Sexual and reproductive health (SRH) is referring to physical and emotional wellbeing and includes the ability to be free from unwanted pregnancy, unsafe abortion, sexually transmitted infections including HIV/AIDS, and all forms of sexual violence and coercion. SRH is the main services packages that prevent and reduce adolescent reproductive health risks and problems. While the government of Ethiopia has undertaken several measures to improve SRH services, there was limited data on utilization among adolescents and associated factors.To assess utilization of SRH services and associated factors among adolescents attending secondary schools in Haramaya District, Eastern Ethiopia.school-based cross-sectional study was conducted among adolescent students aged 15–19 years. A total of 692 adolescents were selected using a multi-stage sampling from two randomly selected secondary schools, each from rural and urban settings, in Haramaya district where 642 provided complete data and included in the analysis. A structured, pretested, and self-administered questionnaire was used to collect data. Data entry was conducted using Epi Data version 3.1 and exported to STATA version 16 for analysis. Bivariable and multivariable binary logistic regression were used to identify factors associated with school adolescents’ utilization of SRH. Statistically significant associations are declared at P-value < 0.05.A A total of 642 completed the survey questionnaire, constituting a response rate of 92.7% (642/692). Male adolescents accounted 63.7% and the mean age of respondents was 17.71 years. Among those who completed the survey, 23.5% (95% CI: 20–26.8) utilized SRH services. Adolescents who were exposed to SRH information (adjusted odds ratio (AOR) = 2.11, 95% CI: 1.22–3.6), aware of SRH service providing facility  and SRH service components , and distance from SRH facilities  were significantly associated with the utilization of SRH services.Nearly one-in-four secondary school adolescents (23.5%) utilized SRH services. Targeted promotion of SRH providing facilities and SRH service components aimed at awareness creation could improve adolescents’ utilization of SRH services. Improved SRH services utilization among adolescents who were far from SRH services providing facilities needs further investigation. Sexual and reproductive health (SRH) refers to physical and emotional wellbeing and includes the ability to be free from unwanted pregnancy, unsafe abortion, sexually transmitted infections including HIV/AIDS, and all forms of sexual violence and coercion. Components of the SRH services are important to reduce adolescent reproductive health risks and problems. This paper reports on the frequency of in-school adolescents who utilized SRH services and the factors that determined it.we collected data from 642 in-school secondary school adolescents, aged 15–19 years, in Haramaya district, East Ethiopia. The data were collected from two schools, one from rural and the other from urban areas, using lottery method from the available schools in the district. A face-to-face interview was conducted to obtain data from the adolescents in the selected secondary schools.Out of 642 in-school adolescents, 23.5% utilized SRH services in Haramaya District. Adolescents who previously received SRH information, who knew SRH services providing facility, aware of SRH service components, and those who live not close to the SRH facilities were utilized the SRH services more often compared to their friends.Nearly one-in-four secondary school adolescents (23.5%) utilized SRH services. Targeted promotion of SRH providing facilities and SRH service components aimed at awareness creation could improve adolescents’ utilization of SRH services. Sexual and reproductive health (SRH) refers to physical and emotional wellbeing and includes the ability free from unwanted pregnancy, unsafe abortion, STIs including HIV, and all forms of sexual violence and coercion. The majority of people become sexually active during the adolescence period putting people in this age group at higher risks of SRH problems . The WorGlobally about 1.2 billion of the total population are adolescents and more than half of this population lives in developing countries . Sub-Sahth birthday, and 3.9 million females aged 15–19 years undergo unsafe abortion every year [Pregnancy, abortion, and STIs (including HIV) are the major SRH risks that result in adverse reproductive health outcomes in adolescents than adults. According to the 2018 Inter-Agency Working Group on Reproductive Health (IAWG) report, AIDS-related death among adolescents are nearly tripled from 21,000 in 2000 to 60,000 in 2014, one-in-four women give birth during adolescence or before they reach their 18ery year . Female Sexual and reproductive health services include access to information and services on prevention, diagnosis, counselling, treatment, and care, and require that all people can safely reach services without traveling for a long time or distance .A study conducted in higher education institutions in Ethiopia revealed that one-third of university students have already been sexually experienced. Of these, nearly two-thirds were found to have sexual experience already before joining higher education institutions evidencing that SRH problems manifest early on and calls for interventions at early adolescence . A rangeThe International Conference on Population and Development (ICPD) in 1994 held in Cairo established a comprehensive SRH service that needs to address and solve the SRH problem for adolescents and youth . NationaDespite that utilization of ASRH are not well explored in Ethiopia, previous studies focused on youth and adult people. A few studies that assessed ASRH services, in turn, are limited to selected service components. TherefoWe conducted a school-based cross-sectional study in Haramaya District which is located at 500 km away from Addis Ababa, the capital of Ethiopia. When this study was conducted, the district has a total population of 313,152 . Only 16.9% of the total population were urban residents with the significant majority residing in the rural parts of the district. There are eight health centers and one general hospital in the study area. The district has 6 secondary schools  and enrolled a total of 6972 students in the 2019/2020 academic year .This study was conducted from March 1 to 15, 2020 in secondary schools at Haramaya District, East Hararge Zone, Oromia Regional State, Ethiopia.The sample size was calculated using a single proportion formula with the assumption of 95% confidence interval, 21.2% proportion of SRH service utilization  from theA multi-stage sampling method was applied to select representative sample of adolescent students. In the first stage, secondary schools in the Haramaya district were clustered into two groups , then from each cluster one school was selected by a simple random sampling method, making a total of two schools. Secondly, school adolescents were stratified by their grade level of attendance. Sample size was then allocated proportionally after obtaining list of students from the respective school administration. Finally, the study participants were selected from each grade by using simple random sampling from the sampling frame of student roster.SRH information Education and counsellingContraceptive servicePregnancy test & careVoluntary counselling and testing (VCT)Sexually transmitted infections (STI) screening, diagnosis, and management servicesSafe abortion careThe dependent variable of interest in this study was adolescent SRH service utilization. The definition of the utilization of the SRH services was ever utilization of any one of the following SRH services:The outcome variable was measured based on self-report using a “yes or no” response to a single question on whether participants have ever utilized any one or many of the specific SRH service components in private and government health facilities.Data were collected using a pre-tested, self-administered questionnaire adapted from the John Cleland’s illustrative questioner for interviewing young people and reviewing works of literature. The tool consisted of three sections: socio-demographic, individual, and health facility characteristics. The original questionnaire was developed in English and then translated into the local Afan-Oromo language. The translated version was then translated back to the original English version for consistency. Six data collectors who were diploma graduates and two supervisors (BSc degree) conducted the data collection following orientation on study objectives, benefits of the study, research ethics and informed consent, and data collection techniques. Before the actual data collection, a pre-test was conducted to identify problems with the assessment tool in a small sample of students, 35(5%), outside the selected study setting. During the data collection, the principal investigator and the supervisors made daily checks to assess completeness of each returned questionnaire and questionnaires with several missing values for important variables have been discarded.Refers to ever utilization of one or more of SRH service components in private and government health facilities . PositivIn this study, accessibility was measured based on adolescent’s