RNAs can interact and form complexes, which have various biological roles. The secondary structure prediction of those complexes is a first step towards the identification of their 3D structure. We propose an original approach that takes advantage of the high number of RNA secondary structure and RNA-RNA interaction prediction tools. We formulate the problem of RNA complex prediction as the determination of the best combination (according to the free energy) of predicted RNA secondary structures and RNA-RNA interactions.We model those predicted structures and interactions as a graph in order to have a combinatorial optimization problem that is a constrained maximum weight clique problem. We propose an heuristic based on Breakout Local Search to solve this problem and a tool, called RCPred, that returns several solutions, including motifs like internal and external pseudoknots. On a large number of complexes, RCPred gives competitive results compared to the methods of the state of the art.We propose in this paper a method called RCPred for the prediction of several secondary structures of RNA complexes, including internal and external pseudoknots. As further works we will propose an improved computation of the global energy and the insertion of 3D motifs in the RNA complexes.The online version of this article (10.1186/s12859-019-2648-1) contains supplementary material, which is available to authorized users. RNAs can link to each other and form complexes having catalytic functions. A well known example is the ribosome  composedPegs and Rubber Bands and an approximation algorithm is proposed ∈E}.OM is the set of pairs of vertices  where v is not in the clique C but there exist edges between it and all the vertices of the clique C, except u which is in the clique C; OM={:v∉C and u∈C,∀v′∈C∖{u}∃∈E and ∉E}. The OM set is used to do the replacement movements.OC is the set composed of all the vertices outside C; OC={v:V∖C}.In the local search phase of BLS, all the possible movements are considered and the one optimizing the most the solution weight is chosen. The available movements are either to add a vertex in the clique or to replace a vertex in the clique with one that is not in the clique. To define the movements, some definitions are needed. Let The perturbation phase aims to modify the current solution to escape a local optimum. The perturbation can greatly degrade the solution, the strength depending on how many times the solution was not improved in the local search phase. The perturbation strategies are based on four movements that are performed several times: to add, replace or remove a vertex of the clique (weak perturbation) and to restart (strong perturbation). The perturbation phase uses a tabu list in order to avoid to pick up again a vertex for a movement if it was removed from the solution some iterations before. The perturbation phase is a main difference with other local search methods and allows to explore more efficiently and faster the search space.The authors show that this heuristic provides improved results for a number of MWCP instances and that this heuristic is usable for large graphs in reasonable time. It makes this heuristic a good candidate to develop a tool for the RNA complex prediction since large sets of inputs can be used.The BLS method is adapted for the constrained MWCP by making some modifications to the initial clique finding phase and to the movements, in order (i) to take into account the different kinds of vertices, (ii) to take into account the constraints related to the secondary structures and (iii) to avoid the weak cliques.C be a clique from the initial graph G. PA be the set composed of all the interaction vertices that are outside C and are connected to all the vertices in C; PA={vI:vI∉C,∀u∈C ∃∈E}.Let OM be the set composed of the interaction vertices pairs  ) such that vI (or vS) is outside C and is connected to all vertices in C except to the vertex uI∈C (or uS∈C).Let OC be the set composed of all interaction vertices that are outside C; OC={vI:VI∖C}.Let Before describing our BLS-CMWCP algorithm, let us give the new definitions about the sets used  or secondary structure vertex pairs  in OM allow only the movements respecting the constraint of having exactly one secondary structure per RNA. In the following, we describe each modification and the differences between our algorithm BLS-CMWCP and BLS: To generate the initial clique: in BLS, the phase consists in selecting randomly a vertex and then to add iteratively vertices if they form a clique, until no more vertex can be added. In BLS-CMWCP, this phase consists in selecting randomly an interaction vertex and then selecting for each RNA a secondary structure vertex that forms a clique. Forming a clique is always possible thanks to the empty secondary structures which are obviously compatible with any interaction vertex.To add a vertex movement: an interaction vertex vI is selected in PA and added into C.To replace a vertex movement: a vertex pair ) is selected in OM, vI (or vS) is added to C and uI (or uS) is removed from C.To remove a vertex movement: an interaction vertex vI is selected in C to be removed.To restart the clique movement: an interaction vertex vI from OC is added to the clique C. Then if the structure vertices of the clique, vS∈C, do not form a clique, they