencing correlates with the presence of CTCF at the TSS of escaping genes on the X chromosome, but not when aXist transgene is integrated in an autosome . More detailed analysis of the possible role of the CTCF-binding sites flanking theJarid1c locus is still intriguing35. On the one hand, deleting 3’ CTCF sites does not preventJarid1c’s capacity to escape. Rather, this promotes illegitimate escape of neighboring genes. On the other hand, flanking GFP transgenes inserted in theHprt locus with CTCF-binding sites did not prevent its silencing uponXist expression40. Taken together, these data suggest that CTCF might not be sufficient to explain escape. It would be interesting to know whether there is a common mechanism of escape (do other escapees display similar behavior when inserted into different X-linked regions?) and whether there are common genomic or epigenetic features or trans-factors involved.How some genes avoid XCI altogether is another interesting question. Several such constitutive “escapees” exist in mice39. The mechanisms allowing such regions to overcome the heterochromatic state and become reactivated remain unclear. In the environment of the silent X, where many chromatin modifiers play a role in locking in a very stable silent state, it will be important to understand the features of regions that can revert from this repressive state and the extent to which this is driven by specific transcription factors, chromatin states, chromosome architectural proteins, or nuclear compartments.In addition to genes that constitutively escape from XCI, tissue-specific escapees that are silenced during XCI in pre-implantation embryos and become reactivated in certain tissues also exist. Whether Xist-binding sites are found in the vicinity of such loci when they are silenced but then disappear in the tissues in which they escape is not yet known. The nature of the regulatory regions and epigenomic features of these escapees was recently hinted at based on allele-specific ATAC seqXist expression is accompanied by global changes in chromatin and gene expression. Genetic dissection of the Xist transcript revealed that a highly conserved region called the A-repeat (on exon 1)41 is necessary for Xist’s gene-silencing role during XCI42. However, the mechanisms responsible for gene silencing and the importance of chromatin-modifying complexes for the initiation of gene silencing are still largely unknown. Most of the studies characterizing Xi chromatin were based on indirect immunofluorescence approaches, which are quite crude assays and might highlight only the players that are more enriched in the Xi territory. Additionally, it was observed that the exclusion of RNA Pol II and histone acetyl marks from the Xi territory precedes gene silencing, indicating that there might be other protein complexes important for the initiation of X-linked gene silencing.As mentioned above,18. One reason for this was that RNA pull downs often retrieve many non-specific interacting proteins that bind nucleic acids in general. Another challenge was the length of the Xist RNA molecule , which renders it difficult to manipulate and to use for capture while maintaining its integrity.One of the obvious first steps needed in order to learn about the mechanisms involved in Xist-mediated gene silencing is the identification of the protein, RNA, and DNA partners of Xist RNA that likely mediate its functions. This represented a technical challenge for many years, as Xist RNA pull down experiments present many difficulties and only recently have systematic approaches yielded conclusive data18. Many of these have turned out to be important for the initiation of gene silencing during XCI. Furthermore, genetic screens were applied to identify factors that impair Xist-mediated gene silencing21.In the past year, several groups have finally been able to isolate factors that bind Xist RNA specifically using a variety of biochemical approaches that rely on the purification of Xist and its binding partners followed by mass spectrometryDrosophila melanogaster43). Spen is a very large protein with several RNA-binding domains. It has been implicated in transcriptional silencing owing to its interaction with the NuRD-MBD3 