self-report of distance they had to travel to health facilities providing SRH services.Data were entered into Epidat version 3.1 and then exported to STATA version 14 for statistical analysis. Descriptive results were presented in tables, figures, and using numerical summary measures for continuous variable. To explore relationship between variables, cross-tabulation was conducted for variables of interest against the outcome variable. SRH services utilization was measured using a dichotomous response (Yes = 1 and No = 0). The positive response was further validated with questions on the type of SRH services utilized. The proportion of SRH service utilization was computed with specific service components. Family wealth index was computed using principal component analysis (PCA) by considering locally available household assets and categorized in to five . Bivariable binary logistic analysis was conducted to select candidate variables for the multivariable binary logistic regression. Variables with P-value less than 0.25 in bivariable binary logistic regression model proceeded into the multivariable binary logistic regression model. Moreover, model fitness was checked by the Hosmer–lemeshow test and multi-collinearity of variables in the final model was also checked using the variance inflation factor (VIF). Variables in the final model with P-value < 0.05 were considered as statistically significant predictor of SRH service utilization. Effect estimates were reported using odd ratio with a corresponding 95% confidence interval.A total of 642 adolescent students have participated in the study which makes a response rate of 92.7%. From the total respondents, 63.7% respondents were male students with the mean age of 17.71 years SD (± 1.19) and more than half, 57.6%, were grade 9 and 10. From the total participants 75.1% were single and 86.1% were Muslims. Regarding the living arrangement of students, most of them, 81.5%, were living with their families  of in-school adolescents utilized SRH services  in Haramaya district. The most frequently used SRH services components were counseling, information, and education, followed by VCT & HIV testing Fig. . Of 132 A binary logistic regression model was used to identify factors associated with utilization of SRH services. Based on the estimates from the bivariable binary logistic regression model, the factors significantly associated with utilization of SRH were age, marital status, living arrangement, having own income, ever had sexual intercourse, ever exposed to SRH information, discussion of SRH issues, perceived SRH risks, know of RH facility, aware of RH services, and being far at distant from the facility. All variables that showed association in the bivariable model are entered into the multivariable binary logistic regression model. Controlling for the effect of other variables in the model, adolescents with the following characteristics were over two-fold more likely to use SRH services: exposure for information , awareness of SRH services, identifying SRH facility whereabouts (AOR = 1.83 95% CI: 1.12–3.0), and far from SRH clinics  showed statistically significant association at p-value < 0.05 . Factors significantly associated with the in-school adolescents’ utilization of SRH services include exposure to SRH information, awareness of SRH services providing facility and service components, and distance from service providing facilities.The level of SRH services utilization among in-school adolescents in the study setting was 23.5%. This finding was consistent with similar studies among in-school adolescents in Ethiopia and elsewhere where SRH services utilization reports ranged from 21.2 to 24.6% –21. HoweThe most utilized SRH services components include information, education, and counseling (80.1%) where previous studies also reported a similar services utilization pattern that was largely limited to information seeking and counseling services . The perAdolescents who previously had exposure to SRH information were significantly associated with current service utilization. Previous studies also reported consistent reports that adolescents who had multiple sources of SRH information including discussion with family members, friends, healthcare providers, and schools were more likely to use SRH services , 27, 28 Adolescents who were aware of the specific SRH service components were more likely to utilize the services than those who did not know. Due to the nature of the study, however, it was not possible to state whether awareness of the specific SRH services components had led to service utilization or the reported awareness was secondary to the service utilization itself. Previous similar studies elsewhere emphasized the importance of adolescents’ awareness of SRH services providing facilities, particularly for rural adolescents . It was Interestingly, in this study, adolescents who were far from health facilities that provide SRH services were two times more likely to utilize SRH services than those who lived close to SRH services providing facilities. This finding was against the reports in previous studies that indicated availability of service providing facilities in close by facilitated SRH services utilization , 19. A sOur study was not without limitation. Pertaining to sensitive nature of the topic studied, respondents may not give honest responses as required. This might underestimate the prevalence of SRH service utilization. We have tried to reduce issues related to confidentiality by making the data collection anonymous and using a self-administered structured questionnaire in the local language for data collection obtain information. This study cannot indicate the direction of the causal relationship.The level of in-school adolescents SRH services utilization was not satisfactory to prevent potentially amenable problems related to and associated impacts of sexual activity and pregnancy related outcomes of adolescents. Targeted promotion of SRH providing facilities and SRH service components aimed at awareness creation could improve adolescents’ utilization of SRH services. Improved SRH services utilization among adolescents who were far from SRH services providing facilities needs further investigation.


NT-ARM), and a peptide encompassing the N0 chaperon module of the P protein. We showed that the chaperone module undergoes a disordered−order transition when it assembles with N0 and measured an affinity in the low nanomolar range using a competition assay. We solved the crystal structure of the complex at a resolution of 2.3 Å, unveiling the details of the conserved interfaces. MD simulations showed that both the chaperon module of P and RNA-mediated polymerization reduced the ability of the RNA binding cavity to open and close. Finally, by reconstituting a complex with full-length P protein, we demonstrated that each P dimer could independently chaperon two N0 molecules.As for all non-segmented negative RNA viruses, rabies virus has its genome packaged in a linear assembly of nucleoprotein (N), named nucleocapsid. The formation of new nucleocapsids during virus replication in cells requires the production of soluble N protein in complex with its phosphoprotein (P) chaperone. In this study, we reconstituted a soluble heterodimeric complex between an armless N protein of rabies virus (RABV), lacking its N-terminal subdomain (N Rhabdoviridae, a large family of nonsegmented negative-sense RNA viruses that includes vesicular stomatitis virus (VSV) [Rhabdoviridae is classified into the order Mononegavirales (MNV) with, inter alia, the families Paramyxoviridae , Pneumoviridae , and Filoviridae (including the zoonotic Ebola and Marburg viruses) [Mononegavirales is now classified with all other known segmented and nonsegmented negative-sense RNA viruses in the phylum Negarnaviricota [Rabies is a zoonotic, incurable brain disease that, despite the availability of an efficient vaccine and post-exposure prophylaxis, continues to kill tens of thousands of people every year according to WHO . Its etius (VSV) , Chandipus (VSV) , and sevus (VSV) . Becauseviruses) ,6. The o0, in the form a complex named the N0−P complex [The RABV genome of 11.9 kb comprises five transcription units, which encode five structural proteins: the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the surface glycoprotein (G), and the large RNA-dependent RNA polymerase (L-RdRP). The multiplication of the virus in appropriate host cells involves (i) the transcription of the genomic RNA to generate messenger RNAs, (ii) the replication of the genome through the intermediate production of positive-sense antigenomic RNAs, which are subsequently used as a template to generate new genomic RNAs, and (iii) the packaging of both genomic and antigenomic RNAs into linear homopolymers of N, forming helical and flexible ribonucleoprotein complexes, named nucleocapsids (NCs) ,8. A uni complex ,14, in w complex .0, but also hijacks cellular components [NTR, aa 1–90) and a C-terminal region (PCTR) consisting of two folded domains, the multimerization domain, PMD (aa 91–131), and the C-terminal domain (aa 186–297), PCTD, which are connected by a flexible linker (aa 132–185) [NTR consists of the chaperon module (PCM—aa 1–40) that binds N0 [NTR binds L-RdRP (residues 