The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73–90), loop 2 (200–214). In NP, loops 1 and 2 formed a 10–15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91–112) that interacted with the RNA grove (linker 360–373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein. The nucleoprotein of Influenza A virus, NP, covers and protects the eight single-stranded viral RNA segments of negative polarity Recently, X-ray structures have shown that NP forms a trimer in the crystalline state In this study, we questioned the role of the flexible elements found in NP structure in promoting RNA binding and oligomerization. To that end, we quantified the flexibility of NP by molecular dynamics (MD) simulations, based on the X-ray structure of the H1N1 protein to which the missing regions have been added. Molecular modeling is an available tool to study protein dynamics, especially of flexible regions unsolved by X-ray crystallography, without the need of labeling high protein concentrations required for NMR studies. The flexibility of the wt protein was compared to the fluctuations of a mutant R361A selected for its location in the RNA binding groove. If the flexibility of the loop regions impacts on the RNA binding groove and mediates RNA binding and protein self-association, differences should be observed between NP and R361A. The simulations highlighted the flexibility of three defined regions in NP monomer that were perturbed either by oligomerization via trimer formation or by the R361A mutation. The first two regions were the basic loop 1 and the protruding element (loop 2) on one face of the protein and the oligomerization loop 3 on the other face of the protein. The flexibility of loops 1 and 2 facilitated RNA binding to wt NP, presumably mediated by a conformational change of these loops as previously proposed We tested (1) the existence of flexible elements included in NP structure and (2) the putative role of these flexible regions in promoting RNA binding and NP oligomerization. To that end, we used NP in both monomer and trimer forms. The simulations first analyzed one NP monomer, based on the available PBD file 2IQH  see exp. The sinThe backbone RMSF of NP trimer and of NP monomer were calculated and compared. β and Cγ) of loop 2 which drove and stabilized the loop-loop interactions in R361A. Thus, the smaller size of the “pinch” formed by loops 1 and 2 that may hold the RNA in place and the lower flexibility of loop 2 reduced the accessibility of the RNA binding groove in the mutant as compared to NP. Indeed, R361A has a lower affinity for RNA and a reduced ability to oligomerize (see below).The RMSF value decreased markedly in loop 2 while it increased somewhat in loop 1 of R361A compared to loops 1 and 2 of NP monomer respectively . In R361To characterize the interactions between loops 1 and 2, we calculated the minimal distance between loops 1 and 2 observed in the wt and mutated proteins during the dynamics . In the In NP, loop 1 was stabilized by a salt bridge between K113, located at the end of the first β-sheet and E73 at the basis of loop 1 . K113 alIn conclusion, the MD simulations suggested that the flexibility of loops 1 and 2 of NP monomer may be required to grasp RNA and promote RNA binding. This feature was also seen in the trimeric form of NP . The fleTo test this model, we expressed the recombinant NP, R361A, wt- E80A- E81A and R361A- E80A-E81A proteins. We determined their RNA binding affinity and oligomerization. The mutation of the two consecutive aspartates E80 E81 into alanines should abolish the salt bridge between E80 and R208 (without the possibility of a compensating interaction between E81 and R208) and facilitate access to the RNA binding groove. We expected improved RNA binding and RNA-induced oligomerization in this triple mutant as compared to that in R361A.E. coli and purified after RNAse treatment by affinity and size-exclusion chromatographies (data not shown). Using low salt conditions (50 mM NaCl), NP eluted in a main peak at (81.6±1.0) ml. NP was eluted at a similar retention volume than R416A mutant, known to be monomeric The wt NP protein and the R361A, wt-E80A-E81A, wt-R204A-R208A and R361A-E80A-E81A, R361A-R204A-R208A mutants were expressed as C-terminal His-tagged proteins in Surface plasmon resonance experiments were carried out to analyze the binding kinetics of NP and its mutants to single-stranded linear RNA. The 24-mer RNA fragment used for this experiment had the same 24-mer sequence, named Flu1, which was used in previous studies The monomeric RNA-free proteins formed NP/RNA complexes of 1/1 stoichiometry, showing that RNA binding precedes NP oligomerization. The oligomerization was followed by dynamic light scattering (DLS) that monitored the change of the hydrodynamic diameter of NP as a function of time after the addition of RNA.h = 7±1 to 16±1 nm was observed and corresponded to RNA-induced oligomerization  and loop 2 (R208) and expressed the mutant R361A-E80A-E81A in an attempt to disrupt the putative salt bridge E80-R208. As expected, this triple mutant had a Kd of 240 nM for RNA as compared to 565 nM for R361A . In addiWe also expressed loop 2 mutants. The double mutation R204A-R208A aimed at avoiding a compensating salt bridge formation between R204 and E80 or E81. The wt-R204A-R208A mutant exhibited a decrease of ca. eight fold compared to the Kd of wt NP, similarly to the affinity loss of wt-E80A-E81A. However in R361A-R204A-R208A, the protein–RNA interactions were weaker than in R361A. We assume that the reduced flexibility of loop 2  and its The R361A-R204A-R208A protein formed oligomers of significantly larger size than those observed with R361A, while R361A-E80A-E81A oligomers were as large as to wt ones within experimental error, Altogether, the drastic increase of the Kd for RNA and the subsequent altered oligomerization of R361A relative to NP may be mediated by a reduced flexibility of loop 2 and hindrance to access to its RNA binding groove and its vicinity. The R361A mutation did not alter RNA polymerase activity although the virus could not be rescued off = 0.004 s−1 for the NP-RNA complex as compared to koff = 0.007 s−1 for the wt-E801-E81A-RNA complex. The importance of the basic loop 1 can be also deduced from epitope mapping of a monoclonal antibody directed against NP The simulations taken together with the experimental data have suggested that flexible loops 1 and 2 are required for NP activity. Movements of loops 1 and 2 could be part of the NP conformational changes induced by RNA binding. We cannot exclude that additional conformational changes may take place in NP in longer timescales. It is likely that the basic loop 1 helps sampling the environment as proposed 2. The system was heated linearly to 300 K over 60 ps. Molecular dynamics simulation in NPT ensemble was used to equilibrate the system and for production run. During the 1.6 ns equilibration phase, the restrains applied of the solute atoms were gradually reduced from 5.0 kcal/mol/Å2 to zero. The pressure and temperature were restrained to 1 atm and 300 K, respectively. Five trajectories of 10 ns each were produced for the wt NP and R361A monomers, respectively. One trajectory of 4 ns was simulated for the NP trimer. The simulated trajectories were further analyzed using the molecular dynamics simulation and the analysis package CHARMM The MD simulations of the NP, R361A monomers and NP trimer were carried out using the program NAMD Root-mean-square fluctuation  of the protein atoms from averaged positions was further minimized and averaged over the residue backbone heavy atoms.Direct correlation function of two vectors, A and B was calculated using <A(0)·B(t)>.An hydrogen bond was considered to form when the distance between the donor and acceptor heavy atoms was less than 3.5 Å. An interaction between two hydrophobic residues was considered if there is a space between their side-chains which cannot accommodate one water molecule.We estimated that the minimum distance between heavy atoms of two hydrophobic side-chains should be less than 6.1 Å.NaCl (99.9% purity), Tris-HCl, glycerol were purchased from Sigma. The RNA oligonucleotides were synthesized on an Expedite 8909 synthesizer and purified by electrophoresis on denaturating acrylamide gels or purchased at Eurofins MWG Open with HPLC purification. The oligonucleotides immobilized on streptavidin sensor chips were biotinylated at their 5′ end. The sequences of the oligonucleotides are listed below:5′ UUU GUU ACA CAC ACA CAC GCU GUG 3′Flu-1: 5′ UUU UUU UUU UUU UUU UUU UUU UUU U 3′rU25: Pfu DNA polymerase with the QuickChange site-directed mutagenesis kit (Stratagene). Nucleotide sequencing was carried out to confirm the presence of the nucleotide substitution. The Escherichia coli BL21 (DE3) cells carrying the plasmids were induced 4 hours by isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C (NP) or 12 h at 28°C  and collected by centrifugation. The pellet was resuspended and sonicated in a lysis buffer (20 mM Tris at pH = 7.4 with NaCl (50 mM or 300 mM), 5 mM imidazole, 1% Triton and 1 mg/ml lysozyme) and treated with 0.15 mg/ml RNAse A at 35°C for 20 minutes in the presence of 10 mM Mg2+. The proteins were purified by IMAC-Ni2+ affinity chromatography followed by size-exclusion chromatography. The NP was purified at 50 mM NaCl . The R416A, R361A, the double mutants wt-E80A-E81A, wt-R204A-R208A and the mutants triple R361A-E80A-E81A, R361A-R204A-R208A were purified at 300 mM NaCl. We used a Superdex S200 column with an enhanced separation for molecular weights in the range 15 to 100 KDa. After purification, the protein concentration was determined by the extinction coefficient ε = 56200 M−1.cm−1 at 280 nm.We cloned the full-length nucleoprotein NP gene of the H1N1 (strain A/WSN/33) with a 6-His-tag at its C-terminus end in the pET22 vector (Novagen) under the