are replaced with other structure vertices. Finally, the remaining interaction vertices of the clique, vI∈C, are removed if they do not form a clique anymore.To generate sub-optimal cliques: contrary to BLS method, we want to return several sub-optimal cliques. In BLS-CMWCP, at each iteration of the local search and of the perturbation phases, any new clique is saved. Then when the solutions are returned, they are sorted according to their free energy.To forbid the weak cliques: during all the search, if a movement leads to a weak clique, it is not considered.Having only the interaction vertices L0 and LMax), the maximum number of non-improving solutions before a strong perturbation is performed (T), the coefficients for accepting non-improving solutions (αs and αr), the coefficient for tabu tenure (ϕ) and the probability for applying directed perturbations (P0). Some parameters were fixed in the BLS heuristic and we used them as such in BLS-CMWCP. We determined the other parameters by performing experiments on a dataset of 30 graphs derived from RNA complexes. We then chose the following parameters: L0=0.1∗|V|, LMax=0.1∗|V|, T=10, αs=0.5, αr=0.5, ϕ=7 and P0=1. The stop condition for the local search occurs either when the optimum clique is found or the maximum number of iterations is reached. In RNA complex prediction we do not know the optimum clique, then the stop condition here is a maximum number of iterations (fixed at 500). This parameter can be set by the user.The BLS heuristic proposed in  has a sen sequences of RNAs, several secondary structures per RNA and several interactions per pair of RNAs. First the energies of the secondary structures and the interactions are computed. The compatibilities between the secondary structures and the interactions are determined and the graph is built. BLS-CMWCP returns constrained cliques from which are derived RNA complexes. If some sequences are identical, symmetrical complexes can occur. These lasts are identified and removed to avoid redundancies in the results. Finally, the RNA complexes are sorted according to their free energy. RCPred is available on the EvryRNA platform.We implemented in C++ BLS-CMWCP and obtained the tool called RCPred (RNA Complex Prediction). RCPred takes as inputs In this section, we present the results we have obtained with RCPred on a large set of RNA complexes. First we detail below the dataset used and how we recovered the secondary structures and interactions. We then give the results of RCPred and compare it with NanoFolder , NUPACK In the following experiments, we use a dataset composed of 90 non-redundant RNA complexes. The dataset is extracted from the database RNA STRAND  that gatFor each RNA, we generated the secondary structures using three tools from the literature for RNA secondary structure prediction, namely BiokoP , pKiss  and RNAsWe present in this section the results obtained by our tool RCPred and the comparison with the results of NanoFolder, NUPACK and MultiRNAFold. As RCPred is based on an heuristic, the results presented here are obtained from 5 executions. Among these tools, only RCPred and NUPACK can return sub-optimal solutions corresponding to different predicted complexes.1-score which is the harmonic mean between the sensitivity and the PPV and the Mathews Correlation Coefficient (MCC) which is a balanced measure between sensitivity and specificity (that measures the proportion of negatives that are correctly identified). These statistics are computed as follows: TP is the number of true positive base pairs, FN is the number of false negative base pairs, FP is the number of false positive base pairs and TN the number of true negative base pairs.To evaluate the quality of predicted complexes, we used the sensitivity to measure the ability of finding positive base pairs and the Positive Predictive Value (PPV) to measure the ability of not finding false positive base pairs. We also used the F1-score results of the 10 first solutions returned by RCPred in average on 5. As we can see, the solutions having the highest F1-scores are in most cases the first returned. However, in many cases, the sub-optimal solutions reach the highest F1-scores, there are even some complexes for which the best prediction is given by the tenth sub-optimal solution. This confirms that better predictions can be found in the sub-optimal solutions and therefore the need to generate them.We first study for each complex the optimal and sub-optimal predictions of RCPred in order to see the relevance of generating several solutions. We report on Fig. 1-score results of the 10 first solutions returned by RCPred in average on 5 executions, with inputs of BiokoP (A), pKiss (B) and RNAsubopt (C). Knowing that the performances of BiokoP, pKiss and RNAsubopt depends on the size of the RNAs, it is expected that they all predict accurate secondary structures for small RNAs. Hence it is not surprising to observe similar results for the smallest complexes (inferior to 62 nucleotides). For longer complexes the results differ. A comparison of these results with the ones obtained when the input secondary structures generated by the three tools are merged  on the longest complexes (of size greater than 550 nucleotides), because of the size limitation of the web server. Also we encounter some difficulties with