40–88) [CTD binds to the polymeric N−RNA complexes (residues 185–297) [NTR is sufficient for keeping N in a monomeric soluble form [0−P complex is recruited at the site of RNA synthesis by the attachment of PCTD to the template [NT-ARM, and an internal loop in the C-terminal domain, named the NCT-ARM, that dock to neighboring subunits in the polymeric form and thereby stabilize the assembled NC [RABV P is an essential multi-functional protein with a modular architecture , which amponents  and counmponents . RABV P mponents ; each pr132–185) ,21,22. Tbinds N0 , while ms 40–88) , whereasble form ,26,27,28n aa 186–7, PCTD, template , the foctemplate , the ribD aa 91–1, and thetemplate , STAT1 [template , PML [33template , nucleartemplate , BECN1 [template , and Cdctemplate . RABV N template . It alsombled NC .0−P complex between a nucleoprotein truncated of its N-terminal arm (VSV NNT-ARM: aa 1–21), VSV N∆21, and a peptide of 60 residues (VSV P60) derived from P that comprised the chaperone module (PCM) and we solved the crystal structure of the complex [∆210−P60 complex was monomeric in solution, but assembled into RNA-free 10-mers circular complexes in the crystal, resembling the recombinant circular NC-like complexes [CM extended in a groove of the C-terminal domain and formed a long α-helix bound at the interface of the N- and C-terminal domains of N. The structure unveiled a mechanism for preventing the assembly of N by interfering with the binding of the NNT-ARM from the adjacent Ni-1 protomer and with the binding of NCT-ARM from the Ni+1 protomer [0, that the phosphorylation of PNTR by casein kinases  had no effect on the interaction with N0 and that dimeric P could bind and chaperone one or two N0 molecules depending of the concentration of N0 in the assembly conditions [0−P complex has since then been obtained for several other RNA viruses in the families Paramyxoviridae [Pneumoviridae [Filoviridae [CM with the NNT-ARM and NCT-ARM binding is a conserved feature within the order Mononegavirales, although the structure and position of PCM on the surface of N varied between the viruses. In VSV crystal structure, the N0 protein was in a close conformation, unable in this state to accommodate the incoming RNA molecules, whereas, by contrast, in the N0−P complex of these other viruses, the N0 molecule was trapped in an open conformation, unable to strongly bind RNA, but ready to grasp an incoming RNA molecule.Previously, working with VSV, we developed a strategy to reconstitute a core N complex . The recomplexes . In the protomer . The proprotomer . We alsonditions . The stroviridae ,44,45, Poviridae , and Filoviridae ,28,47, s∆210−P60 complex [0−P complexes of other viruses [CM and the truncation of the NNT-ARM, the VSV N∆21 protein assembled in a polymeric form in the crystal, suggesting that P60 was not sufficient to maintain the N0 protein in its monomeric state. Second, in the N∆210−P60 crystal structure, the N protein was in its closed conformation, which was identical to its RNA-bound conformation, whereas in the structure of the other N0−P complexes, the N protein was in an open conformation [∆210−P60 complex, the N-terminal part of PCM was bound in same groove of NCTD as the NNT-ARM and in the same orientation. In other known N0−P structures, the chaperone module (PCM) adopted an orientation opposite to that of the N-terminal arm of N (NNT-ARM), against leaving a doubt that the assembly of N could influenced the interaction with PCM [The comparison of the structure of the VSV N complex  with tha complex  and with viruses ,45,46,47ormation ,45,46,47with PCM ,45,46,470−P complex. We used the same strategy for reconstituting the N0−P core complex than for VSV and NiV [0 into rings by further masking the surface of N with the flexible part of the P peptide. We characterized the structure of the complex in a solution by SEC-MALLS and SEC-SAXS and solved the crystal structure. We implemented a fluorescence anisotropy assay to monitor the binding kinetics and equilibrium of the peptide. To better understand the hinge motion mechanism in the N protein and how the binding of P or the assembly into a polymeric N−RNA complex affected this conformational change, we performed molecular dynamics simulations with the different forms of N.To address these questions, we studied the structure and the dynamics of the RABV N and NiV ,43 that D230 and P68 was analyzed with PDBePISA [The amino acid sequences of Lyssavirus phosphoproteins were retrieved from the Uniprot database  and multPDBePISA , structuPDBePISA , and comPDBePISA .The plasmid (pET22b(+)) containing the gene of full-length RABV P (CVS-11 strain) (UniProt P22363) fused to a C-terminal two-amino linker and 6xHis-tag for expression in bacteria was previously described . The cDNE. coli BL21 (DE3). The cells were grown at 37 °C in Luria Bertoni medium containing ampicillin 100 µg/mL (for pET22b constructs) or kanamycin 50 µg/mL (for pET28a and pETM-40 constructs) until the optical density at 600 nm reached 0.6. The protein expression was then induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were further grown at 18 °C for 18 h. The cells were harvested by centrifugation and the pellet was suspended in buffer A phosphine (TCEP)) and supplemented with an EDTA-free Complete Protease Inhibitor Cocktail . The cells were disrupted by sonication and the crude extract was cleared by centrifugation at 35,000× g at 4 °C for 30 min. For proteins containing a 6xHis-tag , the supernatant was loaded onto an Ni−NTA Agarose  column equilibrated in buffer A. The column was washed with ten column volumes of buffer B  and the protein was eluted with buffer C  and dialyzed overnight against buffer D at 4 °C (50 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and 0.2 mM TCEP). For RABV P42 containing a TEV site, the overnight dialysis against buffer D was performed in the presence of the TEV protease at an approximate weight ratio of 100:2 (fusion protein/TEV). After concentration with Vivaspin 20 (3 kDa molecular weight cut-off (MWCO), Sartorius, Göttingen, Germany) or with Amicon   concentrators, the protein was loaded on a S75 Superdex column  equilibrated in buffer D. For N∆23−MBP, the supernatant was loaded onto an amylose resin column  equilibrated in buffer A. The column was washed with ten column volumes of buffer B and the protein was eluted with buffer E . The fractions containing the protein were pooled and concentrated to 3 mg/mL with Amicon concentrators  (Millipore), the protein was loaded on a S200 Superdex column (Cytiva) equilibrated in buffer D at 4 °C. Each protein sample was analyzed by SEC-MALLS  and the concentration was measured by on-line refractometry .All the constructs were transformed into 6-SUMO-TEV-P42-G41C protein in buffer D was incubated for 20 min with a 10-fold molar excess of TCEP and flushed for 1 min with nitrogen gas (N2). A 10-fold molar excess of 5-carboxyfluorescein (5-FAM) maleimide  dissolved in dimethylsulfoxyde (DMSO) was added dropwise and the mixture was incubated overnight in the dark. A 10-fold molar excess of reduced glutathione was added and the solution was loaded on a PD10 desalting column  and eluted with buffer D. The protein was cleaved with the TEV protease at an approximate weight ratio of 100:2 (fusion protein/TEV) and overnight incubation at 4 °C to remove the tag. The labeling efficiency was assessed using liquid chromatography coupled to electrospray ionization mass spectrometry . The labeled peptide P1–42, G41C-FAM was analyzed by SEC-MALLS  and the concentration was measured by on-line refractometry .A solution of concentrated HisΔ23 was mixed with an excess of purified peptide  or of purified PFL and the mixture was incubated for 2 h at 4 °C. The MBP-tag was cleaved with the TEV protease at an approximate weight ratio of 100:2 (fusion protein/TEV) and overnight incubation at 4 °C. The solution was concentrated and loaded onto a S200 Superdex (Cytiva) column coupled to a short amylose resin (NEB) column equilibrated in 50 mM HEPES buffer at pH 7 containing 150 mM NaCl, 50 mM arginine, 50 mM glutamate, and 0.2 mM TCEP to completely remove cleaved MBP.The purified MBP-TEV-N−1. The column was calibrated with globular standard proteins of known hydrodynamic radius (Rh) [−1. The weight-averaged molecular mass was calculated using the ASTRA software (Wyatt Technology).The size exclusion chromatography (SEC) combined with on-line detection using multiangle laser light scattering (MALLS) and refractometry is a method for measuring the absolute molecular mass of a particle in a solution that is independent of its dimensions and shape . The SECius (Rh) . MALLS d∆230−P42-FAM complex at 100 nM in buffer F was mixed with the unlabeled P42-G41C at a final concentration of 15 µM and fluorescence anisotropy was measured with a spectrofluorimeter  with polarizers and thermostated at 20 °C. The excitation and emission wavelengths were set at 495 nm and 520 nm, respectively. In the equilibrium binding assay, the N∆230−P42-FAM complex (100 nM) was mixed with serially diluted unlabeled peptides in black 96-well plates. The samples were then incubated for 24 h at room temperature and fluorescence anisotropy was measured using a plate reader  at 20 °C using an excitation filter at 485 nm and an emission filter at 535 nm. All the experiments were carried out in triplicate. The curve fitting was performed with the software program Dynafit [In the kinetic assay, the N Dynafit .