control of a T7 promotor. The R361A single mutation and R361A-E80A-E81A, R361A-R204A-R208A triple mutations were introduced by using The binding kinetics were performed on a Biacore 3000 apparatus using streptavidin coated sensorchips  prepared as indicated by the manufacturer. Immobilization of the biotinylated oligonucleotide on the streptavidin coated sensorship was carried out in PBS Dh) and the size distribution of scatters in each sample. Hydrodynamic diameters of the particles were estimated from the autocorrelation function, using the Cumulants method. In a typical size distribution plot from the DLS measurement, the x axis shows a distribution of size classes (nm) and the y axis shows the relative intensity of the scattered light. A total of 10 scans with an overall duration of 5 min were obtained for each sample and time point. All data were analyzed in triplicate. The protein concentrations usually were in the range of 5 to 15 µM. The oligomerization was conducted at 20°C in 50 mM NaCl, 20 mM Tris at pH = 7.5.The measurements were performed on a Malvern nanosizer apparatus thermostated at 20°C. The size distribution was calibrated with latex particles of 65 and 200 nm radius before the determination of the apparent hydrodynamic size. The scattering intensity data were processed using the instrumental software to obtain the hydrodynamic diameter , equipped with an ANTi-60 rotor. The ultracentrifugation was performed at 45 000 rpm (147 280 g), 15°C. The absorption at 280 nm was recorded every 5 min and 55 scans were averaged. The data were analyzed using the Sedfit and Svedberg softwares


Rapid intuitive hunches or gut feelings may be a compelling source of evidence for paranoid ideas. Conversely, a failure to apply effortful analytic thinking may contribute to the persistence of such thoughts. Our main aim was to examine for the first time the associations of persecutory thinking with experiential and rational thinking styles. Five hundred individuals recruited from the general population completed self-report assessments of current persecutory ideation, general reasoning styles and personality traits. Persecutory ideation was independently associated with greater use of experiential reasoning and less use of rational reasoning. The correlations were small. Persecutory ideation was also positively associated with neuroticism and negatively correlated with extraversion, agreeableness and conscientiousness. There was no evidence of an interaction between neuroticism and experiential reasoning in the prediction of paranoia, but high experiential reasoning in the context of low rational reasoning was particularly associated with persecutory ideation. Overall, the study provides rare evidence of self-reported general reasoning styles being associated with delusional ideation. Perceived reliance on intuition is associated with paranoid thinking, while perceived reliance on deliberation is associated with fewer such thoughts. The dual process theory of reasoning may provide a framework to contribute to the understanding of paranoid thinking. Two parallel systems are considered to underlie decision-making: an effortless, bounded rationality, rapid judgement and a slow, reflective, conscious, analytic approach e.g. . The typA self-report questionnaire of experiential  and rational  reasoning has been developed . The RatOur aim in this study was to examine experiential and rational thinking in relation to persecutory thoughts specifically. As this was the first examination of the issue we used the method of a cross-sectional investigation in a large non-clinical adult population, based upon the accumulating evidence that each of the separate psychotic experiences is represented by continuous traits in the general population e.g. . It was 22.1Volunteers Required for Psychological Research. We are looking for volunteers to take part in a medical research study being carried out at the university. The research would take three hours and you would be compensated for your time. If you would like to hear more about the research, then please contact us. We send detailed information about the study so that you can consider whether you would like to take part.”“The individuals who responded were then invited to take part in the screening stage. Depending on participant preference, the screening questionnaires were either sent in the post or were made available via a web-link. According to the English Index of Multiple Deprivation 2010, Oxford ranks 131st out of 354, placing it in the top half of most deprived local authority areas in England. The unemployment rate in Oxford is approximately 6.3%, below the national average of around 7.7%. Half of the city's jobs are in the public sector and universities .500 people from the city of Oxford took part in the study. The data were collected during the screening phase for an on-going experimental study (for which only approximately 10% of respondents will be eligible). Our team sent leaflets to local postcodes with the wording: 2.22.2.1The GPTS-Part B measures persecutory ideation, as defined by 2.2.2The REI is a 40-item measure of an individual's preference for two different thinking styles: rational and experiential. Each style is assessed using 20-item scales. Each can be further broken down into 10-item subscales, assessing self-evaluated ability in the given style  and reliance on and enjoyment of the given style . Items are rated on a five point Likert scale where 1 is “completely false” and 5 is “completely true”. Examples of items from the rational scale include, “I have a logical mind”  and “I enjoy solving problems that require hard thinking” . Examples from the experiential scale include, “I trust my initial feelings about people”  and “I often go on my instincts when deciding on a course of action” . Mean scores for each subscale can range from 1 to 5, with 1 indicating a low ability/engagement and 5 indicating a high ability/engagement for each thinking style. Internal consistencies for the both the rational and experiential scales, and all four ability and engagement subscales are high e.g. , as is t2.2.3The NPA is a brief measure of personality. Five dimensions of personality  are assessed by a total of 12 items rated on 5-point scales, with 1 being “very uncharacteristic”, 3 being “moderately characteristic” and 5 being “very characteristic”. Example items include: “planning parties and social events” (extraversion), “feeling stressed or worried” (neuroticism), “preparing for things well in advance” (conscientiousness), “making sure others are comfortable and happy” (agreeableness), and “thinking about philosophical or spiritual questions” (openness). Scores for each personality dimension are formed by summing the scores from the relevant two or three items. Higher scores indicate a higher level of the personality trait. The NPA dimension scores correlate with coefficients of .7 or higher with those in the International Personality Item Pool .2.3Analyses were carried out using SPSS Version 19.0 . There w33.1n = 447), Black Caribbean (n = 3), Black African (n = 6), Indian (n = 7), Pakistani (n = 6), Chinese (n = 4), and other (n = 25). The highest level of education reached by participants were none/GCSE (n = 50), A-level (n = 135), degree (n = 180) and postgraduate (n = 134). 81 participants reported having been diagnosed or treated for a mental illness.The participant group comprised 210 males and 290 females, with a mean age of 45.7 years (S.D. = 20.2). 199 participants were single, 167 were married or in a civil partnership, 50 were divorced, 47 were co-habiting, and 32 people were widowed. The ethnicities of the group were White  = 0.41, p = 0.744, but rational reasoning total scores were lowest in those with the lowest education and highest in those with postgraduate degrees, F  = 13.41, p < 0.001.The measure summary scores for the participants are displayed in 3.3F  = 8.32, p < 0.001, Adjusted R Square = 0.03. Experiential reasoning, B = 0.02, standard error = 0.005, p = 0.004, and rational reasoning, B = − 0.02, standard error = 0.005, p = 0.003, each independently predicted paranoia. They remained significant predictors of paranoia when controlling for age, gender, ethnicity, and education. In an analysis of variance, there was no evidence in the prediction of paranoia scores of an interaction between experiential thinking and levels of neuroticism (p = 0.865).n = 126) = 22.7 (11.1); high experiential/high rational (n = 114) = 21.5 (9.3); low experiential/low rational (n = 131) = 20.9 (9.0); low experiential/high rational (n = 129) = 19.5 (8.5). There was an almost significant group difference in paranoia scores, F  = 2.53, p = 0.056, accounted for by the low experiential/high rational group scoring significantly lower than the high experiential/low rational group (p = 0.007). These groups also differed in levels of neuroticism, F  = 7.66, p < 0.001. The low experiential/high rational group had lower levels of neuroticism than all three other groups (p < 0.05). In forming the four groups it is of interest that there was no association of scoring high or low in rational thinking and scoring high or low in experiential thinking, chi square (d.f. = 1) = 0.22, p = 0.636.Looking at the potential combinations of the two styles, the highest paranoia scores were in the individuals with high experiential/low rational scores, and the lowest paranoia scores were in those individuals with low experiential/high rational scores see . The mean = 81) scored significantly higher for levels of paranoia and neuroticism but lower for levels of rational thinking and extraversion compared with the other study participants (p < 0.05). There was no difference in experiential thinking between those with a history of mental illness and the other participants (p = 0.905).Interestingly we note that the individuals who reported a history of