the tool MultiRNAFold on some complexes.We then compare RCPred with NanoFolder, NUPACK and MultiRNAFold. We report the F1-scores than the other tools in most cases. With the sub-optimal solutions, we can guarantee that at least a predicted structure is close to the referenced one. For almost all complexes smaller than 120 nucleotides, RCPred is able to find accurate predictions with F1-scores higher than 80%. For longer complexes, RCPred, as well as the other tools, becomes less effective with a maximum F1-score around 70%. Similar results and findings are obtained with sensitivity, PPV, and MCC statistics  and reoThe time execution of RCPred varies between 0.05 s in average on 5 executions for the smallest complex and 16.7 seconds for the longest. We are currently working on optimizing the time execution.In RNA complexes, the tertiary interactions are numerous and have an important role in the stabilization of the global structure. There exist databases gathering 3D motifs appearing in single RNA structures, like the Rna3Dmotif database . MoreoveAdditional file 1Supplementary statistical results. Supplementary Figures showing the sensitivity, PPV and MCC results of RCPred compared to the results of NanoFolder, NUPACK and MultiRNAFold. (PDF 61.8 kb)


Panicum mosaic virus (PMV), a prototypical tombusvirus, and its satellite agents , all of which depend on the PMV-encoded RNA polymerase for replication. The atypical RNAs are defined by terminal polyadenylation and truncation events within the 3′ untranslated region of the PMV genome. These modifications are reminiscent of host-mediated RNA degradation strategies and likely represent a previously underappreciated defense mechanism against invasive nucleic acids.The genomes of positive-sense RNA viruses have an intrinsic capacity to serve directly as mRNAs upon viral entry into a host cell. These RNAs often lack a 5′ cap structure and 3′ polyadenylation sequence, requiring unconventional strategies for cap-independent translation and subversion of the cellular RNA degradation machinery. For tombusviruses, critical translational regulatory elements are encoded within the 3′ untranslated region of the viral genomes. Here we describe RNA modifications occurring within the genomes of  Tombusviridae family have genomes lacking a 5′ cap structure and prototypical 3′ polyadenylation sequence. Instead, these viruses utilize an extensive network of intramolecular RNA-RNA interactions to direct viral replication and gene expression. Here we demonstrate that the genomic RNAs of Panicum mosaic virus (PMV) and its satellites undergo sequence modifications at their 3′ ends upon infection of host cells. Changes to the viral and subviral genomes arise de novo within Brachypodium distachyon  and proso millet, two alternative hosts of PMV, and exist in the infections of a native host, St. Augustinegrass. These modifications are defined by polyadenylation [poly(A)] events and significant truncations of the helper virus 3′ untranslated region–a region containing satellite RNA recombination motifs and conserved viral translational enhancer elements. The genomes of PMV and its satellite virus (SPMV) were reconstructed from multiple poly(A)-selected Brachypodium transcriptome data sets. Moreover, the polyadenylated forms of PMV and SPMV RNAs copurify with their respective mature icosahedral virions. The changes to viral and subviral genomes upon infection are discussed in the context of a previously understudied poly(A)-mediated antiviral RNA degradation pathway and the potential impact on virus evolution.Positive-sense RNA viruses in the  Viruses of eukaryotes face constant pressure to evade the intrinsic defenses of the host cell, including nucleic acid sensors, molecular turnover machinery, and induced immune factors. These selective forces favor viral populations that are adept at making “nonself” look like “self” during infection. Positive-sense RNA viruses have an arguable advantage in this regard, primarily due to the coding orientation of their infectious genomic RNAs and their ability to directly engage the host translational machinery.Tombusviridae is a large family of positive-sense RNA viruses with many species capable of infecting evolutionarily diverse host plants and replicating in numerous cell types (7GpppN cap structures or 3′ polyadenylation [poly(A)] sequences  is a prototypical tombusvirus with a 4.3-kb single-stranded RNA genome encapsidated within ∼30-nm icosahedral virions (19––Stenotaphrum secundatum) (Panicum virgatum) .–24.Panicundatum)  and has irgatum) –27. Withrgatum) –  ––31. Thesgatum) ––, 32, 33.gatum) –– 30, 34), 34PanicBrachypodium distachyon , green foxtail millet (Setaria viridis), and proso millet (Panicum miliaceum)  , 35–38. and SPMV , 35. PMVand SPMV , 39–41.Brachypodium transcriptome samples, and full genomes were reassembled from poly(A)-selected data sets deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). For PMV, cloned cDNAs from infected Brachypodium tissues contained major 3′-end truncations encompassing the entire UTR, with added heterogeneous bases and noncanonical poly(A) tails. Polyadenylated PMV, SPMV, and satellite RNA genomes were also isolated from naturally infected St. Augustinegrass tissues, suggesting that this phenomenon is not an artifact of laboratory infections. We also