Δ23−P68 complex were carried out at the High Throughput Crystallization Laboratory of the EMBL Grenoble Outstation (HTX Lab.). The crystals of the RABV NΔ23−P68 complex were obtained at 20 °C in (0.02 M sodium/potassium phosphate and 0.1 M Bis Tris propane at pH 6.5 containing 20% w/v PEG 3350). The diffraction data were collected on the ID23-1 beamline at the ESRF . The NΔ23−P68 complex crystallized in space group P21212 with one complex per asymmetric unit. The single crystals were harvested from the drop, briefly soaked in the reservoir solution supplemented with 25% glycerol and flash frozen in liquid nitrogen at 100 K before data collection. The data were processed using the program XDS [The crystallization screenings of the Ngram XDS  and scalgram XDS . The strgram XDS  using a gram XDS  as a seagram XDS  and Refmgram XDS  and Bustgram XDS . The quagram XDS . The dat11−RNA crystal structure (PDB ID 2GTT) in order to correct a frameshift error present in the original entry. The error was detected by comparing the structure of one N subunit from the N11−RNA crystal structure with the N structure from the newly solved NΔ230−P68 crystal structure (8B8V.pdb) and by performing MD simulations of an N−RNA trimer extracted from the N11−RNA crystal structure (2GTT.pdb), which showed significant unfolding of the N-terminal domains within a few hundred ns of MD. The N11−RNA structure was re-refined using a combination of manual rebuilding in Coot [Δ230−P68 crystal structure, molecular dynamics flexible fitting (MDFF) in ISOLDE [We re-refined the circular RABV N in Coot  based onn ISOLDE , and repn ISOLDE  and Auton ISOLDE . The newTM 200 increase 5/150 GL equilibrated with 50 mM HEPES buffer at pH 7.5 containing 150 mM NaCl and 0.5 mM TCEP. The program Foxtrot [c and Rg values [The small angle X-ray scattering (SAXS) experiments were performed on the BioSAXS beamline BM29 and former beamline ID14-3 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France and on the SWING beamline at SOLEIL, Paris, France. For direct SAXS experiments, the scattering from the buffer alone was measured before and after each sample measurement and used for background subtraction. The 1D scattering profiles were generated and buffer subtraction was carried out by the automated data processing pipeline available at the different beamlines. For SEC-SAXS experiments, the samples were loaded onto a Superdex Foxtrot  was used Foxtrot . The radg values .68, an ensemble of 10,000 conformers was generated using the software Flexible-Meccano [∆230−P68 complex, an ensemble of 6639 models was built by extracting snapshots of explicit solvent MD trajectories (see Methods—MD simulations), using a time step of 1 ns. For the N∆230−P68 complex, several ensembles of 10,000 models were built with the software programs RANCH [For isolated RABV P-Meccano . For thems RANCH , PD2 [71ms RANCH . The thems RANCH  and ensems RANCH . The opt0), in complex with P68 (N0−P68), and in its RNA-bound form (N−RNA). The N0−P68 MD trajectories were additionally used to provide an ensemble of models suitable for fitting the SAXS data using EOM. The N0 and N0−P68 models were based on the N∆230−P68 crystal structure (missing residues in N0 and P68 were constructed as random chains) and the simulated N−RNA model corresponded to a trimer of RNA-bound N protein extracted from the revised version of the 11-mer N−RNA ring crystal structure. All 3 systems were simulated in GROMACS [0), 6.6 µs (N0−P1-68), and 2.5 µs (N−RNA). RMSD, RMSF, and principal component analysis (PCA) were calculated using GROMACS routines.The classical explicit solvent MD simulations were used to study the conformational dynamics of the N protein in its free form , while the latter was fused to a C-terminal two-amino acid linker (LE) and a 6-histidine tag (P68−His6) (w) determined from static light scattering and refractometry measurements (SEC-MALLS) was in agreement with the theoretical molecular masses of the respective monomers  values ranging from 0.05 to 4.0 nm−1 over a concentration range from 3.3 to 11.0 mg.mL−1 (g < 1.3) were linear (−1)   spectroscopy and small-angle X-ray scattering (SAXS) that the entire N-terminal region of RABV P (from aa 1 to 90) is intrinsically disordered both in isolation and within the full-length dimeric P protein . However68−His6) C. We purmonomers D. For RA mg.mL−1 E. The Gue linear F and shoear (−1) G. To optear (−1) H and usedom coil I,J. The = 0.824) H, while atistics I,J. FiguNT-ARM—aa 1–23) in fusion with a TEV cleavable N-terminal MBP tag (Δ23). We purified the protein using affinity chromatography on an amylose resin. We then mixed the preparations of MBP-NΔ23 and purified P68−His6, cleaved the MBP tag with the TEV protease, and purified the NΔ230−P68 complex using SEC on a Superdex S200 column. We showed by SEC-MALLS that the NΔ230−P68 complex eluted as a single, symmetric peak from the Superdex S200 column  of 1.001, indicating a monodisperse species.We expressed a truncated form of RABV N lacking its N-terminal arm  = 1.0 for the interaction between NΔ230 and P68 and CSS = 0.0 for all other contacts), demonstrating the absence of N polymerization in the crystal  was not visible in the crystal structure and a 5-residue loop in NNTD (aa 155–159) was also absent from the crystal structure, suggesting that it was flexible in the absence of RNA as expected, since this loop interacted with one of the bound nucleotides in the N−RNA complex [68 were clearly visible in the electronic density; residues from 4 to 15 were bound in a groove of NCTD with residues from 9 to 12 forming a helical turn, while residues from 20 to 39 formed a long α helix that docked at the interface between NNTD and NCTD .The structural alignments using PDBeFold showed that N complex   values ranging from 0.06 to 5.3 nm−1) (g < 1.4) were linear  and used the software GAJOE to select sub-ensembles of three conformers that reproduced the experimental SAXS curve (g and Dmax distributions of these selected sub-ensembles indicated the presence of two main populations of the NΔ230−P68 complex (68 was located in the RNA binding groove and bound to the surface of N (conformers 1 and 2 in 68 (aa 40–68) of eight acidic residues (Asp + Glu) for only three basic (Lys + Arg) and the presence of basic residues in the RNA binding groove of N. This insertion in the RNA binding groove could also contribute to the chaperon activity of P by competing with RNA molecules  B. The sh.3 nm−1) B. The Gue linear C and theperiment A and shoperiment D, thus itructure E. Again,tructure F and use complex G,H. One olecules . The othΔ230 and P68 resisted separation by size exclusion chromatography  and we chemically labeled this cysteine with fluorescein (FAM). We showed by mass spectrometry that the labeling was complete at more than 95% (Δ230−P42−G41C*), purified it by SEC, and confirmed by SEC-MALLS analysis that it formed a heterodimer. At a concentration of 100 nM of NΔ230−P42-G41C* complex, fluorescence anisotropy was near 0.24, whereas that of the isolated peptide P42-G41C* was near 0.07 , we observed a monotonous decrease in fluorescence anisotropy revealing that the fluorescent peptide dissociated from NΔ230 and that the system evolved toward a new equilibrium . In a second approach, we assumed similar kon and koff rate constants for P42-G41C* and P42-G41C and fitted the dissociation curve in the presence of the competitor peptide to a reversible exchange mechanism (off = 0.0027 ± 0.0001 min−1 (45.10−6 s−1) and kon = (5.7 ± 0.1) 10−3 nM−1 min−1 . The dissociation constant Kd of 0.50 ± 0.03 nM was readily calculated from these rate constants (koff/kon). However, in the conditions used in our experiment, the kinetics were dominated by the off-rate as shown by the identical koff value obtained in both fitting procedures. Thus, the value of kon should be considered a lower bound value, as no significant difference in the goodness of fit was found when larger values were imposed and the Kd value of 0.50 nM represented an upper bound value. Considering an upper limit for a bimolecular protein–protein interaction on-rate (kon) near 5 × 105 M−1 s−1 [d value of 0.09 nM was obtained.The interaction between RABV Ntography B, suggesreaction A. We genthan 95% . We recoear 0.07 B. We meaear 0.07 B, supporilibrium B. In a fechanism A by usin M−1 s−1 , a lowesΔ230−P42-G41C* complex with serially diluted concentrations of unlabeled P42-G41C and P68. The optimal incubation time required for the system to reach equilibrium, mainly depending on the off-rate (koff), was set by a rule of thumb at 24 h (5 × 0.693/koff = 1284 min = 21 h). The curve provided a measure of the relative affinity of unlabeled peptide to labeled peptide. All things being equal, we expected that the half-saturation concentration should be that of the initial concentration of the labeled complex . The curve obtained with unlabeled P42-G41C had a midpoint (C1/2) around 300 nM, whereas that with P68 had a midpoint near 1000 nM  and that the C-terminal part of P68 slightly reduced the stability of the complex. If we assumed Kd* = 0.5 nM for the labeled peptides, fitting the