mental illness (4In a large general population sample, the self-reported use of intuitive gut feelings was associated with higher levels of persecutory thinking, while the use of deliberative analytic thinking was protective. These are novel findings. The associations were small, explaining little of the variance in paranoia. However it should be borne in mind that it is rare for a self-report of general reasoning style to be linked to delusional ideation, and, moreover, the questionnaire items did not ask about reasoning in relation to the kinds of fearful feelings that are typically associated with delusions. A reliance on experiential thinking is likely to be significantly exacerbated in anxiety-provoking situations. Consistent with previous findings, rational and experiential thinking were found to be independent reasoning processes. The combination of experiential thinking without the check of rational thinking was most closely associated with paranoia.There was a failure to find a predicted interaction between experiential reasoning and negative affective state. It was expected that a use of gut feelings in the context of anxiety would be more likely to result in paranoid interpretations of experience. However the assessment of neuroticism was very limited, comprising just two items, neither of which concerned the physiological components of anxiety. An expected main effect of neuroticism and paranoia was found. Higher levels of paranoia were associated with higher levels of neuroticism. This is consistent with the theoretical idea that paranoia and anxious affect are closely related via the process of threat anticipation . PersonaThe clear study weakness was the cross-sectional design. The associations could be explained by an unmeasured confounder, while the direction of the relationships between reasoning styles and paranoid thinking cannot be known. It would also have been of interest to include more clinical variables such as global functioning and emotional disorder. A further cross-sectional study in a group with delusions in the context of psychotic conditions such as schizophrenia is now warranted, although the optimal design would be to look longitudinally at whether the reasoning styles predict the persistence of delusional beliefs. Experimental or clinical studies testing whether manipulating therapeutically the type of reasoning style alters the course of delusions would be especially valuable e.g. . It woul


In vitro, telomerase requires at least one RNA molecule and a reverse transcriptase-like protein. However, for telomere homeostasis in vivo, additional proteins are required. Telomerase RNAs of different species vary in size and sequence and only few features common to all telomerases are known. Here we show that stem-loop IVc of the Saccharomyces cerevisiae telomerase RNA contains a structural element that is required for telomerase function in vivo. Indeed, the distal portion of stem-loop IVc stimulates telomerase activity in vitro in a way that is independent of Est1 binding on more proximal portions of this stem-loop. Functional analyses of the RNA in vivo reveal that this distal element we call telomerase-stimulating structure (TeSS) must contain a bulged area in single stranded form and also show that Est1-dependent functions such as telomerase import or recruitment are not affected by TeSS. This study thus uncovers a new structural telomerase RNA element implicated in catalytic activity. Given previous evidence for TeSS elements in ciliate and mammalian RNAs, we speculate that this substructure is a conserved feature that is required for optimal telomerase holoenzyme function.The stability of chromosome ends, the telomeres, is dependent on the ribonucleoprotein telomerase.  Telomeres at the ends of eukaryotic chromosomes are composed of specific repeat sequences that cannot be completely replicated by the conventional DNA replication machinery . This shSaccharomyces cerevisiae telomerase RNA is predicted to fold such that three relatively long arms emanate from the catalytic center [. The Est1 protein tethers telomerase to telomeres as it interacts with Cdc13, a protein that is bound on the terminal single-stranded DNA occurring on telomeres . The plasmid pTLC1TRP (EST1) and NLYH95 (est1Δ) with the indicated Tlc1 RNA constructs were used. The CDC13 gene disruptions in strains NLYH55 and NLYH59 were made by replacing the genomic locus by the KanMX cassette using a one-step polymerase chain reaction (PCR)-mediated method  plates and the final strain containing the est1Δ::LoxP allele was verified by PCR and Southern blotting. TLC1 deletion in NLYH55, NLYH59 and NLYH95 was done by replacing the genomic locus by the NatR resistance cassette. pVL1107  was constructed by replacing the wild-type (WT) TLC1 allele of the URA3-based vector pADCEN36  or pRS423-EST1 were transformed into yeast strain NLYH80 expressing the various TLC1 variants.Strain NLYH80 containing plasmid pAZ1 was used as base strain for most experiments  or NLYH97 (yku70Δ) expressing the various TLC1 alleles were grown to an OD660 of 0.7 and spotted in serial dilutions on selective SC-Trp-Ura and FOA-Trp media to test for viability after loss of the complementing WT TLC1 plasmid (pAZ1).Cells of strain NLYH80 (TLC1 WT) on 5-FOA plates, cells from indicated passages were grown in liquid media. Genomic DNA was extracted, digested with XhoI, subjected to agarose gel electrophoresis (0.75% agarose), transferred to a nylon membrane and hybridized to a 300 bp fragment containing 280 bp of telomeric repeats derived from pYLPV (After selection for cells that had lost pAZ1(om pYLPV . Probes om pYLPV . Data weTLC1 (a 686-bp NcoI-NsiI restriction fragment). The membranes were rehybridized with U1- and ACT1-specific probes for loading control and quantification. Analyses of RNA levels were performed using a Typhoon FLA9000 apparatus and the Quantity One software.Total RNA was extracted from 10-ml cultures of exponentially growing cells. After resuspension of RNA pellets in nuclease-free water, northern blot analysis was performed as previously described . Briefly7 complex, and this interaction is essential for Tlc1 RNA stability  supplied with 200 mM NaCl. The cell pellets were then frozen in liquid nitrogen and lysis was performed by grinding the pellets in presence of pieces of dry ice in a standard coffee mill (Krups). The cell powder was thawed on ice and one pellet volume of TMG buffer with 200 mM NaCl, 0.1 mM DTT, 0.2% Triton X-100, 0.2% NP40 and protease inhibitors was added. An S-100 was performed in an ultracentrifuge for 2 h at 100 000g (4°C). Immunoprecipitations (IP) used 2 mg of total proteins adjusted to 0.5% Tween-20 and to which 40 U of RNasin per ml of extract  was added. Following an incubation of 2–3 h at 4°C with 500 ng of anti-MYC antibody , 50 μl (bed volume) of TMG-washed Protein G plus/Protein A agarose  were added and everything was incubated overnight at 4°C with gentle agitation. Beads were then washed twice with 0.5 ml TMG plus 200 mM NaCl, 0.1 mM DTT, protease inhibitor and 0.5% Tween-20, and twice with 0.5 ml TMG plus 0.1 mM DTT, protease inhibitor and RNasin added at 40 U per 0.5 ml of TMG. Beads were resuspended in one bed volume of TMG plus 0.5 mM DTT, protease inhibitor and 40 U of RNasin. The actual telomerase activity assay was performed as previously described (Supplementary Table S3) was monitored to determine telomerase activity in vitro. Visualization and quantification of extension products were performed using a Typhoon FLA9000 apparatus and the Quantity One software.Inside cells, telomerase RNA is bound a few nucleotides upstream of its mature 3′-end by the Smtability . We therribed in . Brieflyescribed  on 5 μl TLC1 variants except for the tlc1Δ sample, which consists of NLYH80 that has lost the complementing TLC1 plasmid pAZ1 on 5-FOA. Yeast cultures were grown to an OD660 of 0.4, and fluorescent in situ hybridization (FISH) experiments were performed as previously described in (Supplementary Table S3), and single-molecule detection was achieved by using a wide-field epifluorescence  cells were co-transformed with a plasmid harboring TLC1 alleles (either WT or with mutations in the stem-loop IVc domain) as well as the pAD-A258 (tlc1Δ148-440) plasmid. The RNA expressed from the latter invariably contains the complete and WT Est1 and Est2 binding domains and thus serves as internal positive control (I.C.). Yeast cells (500 ml) were grown up to OD660 of 1.0. Total protein extracts and HA3-tagged Est1 immunoprecipitations were prepared as described in the methods for telomerase assays except that the extracts were incubated with 500 ng of a high-affinity anti-HA  for 2–3 h at 4°C before adding the TMG-washed beads. Total RNA and post-IP flowthrough RNA were prepared as described  and elution was performed in 17 µl elution buffer. The subsequent PCR reaction was done on 5 µl of the purified cDNA with 20 pmol of each P1-FOR and P2-REV primers (Supplementary Table S3). Detailed PCR conditions will be supplied on request. PCR products were then analyzed by electrophoresis on 1.5% agarose-TAE gel.NLYH80 , and that is not predicted to interfere with these Est1-binding sites, also caused short telomeres . To ensure that the mutations did not completely disrupt the overall structure of stem IVc, we probed the local nucleotide flexibility by SHAPE . This technique is particularly relevant to discriminate flexible and reactive regions from relatively constraint RNA regions, such as base-paired stems for example. On all RNAs, the predicted apical loop (nt 623/624) displayed strong reactivity, consistent with their unconstrained position in the loop, which causes clearly visible bands on the gels . This result confirms that the loop nucleotides in the terminal hairpin remained unaffected by any of the mutations. On the WT and the OPM RNAs, nucleotides of the IL  also