complex, more particularly via HDACs 1 and 245 or HDAC346. Spen is proposed to facilitate the initiation of XCI through direct binding to the Xist RNA A-repeat, via its RNA-binding motifs, and by recruitment of HDACs to the future Xi. siRNA-mediated knockdown of HDAC3, but not of HDAC1 or 2, phenocopied the results obtained with Spen knockdown17. In the absence of Spen or HDAC3,Xist RNA coating is normal but is reported to no longer lead to RNA Pol II depletion or Ezh2 enrichment and to result in defective gene silencing. Given its huge size, Spen might interact with multiple different proteins in various complexes. Although a spectrum of Spen-interacting proteins has previously been described46, a Spen-XCI-specific protein complex has not yet been characterized.Thanks to these two types of approach, a set of factors that are necessary for the initiation of Xist-mediated gene silencing has been defined. Amongst these different methods and the different lists of candidate proteins identified, one factor identified in all studies was Spen  and the other indirect (PRC2). However, all of the data to date suggest that none of these PRC complexes are sufficient to induce X-chromosome-wide gene silencing during XCI. Rather, Polycomb and its associated chromatin changes are likely to be involved in maintenance, as has been found in other contexts . This is also supported by studies using inducible Xist transgenes that lack the A-repeat region. These mutants retain the capacity to express Xist and coat the chromosome incis, although to a slightly lesser extent, and they are also able to induce enrichment of H3K27me3 and H2AK119Ub but are not able to induce gene silencing56. Thus, PRC-induced histone modifications are involved in reinforcement and memory mechanisms for gene silencing rather than for initiation.In fact, like PRC2-H3K27me3, PRC1-H2AK119Ub had already been implicated in an early time-window during XCI21. WTAP is a member of a complex responsible for N6-methyladenosine modification on RNA57, or m6A RNA. RNA methylation is an emerging field, and the many possible functions of this RNA modification on RNA stability, translation, or splicing are still being explored58. However, to date, RNA methylation pathways had never been suspected in XCI. WTAP could have a direct role in Xist RNA methylation, improving its stability, or acting as a guiding mechanism for its target sites t sites . Alterna6A methylation. However, it is still not clear whether this RNA modification machinery might also act on mRNAs of X-linked genes upon Xist-mediated recruitment. The exploration of RNA modifications could provide exciting new insights into XCI initiation mechanisms.In summary, the data obtained so far concerning WTAP’s interaction with Xist appears to point to a role in Xist m65, a role for nuclear organization has long been proposed in the process of XCI66. Fluorescence microscopy revealed that Xist RNA accumulation during XCI leads to the rapid formation of a repressive compartment, from which RNA Pol II and transcription-associated factors are excluded67. This repressive compartment is largely made of repetitive elements initially13. Genes become relocated into this compartment as they become silenced, while genes that escape remain at the periphery of this Xist RNA domain. The exact interplay between nuclear organization and XCI has remained an open question, however, and whether gene relocation into the Xist RNA compartment is a cause or a consequence of gene silencing is unclear.Given the large body of early cytogenetic studies on the Xi describing it as a distinct nuclear compartment (the Barr body)70. The active X chromosome, just like the autosomes, is organized into contact domains comprising neighboring chromosome regions called topologically associating domains (TADs) as well as into active and inactive compartments71. XCI triggers the loss of TADs and the formation of two megadomains on the Xi69  as one of Xist’s binding partners retrieved by RAP-MS17 and the fact that its knockdown impairs silencing of specific X-linked genes  led to the proposal that LBR may be necessary to tether X-linked regions to the nuclear periphery and that this environment would somehow facilitate