equilibrium competition binding curves by numerical integration with the software Dynafit [d values of 4.5 ± 0.5 nM  for P42-G41C and of 8.2 ± 0.6 nM  for P68  [Rhabdoviridae) , NΔ230−P68, and N3−RNA, a set of three adjacent subunits extracted from the circular N11−RNA complex (2GTT.pdb), in which we built the flexible parts to generate full-length N molecules. We performed molecular dynamics (MD) simulations to evaluate the effects of P68, RNA binding, and multimerization on the dynamics of N protein   remained flexible and sampled a wide range of conformations , which yielded an improved model of the circular undecameric N−RNA complex (3−RNA) from the re-refined N−RNA complex structure, the MD simulations showed that the N protein structure was stable over several hundred ns in multiple independent trajectories (In known structures of the Nviridae) ,46,47,79iridae)  . For NΔ2rmations , consistrmations . In the ly found , but thesoftware ,63,64 an complex . Using tectories . The rooCM binding, RNA binding, and multimerization modulated the conformational landscape of the N protein, we performed a principal component analysis (PCA) on the MD simulation datasets, using the N residues common to all three systems  corresponded mainly to the opening−closing movement of the RNA binding groove also around the hinge region   with a main basin centered around PC1 ≈ from −2 to −1, while PC1 values for N−RNA were comprised in a shorter range between −3 and 0. This suggested that RNA binding and multimerization moderately impacted interdomain twisting of N as both systems showed PC1 values roughly centered around the same value  and purified the NΔ230−PFL complex by SEC on a Superdex 200 column. We performed a SEC-SAXS experiment by injecting 50 μL of a sample of NΔ230−PFL into a Superdex 200 Increase column and monitored the elution by SAXS. We collected scattering profiles for q values ranging from 0.085 nm−1 to 3.0 nm−1 at regular time intervals during the elution. The complex eluted as a single peak, but the radius of gyration (Rg) determined by using the Guinier approximation at small scattering vector (q) values (qmax.Rg < 1.3) varied across the elution peak  or one (F2) N molecules.The Guinier plots for F1 and F2 were linear and we determined Rm for F2  from the∆230−PFL complex, one ensemble with one N attached to each chain of P (2N−2P) and one with a single N attached to one of the P chain (1N−2P). We then used the GAJOE program for selecting sub-ensembles that reproduced the experimental SAXS curves at positions F1 and F2 in the chromatogram. The curve in the front part of the elution peak (F1) was better reproduced with 2N−2P models (reduced χ2 = 1.9) than with 1N−2P models (reduced χ2 = 2.7) (2 = 1.9) than with 2N−2P models (reduced χ2 = 19.9)  . A repre = 19.9) . A repreribution E,H. Surp P alone ,19. FurtCM) that autonomously maintains the N protein (N0) in an RNA-free, monomeric, and soluble form [0 (RABV N∆23 lacking the NNT-ARM) [CM is sufficient to keep N0 in a soluble, RNA-free monomeric form, it is possible that NC assembly in cells requires additional functional modules of P. Indeed, exogenous peptides encompassing PCM from different viruses, including RABV, have been found to inhibit viral replication in cultured cells, whereas they were able to solubilize N in the cytoplasm [0 molecules near the site of RNA synthesis and/or to trigger NC assembly. The dimerization of P through its multimerization domain (PMD) [0 within the biological condensates, in which viral RNA replication occurs. Finally, full-length RABV P protein forms dimers [0 molecules per P dimer depending on the concentration of N present at the time of the reconstitution of the complex [As previously demonstrated for RABV and other viruses, the N-terminal intrinsically disordered region of P contains the chaperon module  ,43. Howeytoplasm ,43,79,81ytoplasm ,80 and tytoplasm , and eitin (PMD)  was founin (PMD) , althougs dimers ,22, wher complex .∆230−PFL complex are not different from those of isolated PFL in similar experimental conditions [0 molecules were bound to the P dimer, but the radii of gyration are similar. The SAXS data also clearly show that the two bound N proteins are not preassembled within the N0−P complex, but rather remain independent of each other. It is worthwhile noting that if we assume a generic partial specific volume of 0.73 mL.gr−1 for both RABV P and N proteins [g = 4.9 nm, MM = 68.4 kDa), whereas the 2N−2P complex occupies 36% of the volume   . Similar∆23) was unable to assemble into NC even in the presence of RNA. The assembly of N into polymeric N−RNA complexes could only be reproduced in vitro with full-length N, demonstrating that the NNT-ARM is required for NC assembly , although the mechanism of NC assembly and the detailed role played by the NNT-ARM and NCT-ARM remain poorly understood. We hypothesize that for an incoming N protein to incorporate into a nascent NC, the binding of its NNT-ARM onto the last already incorporated N subunit could be required either for anchoring the new N protein and allowing the assembly or for stabilizing the complex once it is formed or both.Additionally, as found for other viruses ,76, the ∆230 and P68 is strong with a dissociation constant in the low nanomolar range. The dissociation constant of the FAM-labeled peptide was below 1 nM and that of the unlabeled peptide measured by competition assay was 4 nM. This strong affinity is consistent with the large amount of surface area that becomes buried upon association with P68 (~2800 Å2). The binding of the N-terminal part of PCM (aa 4–15) onto N∆230 buries about 1200 Å2, whereas the binding of the C-terminal part (aa 20–38) buries about 1600 Å2. For protein–protein and protein−peptide complexes that bury more than 2000 Å2 upon assembly, an average value of 4.0 cal/mol/Å2 was estimated for the ∆Gbinding [2 would predict a ∆Gbinding of 11.2 kcal.mol−1 and a dissociation constant of 6 nM (at 20 °C), which is in good agreement with our measurements. By comparison, a dissociation constant of 1.1 nM has been determined for the interaction between Ebola virus N0 and a peptide of VP35 that contains the chaperone module, whose association buried a surface area of ~2400 Å2. These figures place RABV N0−P complex among the protein–protein complexes that bury the largest surface area upon assembly and have a high binding affinity [0−P complex in the infected cells at the earliest stages of the virus replication cycle, when only small amounts (low concentrations) of viral proteins are present. The nucleoprotein has a strong tendency to nonspecifically assemble onto cellular RNA and thus chaperoning by the P protein must occur as soon as N synthesis begins. However, the N0−P complex is not the final step in the production line of the viral N protein. N0 must be subsequently transferred to nascent viral RNA to form new viral NCs. To drive the assembly of NC, the N−RNA must probably be as stable or even more stable than the N0−P complex and it is noteworthy that binding of the NNT-ARM of the Ni-1 subunit buries ~1500 Å2, while the binding of the NCT-ARM of the Ni+1 subunit buries ~1700 Å2, totaling ~3200 Å2 per N subunit upon NC assembly, which translates into predicted ∆Gbinding value of 12.8 kcal.mol−1 and a dissociation constant of 0.3 nM (at 20 °C). The assembly of NC from the N0−P complex could thus simply be driven by thermodynamics, the reaction evolving toward the most stable state of the system.The interaction between NGbinding , which faffinity . This st68 can transiently insert into the RNA binding groove of N perturbing/preventing the insertion of an RNA molecule and thus provide a putative additional contribution to the N0 chaperon activity of P. This part of RABV PNTD is rich in negatively charged amino acid and is thus suitable for inserting in the positively charged RNA binding groove. However, this part of the peptide is not visible in the crystal structure, indicating either that the interaction is weak and transient or that the peptide binds in different conformations in the RNA binding groove. In the meantime, the presence of this region destabilized the complex, possibly because the decrease in conformational entropy is not compensated by the formation of additional stabilizing interactions.The molecular dynamics simulations revealed that the C-terminal part of P0−PCM core complex from different viruses belonging to different families of the Mononegavirales revealed many similarities in the complex architecture and in the mechanism of action of PCM, but also some differences [CTD and directly competes with the NNT-ARM and NCT-ARM of adjacent subunits. In the Paramyxoviridae , Pneumoviridae (hMPV), and Filoviridae (EBOV and MARV), PCM binds exclusively to NCTD, in the same groove as the NNT-ARM subdomain of the Ni-1 subunit in the polymeric N−RNA complex. In these complexes, the two protein segments (PCM and NNT-ARM) adopt opposite orientations although they bind in the same groove of the N protein [CM extends to the binding of the NCT-ARM of the Ni+1 subunit and thus also directly interferes with its binding to the Ni subunit. In the Rhabdoviridae (VSV and RABV), one part of PCM also binds to NCTD, in the same groove as the NNT-ARM of the Ni-1 subunit and the NCT-ARM of the Ni+1 subunit, while