reacted well, yielding the predicted reaction pattern. In contrast, in the SA5 and SA3 mutant RNAs, these same nt 608–614 did not react, resulting in low band signals. This result is consistent with the prediction that in the mutant RNAs, the internal bulged area is more constrained by base pairing, i.e. the distal hairpin, which in the WT and OPM RNAs comprises nt 614–632, is extended in the SA3 and SA5 RNAs to nt 609–637 . We therefore conclude that at least in vitro, these RNAs fold in ways that are consistent with the in silico predictions. Strikingly, despite the overall similarity of the structures, the reduction of the internal bulge size had a significant impact on telomerase function in vivo. In cells harboring the SA5 and SA3 alleles, telomeres are extremely short . Note that the OPC allele corresponds to the combination of the SA5 and SA3 mutations, while the OPM allele had a one other nucleotide change . These results indicate that the shortened telomere lengths are not a result of lowered expression levels of the RNA, but rather a consequence of reduced telomerase activity.Stem-loop IVc of the budding yeast telomerase RNA is involved in Est1 binding. In fact, sequences around CS2 [nt 646–659, blue on in vivo  . We conclude that telomerase trafficking into the nucleus is not affected by the ΔSL, SA5, SA3 or the OPM mutations . More importantly, while overexpression of Est1 could lengthen telomeres in WT cells . Consistent with an important decrease of telomerase activity conferred by the mutations in vivo, none of the SA3, SA5 or ΔSL mutations could support growth in the absence of Ku, while the OPM allele did not show a synthetic interaction with yku70Δ cells and cells with that version of the Tlc1 RNA grew as WT  and these defects cannot be suppressed by an overexpression of Est1 (data not shown). These results suggest a dramatic reduction of the ability of this RNA to bind Est1 and hence a complete loss of telomerase-mediated telomere maintenance. However, this same ΔSL+IL RNA can be imported into the nucleus at an efficiency that is comparable with WT, even if we observed a much larger cell to cell variability with this RNA than with the other mutated versions . Moreover, cells with this Tlc1-ΔSL+IL RNA behave similarly as cells that lack telomerase altogether  or cause a closing of the IL . The fact that the Tlc1-ΔSL RNA still contains the complete CS2a suggests that telomerase inhibition is not caused by a protein that binds specifically to this single stranded bulge. In support of that notion, the OPC and OPM versions of the Tlc1 RNA behave as WT in all assays, yet they contain four altered nucleotides in the internal bulge area . We also constructed two more TLC1 alleles, each changing two nucleotides in the apical loop , but neither of those mutations had any effect on telomerase (data not shown). It is therefore most likely that TeSS provides for a crucial interaction interface that is required for telomerase function in vivo. Phylogenetic sequence analyses had shown that the actual sequence of this area in TLC1 is not conserved, even amongst the Saccharomyces sensu stricto group of species, yet there are three co-varying base pairs in the apical stem (starred in in vivo as predicted in silico and it needs to be bordered by flexible unpaired nucleotides in the IL (612/613 and 633/634). We therefore propose that this RNA element, the TeSS, needs to be able to reorient in a particular fashion in the mature and active telomerase complex. This proposed induced fit model could allow TeSS to interact with and perhaps stabilize a core protein such as Est2 to remain associated with the pseudo-knot structure or the templating area of the RNA. Given that Est3 can interact with Est2 directly and some evidence suggests that it is able to assemble into an active telomerase complex without Est1, it is also possible that the core complex targeted by TeSS includes both Est2 and Est3 . Funding for open access charge: CIHR.Conflict of interest statement. None declared.


Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures “mitochondrial RNA granules.” ► GRSF1 resides in the mitochondrial matrix and is required for mitochondrial function ► GRSF1 is required for the processing of tRNA-containing and tRNA-lacking precursors ► GRSF1, RNase P, and nascent RNA are part of “mitochondrial RNA granules” ► Mitochondrial RNA granules are functionally linked to RNA processing Mitochondria are involved in many metabolic processes, a primary function being to convert energy from nutrients into ATP through oxidative phosphorylation. These organelles are thought to derive from an ancient endosymbiotic event between an α-proteobacterium and the ancestor of modern eukaryotic cells. Over evolutionary time most of the ancestral bacterial genes have been transferred to the nucleus, requiring >1,000 nuclear-encoded proteins to translocate to the mitochondrion following cytosolic synthesis . As a reThe human mitochondrial genome encodes two ribosomal RNAs, 22 tRNAs, and 13 open reading frames (ORFs) that are transcribed and translated within the organelle. The mitochondrial genetic information is indispensable for the function of respiratory complexes I, III, IV, and V, as exemplified by the large number of human disorders attributed to mtDNA mutations and the role such mutations may play in aging .Mitochondrial transcription occurs on both DNA strands and gives rise to three polycistronic primary transcripts . The priMTCO1 with noncoding RNA (ncRNA) does not have an intervening canonical tRNA. However, the upstream ncRNA sequence has been proposed to adopt a tRNA-like structure, and so the processing of this transcription unit also depends on RNase P  was originally identified as a cytosolic RNA-binding protein with high affinity for G-rich sequences . GRSF1 bHere, we report that an isoform of GRSF1 resides in a novel mitochondrial structure that we have called “mitochondrial RNA granules,” where it plays an essential role in precursor RNA processing.GRSF1 gene encodes two splice variants  or by using lentiviral-mediated shRNA delivery (RNAi2) A. IntereTo gain further insight into the mechanism by which GRSF1 regulates mtRNA levels, we searched for GRSF1 binding partners. For this experiment, GRSF1, along with any interacting proteins was affinity purified from HEK293T cells expressing GRSF1-FLAG, and the resulting protein mixture was analyzed by mass spectrometry . Among tIn addition to RNase P, the coimmunoprecipitation experiment also identified a number of other putative GRSF1 partners, among which were several RNA-related proteins, including components of the cytosolic or mitochondrial ribosomes. This prompted us to test whether GRSF1 itself could be a component of mitoribosomes. However, using isokinetic sucrose density gradients we did not find cosedimentation of GRSF1 with either the mitoribosomal protein MRPL12 or DAP3 B.Together, these data show that GRSF1 interacts and colocalizes with RNase P in mtRNA granules.Phe, 16S rRNA, tRNALeu(UUA/G), MTND1, RNA14, MTCO3, and MTND5 in GRSF1-deficient cells, confirming the data obtained by Nanostring analysis as described above , and MTND1, is one of the most abundant (Leu(UUA/G), or MTND1 showed a decrease in RNA19 in GRSF1 RNAi2-expressing cells . We found that GRSF1 is required for mtRNA expression and protein synthesis in mitochondria. Moreover, downregulation of GRSF1 or MRPP1, a subunit of RNase P, delays the transit of nascent RNA through MRGs, leading to reduced levels of mature mtRNA and reduced mitochondrial protein synthesis. These data strongly suggest that mtRNA processing and generation of functional RNA species are dependent on the integrity of the MRG.MTCO3 and MTND6-ncRNA precursors. The mechanism by which GRSF1 exerts these functions is still unclear, and it remains to be determined whether the protein is able to bind to specific nucleotide sequences present in these precursors.Little is known about the mechanisms and regulation of mtRNA processing. We have identified GRSF1 as a key player in the processing of tRNA-containing and tRNA-lacking precursors. We have shown that GRSF1 binds the MRPP1 and MRPP2 subunits of RNase P, while the third subunit, MRPP3, was shown to colocalize with GRSF1 by immunohistochemistry. In addition, GRSF1 probably also acts in an RNase P-independent manner, since its activity is implicated in the processing of the tRNA-lacking precursors. Indeed, we found that GRSF1 is the first protein to be required for the processing of RNA14-Is GRSF1 also involved directly in mitochondrial protein synthesis, in addition to its role in RNA processing? We cannot rule this out, and the marked decrease in de novo mitochondrial protein synthesis upon GRSF1 depletion is perhaps more dramatic than would be predicted solely from the effects on RNA processing or stability. Moreover, we did see mitoribosomal proteins in the crude immunoprecipitation experiment. However, our sucrose gradient density fractionation showed no evidence of tight association between GRSF1 and mitoribosomal subunits. Further investigations will be required to understand whether GRSF1 could play a direct role in translation, whether a minor fraction of GRSF1 also interacts with the mature ribosomes, or whether the presence of mitoribosomal proteins in the immunoprecipitation corresponds to intermediates in the process of mitoribosome assembly.We show that the MRGs are distinct from mitochondrial nucleoids. Only fewer than 10% of them overlapped with the DNA-containing structures, and these MRGs may correspond to BrU-labeled foci found in close proximity to nucleoids as described by It is likely that MRGs are dynamic structures whose number and composition may vary according to the metabolic state of the cell. Based on the phenotypic consequences of GRSF1 and RNase P knockdown on RNA processing, we postulate that at least part of RNA