Xist RNA-mediated silencing. Impairment of relocalization of the X chromosome might result in impaired X-linked gene silencing owing to a failure to maintain genes within a confinedXist silent compartment77. However, the Xist RNA-coated X in LBR mutants is still devoid of RNA Pol II and becomes enriched in PRC2, and the effect on chromosome-wide silencing/escape was not examined77. The exact role of LBR recruitment in XCI and its potential collaboration with other Xist protein partners will undoubtedly warrant future investigation.Finally, in addition to Xist’s role in X chromosome organization, it has also been suggested that it might help to bring the Xi to the nuclear lamina. In fact, nuclear positioning is one of the oldest hypotheses for differential treatment of the two Xs in the same nucleus, by localization at the nuclear laminaXist interactors as well as factors identified using genetic screens suggests that even the initiation step of XCI involves a variety of mechanisms. SPEN appears to play a central role and has been reported to interact with HDAC3. This could provide an initiation mechanism via histone deacetylation. On the other hand, WTAP, a member of the RNA methyltransferase complexes, also appears to play a key role in the initiation of XCI by methylation of eitherXist RNA itself or X-linked mRNAs. Only a few chromatin factors were implicated, maybe surprisingly, and although PRC1 was identified as a direct interactor of Xist, PRC complexes are likely to be brought in by Xist to enable the maintenance of gene silencing rather than its initiation, which may involve histone deacetylation. Finally, recent studies also point to alterations in chromosome organization and nuclear localization being involved in the establishment of a stably inactive state, since Xist RNA triggers a massive reorganization of the Xi in the nucleus. This flurry of new results in the field of XCI has opened up many questions, which will hopefully be answered through the careful dissection of the genetic and molecular pathways involved.The emerging picture from recent studies using diverse molecular, biochemical, and genetic approaches is that XCI involves not just one but multiple gene-silencing mechanisms. The challenge now will be to dissect the specific functions and inter-relationships of these different layers of control. The recently published panel of


Trypanosoma brucei. Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome.A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of  Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing. Trypanosoma brucei, the causative agent of African sleeping sickness, is distinguished by a single reticulated mitochondrion containing an unusually large amount of mitochondrial DNA (mtDNA), termed kinetoplast DNA (kDNA). The kDNA comprises ~25 maxicircles and ~5,000 minicircles, mutually concatenated into a single network  pan9–. brucei , 10, 12.in vitro (in vivo. One such complex is the mtRNA-binding complex 1 (MRB1) (T. brucei RGG2 (TbRGG2)  . The MRB1 (MRB1) . This co1 (MRB1)  (for a g1 (MRB1) , 21. Oth(TbRGG2) , 22. RNA(TbRGG2) , 19, 23.10.1128/mBio.02288-16.8TABLE S1 TABLE S1, PDF file, 0.05 MB.MRB1 complex subunits. Listed are the proteins which were detected by various labs as comprising the MRB1 complex. Download Copyright © 2017 Dixit et al.2017Dixit et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the MRB8170 and MRB4160 are unique RNA-binding proteins (RBPs), which were recently shown to bind RNA via a novel and hitherto-undefined RNA-binding domain . These pT. brucei. This quantitative binding assay revealed a high preference of both proteins for maxicircle transcripts. Moreover, binding of both proteins influenced the steady-state abundance of mt mRNAs, as demonstrated by the double knockdown (dKD) of MRB8170 and MRB4160. Rapid tandem affinity purification (TAP) confirmed interaction of both proteins with the core and accessory MRB1 subunits GAP1 and TbRGG2, respectively  , 26 to iectively , 27, andectively . FurtherT. brucei  were tested. Phosphorimaging of the cross-linked RNA revealed that UV cross-linking with a radiant energy ranging from 0.8 to 1.6 J/cm2 was more efficient than 0.4 J/cm2 (2 was applied for preparation