the major part of the chaperone module forms an α-helix that binds at the interface between NNTD and NCTD , the N protein is locked in a closed conformation, causing the RNA binding site to be inaccessible to an incoming RNA molecule and suggesting that the protein must open to accommodate an RNA molecule. When initially observed in the VSV N0−PCM structure, the polymeric assembly of N raised the possibility that the closure of the RNA binding groove was a consequence of the polymerization [0−PCM complex, the N protein is unassembled but is also trapped in a similar closed conformation, demonstrating that this state of the protein is independent of the polymerization and is an intrinsic property of the protein–protein complex as confirmed by our principal component analysis of MD simulations.The first crystal structures of polymeric, circular N−RNA complexes revealed that the RNA molecule was embedded in its protein shell, with some bases facing the protein away from the solvent ,41,88. Trization . In the 0−PCM complexes of Paramyxoviridae, Pneumoviridae, and Filoviridae, the N protein is blocked in an open conformation, in which the RNA-binding groove is too widely open to allow the formation of all the bonds evidenced between the protein and the RNA molecule within the N−RNA complexes [0−PCM complex led us to propose that the binding of PCM perturbs the dynamics of the N protein by linking together different subdomains that must move relative to one another when the protein switches between its open and close conformations [NTD and NCTD move relative to each other in different directions. The binding of PCM but also of the NNT-ARM and RNA onto the surface of N restricts these movements in the protein. The binding of PCM blocks N in open or close conformation depending on the viral family, preventing the positioning of an RNA molecule in the groove between NNTD and NCTD. The binding of RNA, NNT-ARM, and also NCT-ARM blocks N in a close conformation that shields the RNA from the solvent, cellular proteins, and the viral polymerase. Different hypotheses have been put forward to explain the opening of the N protein, involving the intervention of P or L to trigger this process. The simulations suggest that the release of PCM and of the NNT-ARM from the surface of N would allow the protein to undergo its intrinsic opening−closing movements and allow the insertion or release of the RNA molecule.By contrast, in the Nomplexes ,88,89,90rmations ,43. The 0−PCM complex, viruses can currently be separated into two different groups, with one group including the Rhabdoriviridae and another group including Paramyxoviridae, Pneumoviridae, and Filoviridae, which likely reflect evolutionary relationships. The formation of the N0−P complex is likely an ancestral feature of the Mononegavirales, which has evolved by divergence in the different families, conserving the essential properties and showing that the great sequence−structure space diversity of the polypeptide chain allied with the high capacity to mutate of these viruses can create important variations around the same “theme”. A striking example is the inversion of the direction of binding of the PCM on N0.On the basis of the structural architecture and tentative mechanism of action of the N


Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome–symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes.Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of  Bradyrhizobium were characterized in a phylogenetic and genomic context and findings suggested that polypartite and monopartite symICE configurations can cause fitness costs in nonnative combinations and the advantages conferred by either are realized only in compatible chromosomes. Therefore, evidence suggests that no symICE configuration is generally more advantageous than the other and supports a hypothesis that selective advantages lie in the flexibility of symICEs in adopting different configurations and recombining in different regions of chromosomes.It has been hypothesized that symbiosis integrative conjugative elements (symICEs) that confer symbiotic nitrogen fixation and with a polypartite configuration have selective advantages over those that have a monopartite configuration. In this study, symICEs of Horizontal gene transfer (HGT) drives genome diversification and innovation. ICEs are agents of HGT that can transfer between bacterial cells and facilitate recombination with chromosomes . CriticaGenome organization constrains diversification . BacteriBradyrhizobium genus has a large and diverse accessory genome (att) sites and vary in their structural configurations as monopartite or polypartite elements. When integrated in a chromosome, elements of a polypartite symICE are located in different regions and mediate large-scale rearrangements during recombination , tRNA-Glu (∼0.77 Mb), tRNA-Gln (∼0.68 Mb), and tRNA-Pro (0.33 Mb) symICEs.The y genome . The symy genome . These sy genome . SymICEsbination . In Bradl symICE . The larBradyrhizobium and Mesorhizobium were examined in a phylogenomic context . For example, strain NK6 was reanalyzed and is predicted to carry a bipartite tRNA-Arg symICE with elements near tRNA-Arg and ybgC. Four strains in clade 2 have one of three monopartite symICEs, though most strains analyzed here lack a symICE. Strains of clade 3 are predicted to have a nif/fix island with some predicted to have remnants of symICEs  with only three exceeding 10% replichore imbalance fig. 1B; . Except attBV and attBY sites  in chromosomes correlate with the phylogenetic pattern of bipartite tRNA-Val symICEs that recombine at these sequences. In 30 of 33 clade 1 strains natively lacking or simulated to lack a symICE, the two attB sites are in separate replichores, not equidistant from the ter region, and facing head-to-head toward the ori (D). In the first integration step, a bipartite tRNA-Val symICE will increase the size of a replichore and add an attP site oriented head-to-head to its partner attB site (A). In the second step, an inversion will occur that redistributes the chromosome between replichores. The amounts of symICE and chromosome involved reflect the locations of attP and attB sites and their distance apart following the first recombination step. To rebalance replichores, the size of the element that remains in the recombined replichore and the size of the element flipped to the other replichore need to offset the net change associated with the chromosome inversion. This raises the possibility that selection may favor the sizes of symICE elements and distance between attPV and attPY, implying that the size of the variable region is important.Location and orientation of  the ori D. In thettB site A. In theattBV and attBY sites are located within the same replichore and arranged head-to-tail (D). If a bipartite tRNA-Val symICE recombined into these chromosomes, the second step will cause an ∼1.0 Mb-sized deletion .Conversely, in 15 of 18 strains in clades 2 and 3, -to-tail D. If a bfig. 2B; . The delfig. 2B; . A similfig. 2B; . Therefohizobium A.att sites targeted by monopartite symICEs are also important. Integration of a monopartite symICE occurs in one step and directly increases the size of a replichore thus requiring offsetting deletions in the same replichore or gains in the other replichore (C and 3B). Five of six strains with monopartite symICEs have natively balanced replichores and simulated excision of their symICE resulted in an imbalance of ∼10%, consistent with changes having had occurred to balance against an increase by symICE integration . We did not consider the effect of the monopartite tRNA-Pro symICE because its relatively small size reduces its effect on replichore imbalance (B). Locations of att sites relative to the replichore in which offsetting large-scale chromosomal changes occur, are predicted to influence the type of monopartite symICE that will be selected for once recombined in a chromosome.The locations of plichore C and 3B.egration . For mos symICEs D and 2C.mbalance B. Locatiatt sites that deviate from common patterns are predicted to differ in their ability to acquire symICEs. The attBV and attBY sites of strain DOA1 in clade 1 are organized such that a large deletion is predicted to occur upon resolution of a bipartite tRNA-Val symICE  searches  and locations of dif loci . To idensearches . Percentsearches .att sites oriented head-to-head or tail-to-tail (att locus and invert regions when involving a bipartite symICE. Monopartite symICEs were simulated to integrate in both orientations relative to the att sites. SkewIT was used to calculate the SkewI index of GC skew (att sites are not oriented head-to-head or tail-to-tail, a deletion was predicted. To identify essential genes, bidirectional best BLAST searches were done using as a query a dataset of essential genes identified based on analysis of a transposon library derived from strain USDA110 (A custom python script and BioPython were used to start genome sequences at the predicted ori, simulate excision of symICEs if natively in a chromosome, and induce inversions if simulating excision of a polypartite symICE integrated at -to-tail . Another GC skew . For bip USDA110 .Genome Biology and Evolution online.evac078_Supplementary_DataClick here for additional data file.