processing occurs in MRGs. This does not exclude the possibility that GRSF1 and RNase P may have additional functions outside MRGs, nor does it exclude the possibility that MRGs may have other functions beyond a role in RNA processing. Based on our hypothesis on the role of MRGs in processing of tRNA-containing polycistronic transcripts, we expected that RNase Z, the enzyme that cleaves tRNA at its 3′ terminus, would be enriched in MRGs; however, this does not appear to be the case. Instead, RNase Z immunostaining is diffuse in the mitochondrial matrix, suggesting that the protein may have additional functions outside of MRGs and/or that its mitochondrial concentration is high enough in the matrix and not limiting for RNA processing in MRGs. Alternatively, RNase Z-induced cleavage of tRNA in precursor transcripts could occur outside of MRGs. Indeed, previous work has demonstrated that tRNAs are cleaved sequentially, initially at the 5′ end by RNase P, then at the 3′ end by RNase Z. Indeed, cells treated with RNAi against RNase P accumulate 3′ tRNA junctions . The MRGIn previous experiments, In conclusion, we have identified a new mitochondrial subcompartment, the mitochondrial RNA granules, where GRSF1 and RNase P act together to ensure mtRNA processing. Further characterization of the proteome and transcriptome of these structures should prove to be very useful in advancing our understanding of mtRNA processing and may reveal other essential posttranscriptional steps involved in mitochondrial gene regulation.2 at 37°C. 143B ρ0 cells were grown in the same medium supplemented with 110 μg/ml sodium pyruvate and 50 μg/ml uridine. Transfection of cells was done using Fugene (Roche). Mitochondrially targeted ECFP (mitoECFP) was from Clontech. Twinkle-EGFP was a gift from J.N. Spelbrink. MRPP1-FLAG and MRPP3-myc were gifts from W. Rossmanith. For RNA interference, 143B cells were transfected with interferin (Polyplus) or lipofectamine RNAiMax (Invitrogen) according to the the manufacturer’s instructions, and cells were analyzed 2–4 days posttransfection. pLKO.1 containing GRSF1 RNAi2 lentiviral vector was obtained from Sigma, and lentiviruses were produced according to the manufacturer’s instructions. After viral infection, cells were always selected for 24 hr with 3 μg/ml puromycin and left to recover for >24 hr before analysis. The same RNAi2 sequence was cloned into pLKO-TetON, a tetracycline-inducible version of pLKO (Addgene). Induction was achieved with 1 μg/ml tetracycline for 2–4 days. Inducible and noninducible versions of RNAi2 showed the same effect on RNA processing and were used indifferently to express RNAi2 (data not shown). RNAi sequences are described in All cell culture reagents and chemicals were purchased from Sigma unless stated otherwise. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 u/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine in 5% COBrU pulse was performed using 5 mM 5-BrU (Sigma) for 60 min. For chase, cells were washed twice with culture medium and incubated in 25 mM uridine for the indicated times. Fixation was made in 4% paraformaldehyde for 10 min. Immunofluorescence was performed in PBS containing 3% BSA, 0.1% Triton X-100, and 20 u/ml RNasin (Promega). Microscopy was performed with ZEISS AXIOZ1 or ZEISS LSM700 confocal microscopes. To quantify the foci-foci colocalization, cells were labeled with antibodies against the protein/RNA of interest and GRSF1 and imaged, and >20 foci in >5 cells  per condition were blindly labeled on the protein/RNA pictures using ImageJ. Pictures were then merged, and the frequency of colocalization with GRSF1 foci was determined.c antibodies were made in our laboratory. FITC, Texas red, or AMCA-coupled secondary antibodies were from Vector.Antibodies used were as followed: BrU/BrdU (Roche 11170376001), Tom20 (Santa Cruz FL-145), PHB (Neomarkers AB-1), OPA1 (BD PharMingen 612606), COX1 (Invitrogen 459600), COX2 (Invitrogen 35-8200), SDHB (Abcam AB14714), DAP3 (Abcam 13893), Actin (Sigma AC-40), and ELAC2 (Proteintech 10071-1-AP). GRSF1 antibodies were a gift from J. Wilusz, mtSSB from M. Zeviani, and HSP90 β from D. Picard. Cytochrome 32P labeled in vitro-transcribed T7 RNA probes in 50% formamide, 7% SDS, 0.2 M NaCl, 80 mM sodium phosphate (pH 7.4), and 100 μg/ml salmon sperm DNA. Hybridization was carried out at 60°C for 3 hr. Membranes were subsequently rinsed in 1× SSC, 0.1% SDS and washed for 30 min in 0.5% SSC, 0.1% SDS. Imaging and quantification were performed with a phosphorimager (Bio-Rad). Probes are described in Total RNA was extracted with TRI reagent (Sigma). RNA (15 μg) was separated on denaturing formaldehyde agarose gels and transferred to nylon membranes. Membranes were hybridized with The NanoString analysis was performed by the genomics platform of the University of Geneva. HDAC, NUP155, and tubulin were used to normalize the data. Probes are described in Cell fractionation was done in MB buffer  using a glass homogenizer (Kontes). Nuclei and unbroken cells were discarded after centrifugation for 5 min at 2,000 g. The mitochondria-rich fraction corresponded to the pellet obtained after a 10 min centrifugation at 13,000 g. For the PK accessibility test, mitochondria were resuspended at a final concentration of 0.5 μg/μl in MB, swelling buffer (10 mM HEPES-KOH [pH 7.4]), or MB + 0.2% (v/v) Triton X-100. PK was added to a final concentration of 50 μg/ml, and the mixture was incubated on ice for 20 min. PMSF (2 mM) was then added to inhibit the PK. After TCA precipitation, samples were analyzed by western blotting.2CO3 (pH 11.5) and incubated on ice for 30 min before a 30 min centrifugation at 100,000 g.For alkaline sodium carbonate extraction, 100 μg of mitochondria was resuspended in 100 mM Na35S-labeled methionine/cysteine (PerkinElmer). Total protein concentration of cell lysates was measured by Bradford (Bio-Rad), and lysates were resolved on a 15%–20% tricine gel, transferred to a nitrocellulose membrane, and analyzed by autoradiography.143B cells were incubated for 20 min in methionine/cysteine-free DMEM (Sigma) complemented with dialysed serum and 2 mM L-glutamine. Cells were then incubated for 1 hr in the same medium in the presence of 100 μg/ml emetine and 100 μCi/ μL In vitro synthesis of radiolabeled GRSF1 isoforms was performed with TNT kit (Promega). Import into isolated mitochondria was performed according to 2, 1 mM EDTA, 1% [v/v] Triton X-100, and protease inhibitor cocktail [Roche]). Coimmunoprecipitation of MRPP1-FLAG with GRSF1-HA was performed on mitochondria from HEK293T cells expressing both constructs. Mitochondria were homogenized in lysis buffer, and pull-down was performed using polyclonal anti-FLAG antibodies (Sigma). After washing and elution in SDS-containing loading buffer, proteins were separated by SDS-PAGE. RNase A treatment was performed by adding 100 μg/ml RNase A (Fermentas) during lysis.Affinity purification and identification of GRSF1-interacting proteins were performed on mitoplasts from GRSF1-FLAG-expressing HEK293T cells as previously described . To conf143B cells were lysed in lysis buffer and layered on a 2 ml 10%–30% (w/v) sucrose gradient as previously described . Samples∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Data are represented as mean ± SEM and are representative of more than three independent experiments. Stars correspond to a two-tailed Student t test, with


The PIWI protein regulates gene expression at the epigenetic and post-transcriptional level with a variety of endogenous small non-coding RNAs. In poultry, the biological function of the PIWI protein and PIWI binding to small RNAs had not been determined. The present study cloned and analyzed the sequences of the PIWIL1 protein. We also characterized PIWIL1 binding to small RNAs from adult quail testis, where the PIWIL1 protein is specifically expressed. Small RNAs showed a strong peak at 24–27 nt in the testicular RNA library, mapped primarily to repeat sequences and were similar to rasiRNAs. MicroRNAs (miRNAs) were abundant in the ovarian RNA library at a peak of 22 nt. PIWI gene was first detected in Drosophila germline stem cells (GSCs) aub mutants give rise to abnormal eggs, and male mutants are sterile. Additionally, both male and female piwi mutants are sterile. In the germline, the three PIWI proteins combine with a class of repeat-associated small interfering RNAs (rasiRNAs) to exert splicing activity similar to RNase H. Thus, rasiRNAs are distinct from other RNAi pathways, such as miRNAs and siRNAs. In animals, miRNAs require Dicer-1 for their production and siRNAs require Dicer-2. However, rasiRNAs require neither Dicer-1 nor Dicer-2 The MIWI, MILI and MIWI2 are three murine PIWI proteins. Knockout of each protein led to male sterility through the suspension of spermatogenesis and apoptosis of germ cells in vitro and relatively easy to manipulate. Thus, we explored the biological function of the PIWI protein and PIWI binding to small RNAs by using deep sequencing technology for the first time. Our findings will indicate the fundamental importance of recording the small RNA profile to understand gene regulation during spermatogenesis.At present, the study of small RNAs in poultry is focused on miRNAs, including the construction of miRNA expression profiles for various tissues, the screening of tumor markers and the identification of reproduction-related miRNAs This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Jiangsu province. The protocol was approved by the government of Jiangsu Province, China (Permit Number: 45). All efforts were made to minimize suffering.Coturnix coturnix) (45 d) from a quail breeding farm in Taizhou, Jiangsu Province, and stored at −70°C. Total RNA was extracted using the TRIzol reagent (Invitrogen), and the concentration was determined using a NanoDrop Spectrophotometer (Thermo Fisher Company).Different tissues, including