of the MRB4160 and MRB8170 iCLAP libraries (We used the iCLAP protocol with the aim of identifying the direct RNA targets of the two accessory MRB1 subunits MRB8170 and MRB4160 in the mitochondrion of . brucei . MRB4160ibraries  and C. Nibraries  and C.10.1128/mBio.02288-16.1FIG S1 2) were separately resolved using SDS-PAGE and transferred onto a nitrocellulose membrane, which was probed using anti-His antibody, recognizing an epitope on the modified TAP tag. MRB4160 was detected at its ~100-kDa size (lanes 1 to 4). **, partially degraded MRB4160 (modified TAP tag) protein. (B) Specificity and efficiency of affinity-purified, UV-cross-linked TAP-tagged MRB8170. The copurified low-RNase I-treated 0.8-J/cm2 UV-cross-linked MRB8170 with modified TAP tag was followed throughout two steps of affinity purification, fractions of which are indicated at the top. The membrane was probed as in panel A. MRB8170 migrated at ~100 kDa (lanes 1 to 7). **, partially degraded MRB8170 (modified TAP tag) protein. (C) Specificity and efficiency of affinity-purified, non-UV-cross-linked TAP-tagged MRB8170. The non-UV-cross-linked TAP-tagged MRB8170 and control parental cell lines were processed and assayed as in panels A and B. MRB8170 migrated at the expected ~100-kDa size (lanes 1 and 6), while there was no signal in the control (lane 3). Labeling as in panel B. (D) TBE-6% urea gel of the amplified iCLAP sequencing libraries from TAP-tagged MRB8170 and MRB4160, plus parental cell lines. The cDNA was size fractionated and amplified separately to reduce any size biases during PCR. Three sizes were cut out and PCR amplified, indicated as high (H) , medium (M) , and low (L) . The H- and M-nucleotide-long libraries were submitted for next-generation sequencing. RT –ve, reverse transcriptase control (lane 7); MRB4160, iCLAP libraries from UV-cross-linked modified TAP-tagged MRB4160 ; MRB8170, iCLAP libraries from UV-cross-linked modified TAP-tagged MRB8170 ; 29-13 negative control , iCLAP libraries from UV-cross-linked parental 29-13 strain (mock affinity purification). After the removal of adaptor sequence, the resulting iCLAP tags were ~30 to 50 bp in size. Download FIG S1, PDF file, 1.3 MB.MRB8170 and MRB4160 iCLAP characterization. (A) Specificity and efficiency of affinity-purified, UV-cross-linked TAP-tagged MRB4160. The copurified UV-cross-linked RNA-MRB4160 complex after two-step affinity purification was confirmed by SDS-PAGE and Western blot analysis. The high-RNase I-treated samples from four different UV doses  was RNase I digested into 60- to 120-nucleotide (nt)-long fragments, reverse transcribed, and subjected to next-generation sequencing . The seqTo analyze the binding of MRB8170 and MRB4160 on maxicircle-derived transcripts, we divided the iCLAP tags into two categories according to their generation from preedited and fully edited transcripts . Since pTo dissect RNA interactions of RBPs that are part of large stable protein complexes, such as MRB1, it is necessary to use extended RNase I digests to generate small RNA fragments. Our protocol produced iCLAP tags ~30 to 50 nt long after the removal of the adaptor sequences. However, a drawback of the short read length is that iCLAP tags mapping to fully edited sequences can also be derived from partially edited mRNAs still undergoing the process. Vice versa, iCLAP tags mapping to preedited sequences can originate from RNAs not yet edited, or from already partially edited transcripts. Thus, in both cases, it is impossible to quantitate the amount of reads originating from partially edited mRNAs, which creates a bias in the numbers of preedited and fully edited iCLAP tags . Approxi10.1128/mBio.02288-16.2FIG S2 x axis. (C) Normalized bar plot with respect to gene length of never-edited transcripts. Three bar plots show the normalized data (using DEX-seq) with respect to gene length of MRB8170 iCLAP tags, MRB4160 iCLAP tags, and RNA-seq reads mapped to never-edited transcripts of the mt mRNA indicated on the x axis. Download FIG S2, PDF file, 1 MB.iCLAP and RNA-seq binding to maxicircle. (A) Pie chart of uniquely mapped iCLAP tags (~30 to 50 nt long) and RNA-seq reads mapped to the maxicircle genome. Percentage of MRB8170 iCLAP tags, MRB4160 