From the development to disease, long non-coding RNAs (lncRNAs) regulate chromatin landscape ultimately influencing gene transcription, X-chromosome inactivation, RNA-mediated epigenetic inheritance, DNA methylation and genome stability through maintenance of active and silent chromatin state. LncRNAs function through establishing and maintaining stable RNA-chromatin interactions at a specific genomic locus through RNA-protein and/or RNA-DNA interactions. Growing evidence underscores the significance of lncRNA sequence, structure and epitranscriptomic modifications as potential mediators of RNA-chromatin interactions. Whether lncRNA sequence, structure and epitranscriptomic marks, either alone or in a particular combination, act as specific molecular code governing RNA-chromatin interactions needs to be elucidated. The future of lncRNA research and its therapeutic success relies on mapping the interplay among lncRNA sequence, structure and epitranscriptome. Further, it is important to understand how these features, alone or in a particular combination, stabilize RNA-chromatin interactions ultimately influencing gene expression and function.Bisserier et al. provide novel insights into potential roles of exosomal lncRNAs in determining the effects of radiation after spaceflight. As exosomal RNAs are known to mediate cellular communication, Bisserier et al. provide new experimental data on exosomal RNAs obtained from peripheral blood plasma of three astronauts who flew on various shuttle missions between 1998 and 2001. In their transcriptome analysis of exosomal RNAs during spaceflight, they report several differentially expressed lncRNAs in exosomes isolated from astronauts at different timepoints during their spaceflight. These results also provide new insights into the diagnostic and prognostic value of astronauts-derived exosomal lncRNAs as emerging ncRNA biomarkers and highlight the potential role for exosomal lncRNAs in health risks associated with spaceflight.As we enter the space age, it is important to understand how the non-gravitational environment affects our health  In this Somasundaram et al. provides an in-depth analysis of lncRNAs with specific focus on their role in establishing and maintaining biomolecular phase-separation. Formation of membrane-free ribonucleoprotein condensates resulting in intracellular compartments by liquid–liquid phase separation or condensation is a critical phenomenon that compartmentalizes lncRNA and other macromolecules separate from others. In this Review article, Somasundaram et al. further provides a deeper understanding of phase separation, physio-chemical properties of such biomolecular condensates, their composition and the role of RNAs in driving the formation and stabilization of biomolecular condensates. Particularly, the Review highlights several lncRNAs, which are known to act as “the anchor” using their specific intrinsic sequence motifs within the condensates. Furthermore, the authors provide some recent evidence for lncRNAs working through phase separation in association with diseases, such as cancer, further highlighting the significance of condensate biology in cellular physiology and argue why better understanding of condensates would enhance therapy. Taken together, this review article is an excellent addition and an insightful summary of new developments in the field of ncRNA and phase separation.For a biochemical reaction to occur in a cell, the interacting partners must be present in the close proximity . To addrLee O. Vaasjo summarizes chemical changes to RNA as a critical regulator of mRNA regulation. RNA modifications, particularly in the form of N6-methyladenosine (m6A) methylation, have been shown to be integral for RNA metabolisms of mRNAs and for several lncRNAs. For mRNAs, m6A modifications are specifically regulated through distinct mechanisms occurring within 3′-UTR or coding regions. However, little is known on how m6A modifications are regulated within 5′-UTRs of mRNAs. In this Research Topic, Vaasjo summarizes elegantly how m6A methylation regulates 5′-UTRs of mRNA through lncRNAs. Specifically, the author provides recent examples on how several lncRNAs can target specific sites within 5′-UTRs of mRNAs to regulate m6A patterning within 5′-UTRs of mRNAs. Furthermore, the author highlights a critical epigenetic crosstalk mediated by lncRNAs through direct interactions of lncRNAs with chromatin remodeling proteins in determining m6A pattern within 5′-UTRs of mRNAs. This Review highlights an important crosstalk occurring between lncRNAs and the m6A modifying machinery in the nucleus; addressing one of the key mechanisms by which lncRNAs not only working at the level of gene expression but also at the epitranscriptomics level thereby regulating the expression of coding transcriptome. This Review can be regarded as an excellent summary of novel findings that underscores an important interplay between the non-coding (ncRNA) transcriptome and the coding (mRNA) transcriptome through complex regulation of epitranscriptomic mechanisms.To date, over 170 RNA modifications are known across organisms , which oZiemann et al. tackles the problem of identifying ncRNAs and RNA-binding proteins (RBPs) that may be associated with hearts of dilated cardiomyopathy (DCM). Ziemann et al. report significant changes in expression of several genes encoding ncRNAs, such as ribosomal RNAs (rRNA), and further classify that downregulation of several RBPs as a specific feature of hearts in DCM. Although roles for majority of ncRNAs remain unexplored in mature adult tissues, such as in the heart, Ziemann et al. elegantly show that dysregulated expressions of several RBPs are present in hearts of DCM. In particular, the changes were observed in expression for gene sets of mitochondrial (mt)-rRNA processing, aminoacyl-tRNA synthases, and mitoribosome subunits for in-situ protein synthesis. The authors identified several enzymes such as mitochondrial rRNA methyltransferases  that regulate rRNA epitranscriptome and mitochondrial ribonucleases  as differentially regulated genes in DCM. Whether these changes in expression to rRNA methyltransferases and RNases have transcriptome-wide impact on coding and non-coding RNAs needs further investigation. Interestingly, Ziemann et al. report downregulation of several mtRBPs, including Slirp, Gadd45GIP1, Dap3, Ptcd1-3, Lrpprc, Grsf1 etc, which are involved in rRNA modification and formation of mitoribosome. However, how these RBPs influence the pathophysiology of DCM remains unknown. Together, these new results from Ziemann et al. provide important new evidence indicating changes in expression of enzymes and proteins potentially involved in ribosomal epitranscriptome in DCM hearts.Mitochondria are dynamic powerhouses of the cell that are important for many cellular activities . As the In summary, this Research Topic provides an important overview on RNA-based molecular mechanisms that are not only of fundamental importance but also of therapeutics. Research articles as well as Reviews published in this Research Topic cover a broad range of mechanisms involving RNA-chromatin interactions. These mechanisms provide important insights to uncover ncRNA function and a deeper understanding of RNA-chromatin interactions will undoubtedly begin a new era in RNA-based therapeutics.