the heart, liver, kidney, brain, testis, ovary, pectoral muscle and lung, were obtained from adult Common quails (PIWIL1 gene was amplified from adult quail testis using the following primers: F-5′ATGACAGGAAGAGCTAGAGCC and R-5′TTAGAGATAGTAAAGTCTG. RACE was performed using the SMARTer™ RACE cDNA Amplification Kit (Clontech) with the GSP1 (5′-GGTAGCTCCCTTAATCCGTGCAAAGCC) and GSP2 (5′-CGAGCAATCACACACTGACTTGGAATGGGAC) primers.Using the mRNA sequence of the red jungle fowl (GeneID: 416804), the CDS of the PIWIL1 cDNA fragment corresponding to amino acids 697–867 was cloned into the pET-32a vector for bacterial overexpression of the peptide. The solubilized inclusion bodies were used to immunize two New Zealand rabbits. Antibodies were affinity purified by immunoadsorption . Immunoprecipitation of the PIWIL1-RNA complex in adult quail testicular extract was performed using the PIWIL1 polyclonal antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). The binding RNA was prepared from the RNP complex.A PIWIL1 mRNA sequence were labeled with DIG-dUTP using the DIG DNA labeling kit . Total RNAs were fractionated on a 1.2% formaldehyde denaturing gel (25 µg/lane). After electrophoresis, the gel was transferred by capillary action with 20× SSC onto a nylon membrane (positively charged) following UV crosslinking . The probe/hybridization mixture (3 ng/ml) was added and incubated overnight with gentle agitation. After hybridization and stringency washes, the membrane was incubated for 30 min in blocking buffer according to the DIG detection Kit II . The membrane was incubated with anti-DIG-AP at a dilution of 1∶20,000 for 30 min at room temperature and washed with 0.1× SSC/0.1% SDS solution followed by incubation with CDP-STAR at a dilution of 1∶100 for 5 min at room temperature. The membrane was exposed to X-ray film for 1 min and fixed.DNA probes complementary to the Tissues were homogenized in 3–5 volumes of lysis solution with PMSF  and centrifuged at 13000× g for 5 min at 4°C (Eppendorf 5415R centrifuge). The concentration of the liquid supernatant was determined using the BCA method . The total protein was separated by 10% SDS-PAGE (50 µg/lane) and electro-transferred to a PVDF membrane . The transferred membrane was rinsed and blocked with 5% BSA overnight at 4°C. The membrane was incubated with rabbit polyclonal anti-chicken PIWIL1 antibody at a dilution of 1∶8000 for 1.5 h at room temperature. After rinsing, the membrane was incubated with IgG anti-rabbit secondary antibody coupled to peroxidase  at a 1∶3,000 dilution for 1.5 h at room temperature. After a 10 min incubation with the chemiluminescent substrate, the membrane was exposed to film (Kodak).The small RNA fraction between 18 and 30 nt was isolated by 15% denaturing PAGE and ligated with proprietary adaptors. The ligated RNA was then converted to cDNA by RT-PCR, and the cDNA was sequenced on Illumina Genome Analyzer II× following the protocol of the Illumina TruSeq Small RNA Preparation kit . The initial sequencing results were converted into sequence data by base calling to generate raw data.Gallus gallus v.2.1) and known databases, such as Rfam (10.1) and miRBase (18.0), small RNAs could be annotated into different categories. Those small RNAs that could not be annotated were used to predict novel miRNAs. The data analysis of small RNAs fraction of 23–32 nt was carried out according to the previous research method The raw data underwent data cleaning, which included discarding the low quality reads and several kinds of contaminants. The identical reads in the raw data were clustered into unique families. By mapping clean reads to a reference genome (PIWI cDNA from adult testis containing a 142 bp 5′ untranslated region (UTR), a 2.59 kb open reading frame (ORF) and a 667 bp 3′ UTR followed by a poly(A) tail and named it PIWI-like 1, or PIWIL1. The ORF predicted that the encoded PIWIL1 protein contained 864 amino acid residues , and the small RNA library was constructed. Simultaneously, the small RNA fraction (18–30 nt) was isolated from total RNA in adult quail testis and ovary. We obtained 21,131,183 reads from the IP product, 11,350,879 reads from the testis and 10,495,986 reads from the ovary. The ovarian RNA library showed a peak at 22 nt corresponding to miRNAs . The tesGallus gallus genome and miRBase (18.0), miRNAs were identified from all three small RNAs libraries and divided into seven groups in order of high-to-mid confidence  depository. However, the ratio of all known miRNA copies in the ovarian RNA library ranged up to 17.7% (1.9% for the testicular RNA library and 0.1% for the IP product), suggesting that miRNAs were the dominant species of small RNA , 11,121,041 (97.98%) and 8,190,484 (78.03%) clean reads from the IP product, testis and ovary, respectively. Using BLAST to search against the nfidence . In the mall RNA .To investigate the differential expression of the miRNAs, we normalized and compared all unique miRNAs using IDEG6. The miRNA counts showed a significant difference between the testicular and ovarian RNA libraries . For exaGallus gallus genome were analyzed and found to contain 566,212 (4.99%), 928,311 (8.84%) and 690,891 (3.27%) clean reads from the testis, ovary and IP product, respectively. As shown in Gallus gallus genome for one hit, 12.25% of unique reads were located in repeat sequences and 14.86% of unique reads corresponded to exons.In the testicular RNA library, the length distribution of small RNAs revealed a strong peak at 24–27 nt, similar to rasiRNAs and piRNAs. To identify the genetic characteristics of these small RNAs in quail, the sequences in the 23–32 nt range that mapped to the An examination of the unannotated unique reads that mapped to 1–5 discrete loci revealed a non-uniform distribution in the genome , and S4.PIWI encodes a nucleoplasmic protein whose activity modulates the number and division rate of germline stem cells In Drosophila, Gallus gallus genome. Given the unique peak at 24–27 nt in the testicular RNA library, very few unique reads were annotated, suggesting that the remaining reads likely belonged to a novel class of small RNAs. However, a less extreme 5′ uracil bias had been noted in unknown unique reads, which differed from the extreme 5′ uracil bias for piRNAs Gallus gallus genome more than once. The unique reads were mainly concentrated in chromosome W followed by chromosome Z from the testis and ovary, which was consistent with the distribution of repeat sequences. It appeared that the unknown unique reads may be involved in the transcription or translation of repeat sequences in the germline. Given the nucleotide length, testicular specificity, PIWI protein interaction and genomic origin, we hypothesized that the unknown unique sequences were quail rasiRNAs. However, many of the unknown unique sequences from the testis mapped to chromosome W in our study. At present, we are unable to explain this phenomenon. Whether there is a relationship between PIWIL1 binding to small RNAs and the sex determination mechanism requires more experimental evidence.In mammals, the PIWI protein and piRNAs are RISC components that regulate spermatogenesis. To determine the mechanism of PIWI-mediated piRNA pathway in quail, we obtained the PIWIL1-RNA complex from testicular extract of adult quail and purified the associated RNAs. The length distribution of PIWIL1 binding to small RNAs showed a predominant peak at 24–25 nt, which was similar to the length distribution of rasiRNAs in Drosophila OSK and VASA, which differs from the known translational repression role of the miRNA pathway. In support of this point, MIWI also appears to positively regulate gene expression in mice As we all known, the PIWI subfamily protein plays an important role in the germline but its molecular mechanism remains unclear. It was reported that the functional role of PIWI protein in Drosophila germline determination is due to their capacity to interact with miRNAs Reproduction-related miRNAs have been the recent subject of intense interest. Sexually dimorphic miRNA expression has been reported during the chicken embryonic gonadal development, suggesting that miR-202* may function in regulating testicular development Figure S1Immunoprecipitation. The native PIWIL1 protein was captured by the anti-PIWIL1 antibody from the adult testicular lysate and then detected by Western blot (Lane 1). Brain served as the PIWIL1-negative control (Lane 2). Pre-immune serum from rabbits (purified) served as the antibody-negative control (Lane 3). The adult testicular lysate served as the PIWIL1-positive control (Lane 4).(TIF)Click here for additional data file.Figure S2Nucleotide bias of the small RNAs. For each position, the proportion of A, U, G and C found in the unannotaed unique reads is represented.(TIF)Click here for additional data file.Figure S3Chromosome distribution of unannotated unique reads in the ovary. The number of bases in exons of Refseq genes and repeats are plotted as a percentage of overall chromosome length. The numbers of piRNAs are normalized to chromosome length and plotted.(TIF)Click here for additional data file.Figure S4Chromosome distribution of unannotated unique reads in the IP product. The number of bases in exons of Refseq genes and repeats are plotted as a percentage of overall chromosome length. The numbers of piRNAs are normalized to chromosome length and plotted.(TIF)Click here for additional data file.Table S1Distribution of small RNA among different categories in three libraries .(DOC)Click here for additional data file.Table S2Distribution of small RNA among different categories in three libraries .(DOC)Click here for additional data file.Table S3Differential expression of miRNAs in testis and ovary. All unique miRNAs from testis and ovary libraries were normalized and used to detect their differential expression using IDEG6.(XLS)Click here for additional data file.


Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms.  Apoptosis mainly occurs following activation of the caspase cascade by the mitochondrial or apical pathways M. tuberculosis-infected humans sst-1 and slca11 (formerly nramp-1), the best characterized murine genetic loci determining susceptibility/resistance to mycobacterial infections, were associated with the degree of macrophage apoptosis M. tuberculosis or stimulation with PPD activate both the apical and mitochondrial apoptotic pathways by inducing TNF-α production, caspase activation and calcium influx, M. tuberculosis and lipoarabinomannan (ManLAM) inhibit these pathways by inducing IL-10, activating Akt and blocking calcium influx Apoptosis is a process associated with multiple infectious diseases including tuberculosis M. tuberculosis also activate apoptosis M. tuberculosis that promote apoptosis has not been performed.Several mycobacterial molecules of diverse chemical nature were reported to induce apoptosis in various types of host cells. In particular, the 19 kilodalton (kDa) lipoprotein was shown to trigger murine and human macrophages apoptosis through TLR-2 via nitric oxide dependent and independent pathways M. tuberculosis strain H37Rv, and unexpectedly discovered this primary apoptotic factor was stable mycobacterial RNA fragments and not a protein or lipid. Additional experimentation revealed the nature of these stable RNA molecules and that their induction of apoptosis was via a caspase-8 dependent, and TNF-α and caspase-1 independent mechanism that affected the ability of human monocytes to control mycobacterial infection.To further address this issue we purified the major apoptosis-inducing product released by virulent M. tuberculosis (MOI of 5∶1) induces early apoptotic events that are defined by annexinV positive and propidium iodide negative staining M. tuberculosis. Thus, it was hypothesized that induction of apoptosis in human monocytes was due to a secreted product of M. tuberculosisin vitro cultures of M. tuberculosis H37Rv was shown to induce monocyte cell membrane damage similar to the live infection  present in fraction 7 was initially determined by assessing the loss of stimulation after differential enzymatic digestion with Proteinase K, DNaseI, or RNaseV1 . UnexpecM. tuberculosis H37Rv CF is enriched in RNA, which was the sole inducer of human monocytes cell membrane damage.To confirm that RNA was responsible for the biological activity, fraction 7 was digested with DNaseI and Proteinase K, extracted with phenol/chloroform/isoamyl alcohol (25∶24∶1) to remove contaminating lipids and undigested proteins and the resulting RNA was gel purified from a denaturing urea-polyacrylamide gel . Gel purM. tuberculosis RNA present in the CF predominantly consisted of tRNA and rRNA with lengths between 30 to 70 bases , presumably autolytic, cultures. After extensively washing cells with GAS medium, CF was collected at different time points (0 to 28 days) and analyzed by SDS-PAGE and ethidium bromide staining. Interestingly, the presence of M. tuberculosis RNA paralleled M. tuberculosis protein secretion into the CF. Specifically, the RNA began to accumulate between two and three days of culture (early log phase), becoming more abundant as the culture progressed , intermediately activated caspase-3 (DEVD-FMK) and had negligible effect on caspase-1 (YVAD-FMK), as determined by flow cytometry  after four days of incubation revealed a greater than two fold increase in the number of bacilli associated with RNA treated vs. untreated monocyte cultures  mediated signaling in vitro experiments with mycobacteria-infected macrophages but also in patients with active disease M. tuberculosis RNA.The activation of specific caspases is central to the induction of apoptosis and classically differentiates between the mitochondrial and apical pathways M. tuberculosis RNA fragments. In contrast, bacterial RNA interactions with cryopyrin activate caspase-1 M. tuberculosis RNA fragments. It must also be considered that tRNA and rRNA undergo multiple species-specific modifications M. tuberculosis RNA fragments need to be considered in future studies such as Protein Kinase R (PKR) that is activated in macrophages infected with M. bovis BCG M. tuberculosis RNA fragments must also be considered with respect to accumulation within the host. The complex secondary structure of the tRNA and rRNA might protect against host as well as bacterial RNase activity, and allow for its stable accumulation in the granuloma.The RNA fragments characterized in this study possess numerous dsRNA hairpin structures, and studies with other viral and bacterial dsRNAs demonstrate that interactions with TLR-8 or cryopyrin led to caspase activation M. tuberculosis as the primary apoptotic factor was unexpected. The possibility that release of RNA fragments is due to lysis of M. tuberculosis was investigated by following the kinetics of RNA release. Such analyses revealed that accumulation of the secreted RNA fragments paralleled the kinetics of M. tuberculosis protein secretion. RNA was observed in the CF as early as 2 days, the same point in time used to distinguish truly secreted proteins from those resulting from autolysis Mycobacterium spp. is involved in DNA transfer or conjugation M. tuberculosis genome in vivo mycobacterial infections M. tuberculosis infection when stimulating with mycobacterial instead of a synthetic RNA.Multiple classes of pathogen derived macromolecules are defined as modulators of innate immunity, including RNA and DNA Multiple studies have evaluated how different types of macrophage cell death affect mycobacterial survival M. tuberculosis is the bacterium's ability to interact with the host immune response in a manner that allows for its own survival and at the same time causes active disease in only a relatively small percentage of the individual it infects. With this perspective it is not surprising that this pathogen has evolved a means to take advantage of the eukaryotic mechanism of programmed cell death. Likewise, the involvement in this process of a bacterial product that is released or secreted only during active growth is not surprising. Thus, the continued elucidation of the exact signaling mechanisms exploited by M. tuberculosis to induce apoptosis as well as the process the bacterium utilizes to release RNA fragments will allow further definition of tuberculosis pathogenesis and the biology of this highly successful pathogen.A hallmark of M. tuberculosis H37Rv  was grown as a pellicle in Middlebrook 7H9 medium  supplemented with glycerol , oleic acid-albumin-dextrose-catalase (OADC) (Becton Dickinson), and Tween 80 (Becton Dickinson). Cultures were collected and M. tuberculosis cells washed twice with phosphate buffered saline (PBS). To disrupt bacterial clumps, M. tuberculosis was suspended in RPMI-1640 with 20% glycerol and probe sonicated  five times at 2.5 W output for 1 min at 4°C. The cell suspensions were stored at −70°C until use. CFUs were determined prior to monocyte infections by plating 100 µL of serial dilutions of an M. tuberculosis stock on Middlebrook 7H10 agar (Becton Dickinson) supplemented with glycerol and OADC. Colonies were counted after two weeks of incubation at 37°C.To prepare bacilli for monocyte infections, M. tuberculosis strain H37Rv was grown in glycerol alanine salts (GAS) medium under constant rotation in roller bottles for two weeks at 37°C and culture filtrate (CF) was obtained as previously described For isolation of biologically active fractions, 2 to a final concentration of 0.4 mg/ml and digested overnight at 37°C with RNase-free DNaseI (Sigma-Aldrich), followed by RNase-free Proteinase K (Sigma-Aldrich) for 8 h at 37°C at a final concentrations of 0.04 mg/ml. The digested material was partitioned with an equal vol of phenol∶chloroform∶isoamyl alcohol (25∶24∶1) followed by an equal vol of chloroform∶isoamyl alcohol (24∶1). RNA was precipitated from the final aqueous phase with 0.1 vol of 3 M sodium acetate (pH 5.3) and 2.5 vol of ethanol, and collected by centrifugation . Final purification was performed by preparative gel electrophoresis using a denaturing urea poly-acrylamide gel M. tuberculosis RNA and standards by denaturing urea poly-acrylamide gel electrophoresis and staining with ethidium bromide. To assess for the presence of LAM, samples (1 µg) were subjected to Western blot analysis using the LAM specific monoclonal antibody CS-35 as the probe To prepare highly purified RNA, the material eluted from DEAE-sepharose with 500–1000 mM NaCl was suspended in 0.1 ml of 10 mM ammonium bicarbonate, 1 mM MgClM. tuberculosis H37Rv was centrifuged at 3,000 rpm and washed three times with medium. Bacterial pellets were suspended in GAS medium and used to inoculate 400 ml cultures. CF was collected at different