iCLAP tags, and RNA-seq reads mapped to preedited and fully edited maxicircle transcripts. Preedited, black; fully edited, gray. Never-edited transcripts were excluded for the analyses. Our protocol successfully obtained ~30- to 50-nt-long iCLAP tags, after the removal of the adaptor sequences. A drawback of the short read length of iCLAP tags is the resulting inability to quantitate the amount of reads originating from partially edited mRNAs; therefore, in both cases, it is impossible to quantitate the amount of reads originating from partially edited mRNAs, which creates a bias in the number of preedited and fully edited transcripts in the iCLAP pie chart. (B) Percentage of RNA-seq reads uniquely mapped to the three classes of maxicircle mRNAs. Three bar plots showing the percentage of uniquely mapped RNA-seq reads to pan-edited transcripts in the preedited and fully edited regions, plus the total of the two (preedited and fully edited), respectively, for each of the mtRNAs indicated on the Copyright © 2017 Dixit et al.2017Dixit et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the COX3, ND7, ND8, A6, CR3, RPS12, ND9, ND3, and CR4), minimally edited  and never-edited  transcript categories. For those transcripts undergoing editing, preedited and fully edited iCLAP tags were combined. The distribution of MRB8170 and MRB4160 iCLAP tags on mtRNAs was compared to their expression level determined by publicly available T. brucei RNA sequencing (RNA-seq) data, which also include transcripts originating from the organelle  correlates with their occurrence in the RNA-seq data (2.2%)  and C. ICOX3 but have minimal binding to ND9, ND3, and CR4. Interestingly, the extent of binding correlates with the number of U insertions/deletions needed to be fully edited  of MRB8170 and MRB4160 in editing, we quantified the binding of both proteins to nine individual pan-edited transcripts using iCLAP tags mapping to preedited and fully edited mRNAs . In agrey edited . Visual nd RPS12  and S4. 10.1128/mBio.02288-16.3FIG S3 ND3, ND9, CR4, COX3, and RPS12). Genomic browser snapshot of iCLAP tags and RNA-seq reads mapped to individual pan-edited transcripts, for which the preedited and fully edited versions are shown separately. The unique cDNA count is depicted on the y axis, and the mapped tag position along a given transcript is shown on the x axis. MRB8170 iCLAP tags are in red, MRB4160 iCLAP tags are in blue, control iCLAP tags are in black, and RNA-seq reads are in yellow. ND3, ND9, and CR4 lack significant iCLAP tags from both MRB8170 and MRB4160 to preedited sequence. The other 6 pan-edited mRNAs  shared various orders of iCLAP tags from MRB8170 and MRB4160, positively correlating with their editing extent. Download FIG S3, PDF file, 1.5 MB.MRB8170 and MRB4160 binding to a subset of pan-edited transcripts . Labeling as in FIG S4, PDF file, 1.6 MB.MRB8170 and MRB4160 binding to a subset of pan-edited transcripts  in MRB8170/MRB4160 dKD cells. Control cells were depleted of ATM1 mRNA, encoding an inner membrane transporter that does not affect mt gene expression . Indeed,Taken together, iCLAP and knockdown data support a role for MRB8170 and MRB4160 in flagging mRNAs for editing, as their absence reduces the abundance of edited transcripts in the procyclic stage.ND4, ND5, and MURF5 mRNAs were represented in more than 90% of the iCLAP tags mapping to never-edited transcripts, while the remainder were derived from ND1, COX1, and MURF1  are part of the main figure  and pan-edited (preedited RPS12) mRNAs. As seen before in the iCLAP data, MRB4160 failed to bind to never-edited transcripts  in cell lines expressing tagged MRB8170-mTAP or MRB4160-mTAP and a parental cell line lacking the mTAP-tag (mock IP). Immunoprecipitated RNA was reverse transcribed, and qPCR was performed using primers recognizing preedited and fully edited versions of pan-edited nscripts  and D.T. brucei containing mTAP-tagged MRB8170 or MRB4160, as well as the parental control cell line, were mixed with the beads. In order to validate this new protocol for its pulldown efficiency, the eluates were first probed with antibodies against GAP1 and TbRGG2 from MRB1, which are known to stably interact with MRB8170 and