We performed mutation analysis of ribonucleic acid sequencing (RNA-seq) and whole mitochondrial genome data from tissues collected from metastatic lesions of a male patient in his 60s with metastatic renal cell carcinoma. We confirmed the common mutation sites of a mitochondrial gene containing the T3394Y, R11,807G, and G15,438R sites. Pathway analysis, using RNA-seq data, confirmed the common mutant pathway between renal cell carcinoma and metastatic adrenal carcinoma as cytokine–cytokine receptor (CCR) interaction. However, no common nuclear gene mutations were identified. The two similar sequences suggest that mtDNA-mutation-driven metastasis to the adrenal gland may affect CCR interactions and cause cancer cells to thrive.ND1) gene. The R11,807G (p.T350A) site transitioned from threonine  to alanine . Additionally, the G15,438R or A (p.G231D) site transitioned from glycine  to aspartic acid  in cytochrome b (CYTB). Furthermore, pathway analysis, using RNA-seq, confirmed the common mutant pathway between RCC and adrenal carcinoma as cytokine–cytokine receptor (CCR) interaction. Confirmation of the original mutation sites suggests that transfer to AG may be related to the CCR interaction. Thus, during metastasis to the AG, mitochondria DNA mutation may represent the initial origin of the metastasis, followed by the likely mutation of the nuclear genes.This study aimed to clarify whether genetic mutations participate in renal cell carcinoma (RCC) metastasis to the adrenal gland (AG). Our study analyzed whole mitochondrial gene and ribonucleic acid sequencing (RNA-seq) data from a male patient in his 60s with metastatic RCC. We confirmed common mutation sites in the mitochondrial gene and carried out Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using RNA-seq data for RCC and adrenal carcinoma. Furthermore, we confirmed the common mutation sites of mitochondrial genes in which the T3394Y (p.H30Y) site transitioned from histidine  to tyrosine  in the NADH dehydrogenase subunit 1 ( We performed genetic analysis to investigate metastasis, using cancer tissues that had metastasized from the kidneys  to the adrenal gland (AG). This study aimed to clarify whether genetic mutations play a role in metastasis from RCC to the AG. To this end, we have been conducting research on both nuclear genes and mitochondrial DNA (mtDNA). Previously, we reported that mitochondrial DNA mutations are involved in the development and metastasis of RCC ,2. The kThe AG is an organ approximately 2–3 cm in size, located above the kidney, as shown in Currently, drug treatments for metastatic RCC are diverse and stratified by risk factors using models such as those from the Memorial Sloan–Kettering Cancer Center and the International Metastatic RCC Database Consortium. However, it is difficult to select appropriate cases for study ,14,15,16ND1) regions of mitochondrial DNA in 62 patients [MT-ND1 or D-loop mutations, we found that RCC patients with both D-loop and MT-ND1 mutations exhibit more advanced cancer-specific survival. Thus, differences in nuclear gene expression and mitochondrial genome mutation profiles between primary and metastatic lesions may aid in the selection of various therapeutic agents. Therefore, we performed mutation analysis of RNA-seq and mitochondrial genome data using surgical specimens of tissues from patients with metastatic RCC collected from metastatic lesions. In the current study, we aimed to verify whether these genes are associated with metastasis. In addition, we sought to identify a more effective molecular-targeted drug for metastatic RCC by investigating pre-administration information, selected drugs, and their effects, as well as the prognosis relative to the administration method based on genomic analysis. We believe that these results provide valuable insights regarding the selection of effective drugs for the treatment of metastatic cancer, as well as the relationship between cancer-specific mutant genotypes and metastatic destinations.Previously, our group analyzed the recurrence and survival of the D-loop and NADH dehydrogenase subunit 1  of Tokai University, Japan . Informed consent was obtained from the patient.The surgical specimens of the cancerous and normal kidney tissues were collected, immersed in RNAlater , and stored at −80 °C. The cancerous and normal kidney tissues for study were then collected from the surgical specimens by macroscopic judgment. In addition, a blood sample was collected from the peripheral veins the day before surgery. DNA from blood sample was used as the control sequence.Total DNA and RNA were extracted from cancer tissues using TRIzol , followed by RNeasy  . The RNATissue and blood samples were then incubated with 200 g of proteinase K in buffer (10 mM Tris-Cl (pH 8.0), 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulfate) at 60 °C overnight, and later extracted with phenol–chloroform  twice at 11,000 rpm for 10 min at room temperature (25 °C). DNA was precipitated by the addition of 0.1 volume of 3 M Na-AcOH and 2.5 volumes of ice-cold ethanol. Then, after centrifugation at 15,000 rpm at 4 °C for 20 min, the DNA pellets were rinsed with 70% cold ethanol, dried, and dissolved in TE buffer (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA). The total mtDNA was then amplified by polymerase chain reaction (PCR) with the primer sets , which cPCR was performed using KOD FX Neo  under the following conditions: melting at 98 °C for 5 s, annealing at 63–66 °C for 15 s, and then extension at 68 °C for 20 s, for a total of 35 cycles. The PCR products were treated with EXOSAP-IT  and immediately sequenced using a Big Dye Terminator v3.1 Reaction Kit  and an ABI 3500xL DNA sequencer .Complete mtDNA sequence data from the patient with localized RCC were assembled using Sequencher 5.0 software . The mutation sites were determined using NC_012920.1 as the reference sequence.The raw reads were obtained from the HiSeq 2500, and after completion of the run, data were base-called and demultiplexed . FASTQ format files in Illumina 1.8 format were considered for downstream analysis .Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina), according to the manufacturer’s instructions ,35.The libraries were sequenced on a HiSeq 2500 sequencing platform using HiSeq SBS Kit v4 reagents with 2 × 101 cycles.The adapter sequences and low-quality regions were trimmed using Cutadapt (v1.1)  and Trimmomatic v0.32 , respectively. After pre-processing, the reads were mapped to the human reference genome GRCh38 using TopHat (v2.0.14)  . DiffereHomo sapiens genes by enrichment analysis. The listed genes were submitted to the DAVID functional annotation database (http://david.abcc.ncifcrf.gov/) (accessed on 7 December 2021), providing a broad and unguided test against primarily Gene Ontology (GO) groups as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [p-value is <0.05.We created lists annotated to pathways ,33,39,40ND1 in normal cells was mutated to the heterosite of T3394Y (Y: Pyrimidine) in cancer cells. Site 15,438 changed from G in cytochrome b (CYTB) to R or A. In addition, site 11,807 of the NADH dehydrogenase subunit 4 (ND4) gene changed to G from the R heterosite  site transitioned from glycine  to aspartic acid , while the R11,807G (p.T350A) site transitioned from threonine  to alanine . Furthermore, the T3394Y (p.H30Y) site transitioned from histidine  to tyrosine  . HoweverCXCL1, 5, 6, and 8; CXCR2: CXCL2 and 3; CXCR3: CXCL4; and CXCL4L1) were affected  was also impacted , and oral intake of steroids was initiated. Radiotherapy was administered for the bone metastases. As of October 2021, multiple lung metastases and a right adrenal metastasis-maintained shrinkage, as well as multiple bone metastases, were shown in osteosclerotic images.This study aimed to clarify the involvement of genetic mutations in the metastasis of RCC to the AG. We analyzed the total mitochondrial DNA and RNA sequences of the renal and adrenal cancer tissues in a patient with RCC metastasis to the AG. By analyzing mitochondrial and nuclear DNA from two cancer sequences (K58-R and K58-M), we were able to confirm whether a common gene for cancer metastasis exists. Specifically, three common mutation sites were identified: T3394Y, R11,807G, and G15,438. T3394C mutation is reportedly associated with colon cancer and leukemia ,42. HoweND1, Hayashi et al. showed that mtDNA mutations are involved in cancer metastasis by creating cybrid cells via cytoplasmic transplantation using mouse cells [ND1 had metastasized from the primary RCC to the AG. Mitochondria are likely to be involved in adrenal metastasis, as RCC developed earlier in this patient. Furthermore, in our report, there was a correlation between the recurrence of ND1 and RCC (61 patients) and cancer-related death. These data are provided as part of our supplementary data.Regarding se cells . Complexse cells . This coCXCL1-11 (excluding CXCL7), as well as 13 genes of the CXC subfamily. It was also determined that the CXC subfamily that comprises the pathway is affected by three genes: CXCR1, CXCR2, and CXCR3. Zeng et al. found that the transcriptional levels of CXCL1/2/5/6/9/10/11/16 in RCC tissues were reduced, while those of CXCL3/7/12/13 were elevated. CXC1/5/9/10/11/13 were shown to have a significant correlation with the pathological stage of RCC. RCC patients with low transcriptional levels of CXCL1/2/3/5/13 have a better prognosis [CXCR3 gene, the transforming growth factor (TGF)-β family  pathway is also impacted. Specifically, the expression levels of bone morphogenetic protein (BMP) receptors Acvr1, Bmpr1a, and Bmpr1b in the BMP signal transduction system were decreased. BMP signaling can promote tumor growth and suppress endometrial cancer. Fukuda et al. demonstrated the tumor-promoting effects of BMP signaling in endometrial cancer cells by inducing cancer stemness, EMT, and migration [Moreover, in the pathway analysis based on RNA-seq, the common mutant pathway cytokine–cytokine receptor interaction between RCC and adrenal carcinoma was confirmed. In particular, it strongly affected rognosis . Oldham rognosis . Furtherigration . Severaligration .ID1 [TGF-β is a major inducer of the epithelial–mesenchymal transition (EMT) ,49. The ID1 ,64,65,66Molecular-targeted drugs are used to treat metastatic RCC. Improvements in survival rates have been reportedly compared, and treatment strategies for metastatic RCC are undergoing major changes. However, appropriate risk assessment, prognosis prediction, and optimal administration of molecular-targeted drugs for metastatic RCC in Japan have not yet been established. Therefore, it is necessary to consider information on a large number of patients to establish an effective treatment strategy.Based on mitochondrial gene analyses, we confirmed the common mutation sites between the original RCC and metastatic adrenal carcinoma. The three sites, G15,438R, R11,807G, and T3394Y, helped us confirm that K58-R and K58-M have similar sequences. Moreover, a common mutant pathway, CCR, was identified between RCC and adrenal carcinoma. However, no common nuclear gene mutations were identified. Furthermore, in this study, we were not able to identify specific genes associated with metastasis. Nevertheless, the two similar sequences suggest that mtDNA-mutation-driven metastasis to the AG may affect CCR interactions, allowing cancer cells to thrive.