time points of incubation and processed. Alternatively, CF was precipitated overnight at −20°C with three volumes of ice cold acetone. The precipitated material was collected by centrifugation and analyzed by SDS-PAGE To evaluate the kinetics of mycobacterial RNA release into the CF, low passage AAC CGC ATC CTT CTC iT-3′; lower case letters ribonucleotides, capitalized letters deoxyribonucleotides, ]. The ligation products were gel purified, phosphorylated at the 5′ end with T4 polynucleotide kinase  and ligated to the 5′adaptor sequence [5′ TAC TAA TAC GAC TCA CT aaa 3′ (Dharmacon)]. Synthesis of cDNA from adaptor-ligated RNA was accomplished using the Thermoscript kit (Invitrogen) and the reverse primer (5′ GAC TAG CTG GAA TTC AAG GAT GCG GTT AAA-3′). Double stranded DNA was generated by PCR amplification with the reverse primer, and the forward primer (5′CAG CCA ACG GAA TTC ATA CGA CTC ACT AAA-3′). An aliquot of the PCR products was digested with EcoRI (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted. Overhangs of poly-A were added with Taq Platinum polymerase in the presence of dNTPs. The DNA fragments were ligated with pCR 2.1 TOPO vector (Invitrogen), transformed into TOP10 E. coli (Invitrogen) and individual clones grown for plasmid isolation. The cloned DNA fragments were sequenced through Macromolecular Resources Facility (Colorado State University). Plasmid sequences were analyzed with the Vector NTI software  to identify the 5′ and 3′ adaptor sequences, and the intervening regions were searched against the M. tuberculosis genome, via BLAST analysis.Gel purified RNA was cloned as described by Elbashir and Lau for small interfering (si)RNA g, and cell viability determined by trypan blue exclusion. Cells were suspended at 1×106 cells/ml in RPMI-1640 medium (Gibco BRL) without antibiotics and supplemented with 0.5% heat-inactivated autologous serum (AS). The ratio of monocytes was determined by flow cytometry with anti–CD14-PE staining . CD14+ cells (2×105/well) were plated in 48-well flat-bottomed culture plates  for 4 h at 37°C, and non-adherent cells removed by repeatedly washing with prewarmed PBS with 0.5% AS. RPMI-1640 supplemented with 10% AS (1 ml) was added to each well. The number of adherent cells was determined by mechanical removal and counting in a haemocytometer.Venous blood (120 ml) was obtained from healthy volunteers after signing an informed consent form describing the protocol for human subjects, which was approved by the Ethics Committee of the Facultad de Medicina, Universidad de Antioquia. The blood was defibrinated by continuous agitation with glass beads, centrifuged, and the buffy coat suspended in 3 vol of PBS. Mononuclear cells were separated on Histopaque, density 1.077 g/ml (Sigma-Aldrich) by centrifugation at 400× M. tuberculosis at a multiplicity of infection of 5∶1 for 4 h. Extracellular bacilli were removed by repeatedly washing with prewarmed PBS. RPMI-1640 supplemented with 10% AS (1 ml) was added to each well. The CF and RNA preparations were added at 1 µg/ml protein or nucleic acid concentration, respectively. Cultures were incubated at 37°C, 5% CO2 for 96 h, and the number of viable bacilli determined by plate counting after lysis of host cells with water containing 0.1% saponin Adhered monocytes were infected with viable 6 cells with 2 µg/ml of anti-human CD95 (BD Pharmingen).For analysis of apoptosis induction or cytokine production the CF, individual CF fractions, and purified mycobacterial RNA or rabbit mRNA (Sigma) were added at 1 µg/ml protein or nucleic acid concentration, respectively. PPD was used as a control at 10 µg/ml. As a control, induction of human monocytes apoptosis was performed by incubating 102. Brefeldin A (1 µg/ml) was subsequently added and the cultures were incubated for 6 h. Cells were washed twice with PBS, fixed with 2% paraformaldehyde in 0.1 M NaH2PO4 for 20 min at room temperature, and harvested. Intracellular cytokine labeling was achieved by washing the cells once with permeabilization buffer , and incubating in 200 µl of permeabilization buffer containing 2.5 µg mAb anti-TNF-α-FITC and 2.5 µg anti-IL-10-PE, or IgG2a-FITC and IgG2b-PE as isotype controls for 30 min at 4°C. Cells were washed three times with PBS containing 1% PHS, 1% BSA, and 0.1% sodium azide, pH 7.4. As control for the intracellular signal, non-permeabilized cells were stained with the TNF-α and IL-10 specific antibodies. Thereafter cells were analyzed by flow cytometry with a FACS EPICS XL . The percentage of positive cells and the mean fluorescence intensity were determined using Windows Multiple Document Interface 2,8 software .Evaluation of intracellular cytokines was based on previous methods Cells were cultured for 48 h in the presence of the mycobacterial products and incubated for 30 min at RT in darkness with 5 µl of FITC-labeled annexin-V , 10 µl of propidium iodide at 1 µg/ml , and 100 µl of PBS. Cells were washed with PBS and analyzed by FACS To assess whether apoptosis was dependent on TNF-α signalling, monocytes were incubated for 1 h with purified anti–TNF-α , followed by the addition of the different stimuli for 36 h. Thereafter, annexinV staining was evaluated.M. tuberculosis products to the adhered monocytes. The activation of caspase-1, 3, and 8 was determined by flow cytometry as previously described Caspase activation was measured at 48 h post addition of Experiments were performed a minimum of three independent times. Data were analyzed with GraphPad Prism, version 4 . Comparisons between treatments were performed by one-way ANOVA. Statistical significance was tested at p<0.05 as the critical value. Data are presented as the mean ± SEM.Figures S1Asp.Contig alignments between sequenced RNA fragments and 16S rRNA, 23S rRNA or tRNA Contigs were generated using Vector NTI software to align sequences from the cloned RNA fragments with the respective H37Rv sequences obtained from Tuberculist. Depicted below are the contigs obtained for 16S rRNA (A), 23S rRNA (B) or tRNAAsp (C).(PPTX)Click here for additional data file.Figure S2Extracellular mycobacterial RNA fragments accumulate in the CF with similar kinetics as the rest of the mycobacterial secretome. Low passage M. tuberculosis H37Rv was thoroughly washed three times in GAS media by centrifuging at 3000 rpm and decanting the supernatant. At different time points (indicated below figure) CF was harvested, filtered through a 0.2 µm filter and the CF was concentrated by centrifuging at 3000 rpm in an Amicon with 10 kDa cutoff membrane. Protein concentration was determined by BCA assay and 4.8 µg/lane were analyzed by SDS-PAGE plus silver (left gel) or ethidium bromide (right gel) staining.(PPTX)Click here for additional data file.


The development of chemoresistance still is a major problem in cancer therapy. Mainly, impact of chemotherapy is based on the upregulation of ATP-binding cassette (ABC) transporters, which correlates with bad prognosis and less chemotherapeutic success. Previously, we could show that the HMG-CoA reductase inhibitor simvastatin is able to inhibit the most prominent ABC transporter ABCB1. Additionally, we demonstrated that simvastatin coadministrated with the anthracycline doxorubicin led to increased apoptosis in neuroblastoma cells, and that these effects were comparable with the potential of verapamil, a first generation inhibitor of ABCB1 [The neuroblastoma cell line SH-SY5Y was used for our analyses. Alterations in the protein level of ABC transporters were demonstrated by Western blot analyses and in more detail with FACS. RNA from SH-SY5Y cells treated with simvastatin for 6 and 72 hours was isolated, and the mRNA levels of various ABC transporters were quantified by real-time PCR.Simvastatin exposure led to a concentration-dependent decrease of ABCB1. Similarly, FACS analyses demonstrated a significant decrease of ABCB1 expression on the cell surface. Moreover, doxorubicin-induced elevations in ABCB1 cell surface expression were reversed by simvastatin. However, compensation of ABCB1 by other ABC transporters like ABCC1 and ABCC4 could not be detected. Conversely, ABCG2 showed upregulation on protein and mRNA level.Here we show that simvastatin is able to modulate ABCB1 expression. This potential seems to be divided in immediate and long-term effects. Expression of ABCB1 was downregulated on protein as well as on mRNA level after long-term incubation and partially compensated by other ABC transporters. Moreover, we also observed short-term regulation of the cell surface expression without any changes in total cellular level which might be based on impact of simvastatin in ABCB1 turn-over. Based on our findings, we suggest that simvastatin is a promising candidate as an adjuvant chemotherapeutic drug to impair transporter-mediated multidrug resistance.