MRB4160 + RNA binding of the cytoplasmic mRNA-binding protein DRBD18 (We modified the protocol for UV cross-linking and subsequent pulldown of RBPs using oligo(dT) magnetic beads  in T. brin iCLAP . The modin iCLAP . Oligo. The untrimmed autoradiography film is shown. Download FIG S7, PDF file, 1.6 MB.MRB8170- and MRB4160-associated interactome. (A) Western blot analysis of total extracts (lanes 1 and 2) and oligo(dT) eluates (lanes 3 and 4) from MRB8170/MRB4160 and ATM1 RNAi-induced knockdown cells. GAP1 and MRP1 were detected in parallel using the same polyvinylidene difluoride membrane to serve as a loading control for the oligo(dT) experiment. (B) Copyright © 2017 Dixit et al.2017Dixit et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro cross-linking immunoprecipitation (CLIP) assay, using extracts from MRB8170/MRB4160-depleted cells lysed under mild conditions. The lysate was divided into four equal aliquots and subsequently supplemented with recombinant glutathione S-transferase (GST)-tagged MRB8170 (10 and 20 µM), bovine serum albumin (BSA) (20 µM), or buffer alone. The supplemented supernatant was incubated and subsequently in vitro UV cross-linked. TbRGG2 antibody-coated magnetic beads were used to pull down the protein-RNA adducts, followed by 5′ radioactive labeling of the bound nucleic acid. Upon resolution in an SDS-PAGE gel, the immunoprecipitated RNA-TbRGG2 complex was transferred onto a nitrocellulose membrane. The resulting autoradiogram depicted a direct relationship between the supplemented recombinant GST-MRB8170 in the supernatant and the amount of RNA bound to TbRGG2  tail implicates the polyadenylation mediator complex (PAMC) as yet another player in maintaining the steady-state level of some maxicircle transcripts  they interact with MRB1 core proteins, and their simultaneous depletion compromises the overall integrity of MRB1; (ii) they share a number of RNase-resistant interacting partners outside MRB1 that belong to other processing complexes; (iii) they bind both preedited and edited mRNAs; and (iv) their simultaneous depletion affects the steady-state abundance of all three categories of maxicircle mRNAs. Below, we elaborate on the basis for these conclusions, ultimately proposing a model of how MRB1 functions in shaping the mt transcriptome.in vivo mt mRNA interactome pulldown experiments were carried out in the presence and absence of both MRB8170 and MRB4160. In the latter samples, TbRGG1, TbRGG2, MRP1, and Nudix hydrolase showed a substantial reduction in poly(A) RNA binding. These results allow us to postulate that TbRGG1, TbRGG2, MRP1, and Nudix hydrolase require the assistance of MRB8170 and MRB4160 to bind mRNA. The in vivo data were further supported by the observation that addition of recombinant MRB8170 was sufficient to enhance poly(A) RNA binding of TbRGG2 in vitro. Taken together, we provide strong evidence that MRB8170 and MRB4160 enhance the activity of other mt RBPs, presumably by attracting or stabilizing them to transcripts already decorated by one or both of these paralogs.In agreement with a previous study, our data show that the MRB1 core component GAP1, as well as the accessory protein TbRGG2, is a stable interacting partner of MRB8170 and MRB4160 . Moreovein vivo and in vitro results, the preferential binding of MRB8170 and/or MRB4160 onto preedited mRNAs marks the initiation of RNA editing, followed by binding of TbRGG2 via its RRM domain  and placed in a petri dish 5 cm from the light source for UV irradiation (0.8 J/cm2 at 254 nm for iCLAP library preparation) in a Stratalinker 1800 machine (Stratagene). After a quick spin, the cells were snap-frozen in liquid nitrogen and stored at −80°C until further use. Cell pellets (~1.0 to 1.5 g [dry weight]) were resuspended in 5 ml of lysis buffer  containing Complete EDTA-free protease inhibitor cocktail for 10 min on ice. The cell suspension was lysed and spun down by centrifugation . The supernatant was treated with Turbo DNase (Life Technologies) and RNase I at 37°C for 3 min and then incubated on ice for 3 min as recommended in the published protocol  and then to fully edited  iCLAP cDNA libraries were sequenced using Illumina Hi-Seq 2000 . Raw reads were trimmed of 3′ adaptor sequences (Tag cleaner version 0.16), and PCR duplicates were collapsed (Fastx collapser version 0.13). The remaining reads were ~30 to 50 nt long. The reads were divided into individual replicates using 4-nt experimental barcodes and mapped first onto preedited (GenBank sequence accession no. y edited  sequence10.1128/mBio.02288-16.9TEXT S1 TEXT S1, DOCX file, 0.05 MB.Extended experimental procedures. Download Copyright © 2017 Dixit et al.2017Dixit et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E-MTAB-4934.All the iCLAP sequences are available at ArrayExpress with accession number


Several amino acid substitutions inhibited production of capped, full-length mRNA and instead resulted in accumulation of short transcripts of approximately 40 nucleotides in length, confirming that RSV CRV has a role in capping. In addition, all six variants tested were either partially or completely defective in RNA replication. This was due to an inability of the polymerase to efficiently elongate the RNA within the promoter region. BI-D also inhibited transcription and replication. In this case, polymerase elongation activity within the promoter region was enhanced, such that the small RNA transcribed from the promoter was not released and instead was elongated past the first gene start signal. This was accompanied by a decrease in mRNA initiation at the first gene start signal and accumulation of aberrant RNAs of varying length. Thus, in addition to its function in mRNA capping, conserved region V modulates the elongation properties of the polymerase to enable productive transcription and replication to occur.The large polymerase subunit (L) of non-segmented negative strand RNA viruses transcribes viral mRNAs and replicates the viral genome. Studies with VSV have shown that conserved region V (CRV) of the L protein is part of the capping domain. However, CRV folds over and protrudes into the polymerization domain, suggesting that it might also have a role in RNA synthesis. In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D. Effects were analyzed using cell-based minigenome and  Respiratory syncytial virus (RSV) is a leading cause of respiratory illness in infants, elderly, and immunocompromised individuals, yet treatment is limited to supportive medical care. The large polymerase protein (L) of RSV is essential for transcribing viral mRNAs and replicating the genome, and is an attractive target for antiviral intervention. Previous studies from related viruses have shown that a region of the L protein co-transcriptionally adds a cap to nascent mRNAs. In this study, we showed that perturbation of this region of the RSV L protein resulted in capping failure, as expected. In addition, we found that this region altered how efficiently the polymerase could elongate the RNA, impairing recognition of essential signals. Thus, this region of the RSV L protein has more than one function during viral multiplication, making it a particularly appealing target for antiviral development. Pneumoviridae in the Order Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs)  GTP or [α-32P] ATP in a final volume of 50 μl. For reactions containing BI-D, BI-D was diluted in DMSO and added to reactions at the concentrations indicated. DMSO was maintained at a consistent concentration in each reaction. Reactions were incubated at 30°C for 2 h, followed by incubation at 90°C for 3 minutes to inactivate the polymerase, and then diluted in an equal volume of stop buffer . To resolve 2 nt products, the reactions were analyzed on a 25% polyacrylamide gel containing 7 M urea in Tris-taurine-EDTA buffer. Longer RNA products were resolved by electrophoresis on a 20% polyacrylamide gel containing 7 M urea in Tris-borate-EDTA buffer. The nucleotide lengths of the RNA products were determined by comparison with a molecular weight ladder generated by alkaline hydrolysis of [γ32P] ATP end-labeled RNA oligonucleotides representing the anticipated 25 and 23 nt RNA products produced from the +1 and +3 sites, respectively or by alkaline hydrolysis of [γ32P] ATP end-labeled 1–40 RNA. Bands were visualized by autoradiography and quantified using Licor Image Studio Lite.RNA synthesis was reconstituted eviously , 7, but