find polyadenylated PMV and SPMV RNAs associated with purified virions from transcript-inoculated proso millet hosts, demonstrating that these altered genomes retain packaging signals for effective transmission. The modified RNAs share hallmarks with products of host-mediated RNA decay pathways and may be the result of previously underappreciated host antiviral responses to infection.Here, we present the serendipitous discovery that the genomes of PMV and its satellite agents undergo 3′-end modifications within the infected host cell. Viral and subviral RNAs were detected in poly(A)-selected Brachypodium distachyon as an alternate host to study the misregulated transcriptome resulting from PMV and PMV-plus-SPMV (PMV-SPMV) infections , using mRNA-enriched libraries  , 23. It To further validate and determine the sequence(s) of these apparent poly(A) features within the viral genomes, we sequenced the 3′ ends of PMV products amplified using the oligo(dT)-primed cDNA. Sanger sequencing analyses of 12 individual clones revealed that most products contained several A- or U-rich stretches of added bases (∼30 bases or more) downstream of the viral sequences, with few containing heterogeneous sequences of unknown origin. We also noted several truncations at approximate nucleotide position 4000 within the 3′ UTR of the viral RNA, followed by stretches of A/U-rich bases -selected data set corresponding to the PMV and SPMV viral and subviral genomes. Using the reference genomes for PMV (accession no. NC_002598.1) and SPMV (accession no. NC_003847.1) as search queries, we identified abundant viral and subviral raw reads from the PMV-infected (accession no. SRX747740) and PMV-SPMV-infected (accession no. SRX747746) Brachypodium SRA data sets. The SRA search parameters and results are summarized in After completion of the PMV-eq study , using teq study  and 3A. 10.1128/mBio.01900-19.3TABLE S1Table S1, DOCX file, 0.02 MB.Summary of Sequence Read Archive (SRA) mining search parameters and results. Download Copyright © 2019 Pyle et al.2019Pyle et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Brachypodium transcriptome, we identified ∼20,000 reads with strong sequence similarity to the PMV reference genome (de novo assembled into a single contiguous consensus sequence (contig). This contig was 4,397 nt in length, 71 bases longer than the PMV reference genome, with an extension of 23 bases from the 5′ end of the viral genome. This 5′ extension is identical to the sequence from nt positions 125 to 147 in the original PMV clone and possibly reflects an artifact of the contig assembly process. The wild-type 3′ end was present in the assembled contig, followed by 26 adenine residues downstream of the 5′-ACCAGGCCC-3′ terminal motif . The C848A and U3752C substitutions result in synonymous leucine-to-leucine mutations in the p48 and p26 proteins, respectively. The A1799C substitution results in a nonsynonymous threonine-to-proline mutation at amino acid position 591 in p112. The origin of this point mutation in the major PMV replication-associated protein is unclear, but we hypothesize that it could be an artifact of RNA sequencing and/or base calling algorithms. Alternatively, it could be a valid mutation in the genome that occurred during PMV replication in plants and whose functional consequences remain to be determined. Nevertheless, the blastn search yielded significant hits to related panicoviruses  and the 3′ ends of the PMV satellite RNAs, which originate from recombination events with the helper virus genome during infection  than in the PMV data set, a single contig of 836 nt in length corresponding to SPMV was reconstructed using these reads for the de novo assembly. In a manner similar to that of PMV, the SPMV contig was 10 bases longer than the SPMV reference sequence and contained many mutations throughout the genome, including multiple deletion and substitution events  and the assembled SPMV contig from an mRNA-enriched Brachypodium transcriptome dataset (SPMV_contig). The SPMV p17 capsid protein ORF and p6.3 putative ORF are highlighted in blue and yellow, respectively. The 3′ terminus of the reference genome and nonviral sequences at the 5′ end of the assembled contig are underlined. Mismatches between the sequences are indicated by the absence of a vertical line. Dashes indicate sequence gaps. Sequence limits are indicated on either side of the alignment panel. Lowercase “G” bases in grey for SPMV_ref represent a region of the query that was automatically filtered for low sequence complexity according to the NCBI blastn suite parameters. Download FIG S1, JPG file, 1.4 MB.Sequence alignment of the SPMV reference genome  events in vivo , we nextin vivo , cDNA frin vivo . Most ofin vivo  11). Th. Thin vi10.1128/mBio.01900-19.2FIG S2Brachypodium transcriptome data set. Individual reads from the PMV-SPMV-infected Brachypodium data set were compared against the 3′ UTR of the PMV reference genome (NC_002598.1). The 3′ end of the PMV capsid protein ORF is shown in green. The region corresponding to the 3′ cap-independent translation enhancer is underlined in blue. Base mismatches or insertions are indicated as “Conflict” or “Insertion,” respectively. Residue labeling follows the standard expanded nucleic acid letter code to indicate the nature of each mutation. Download FIG S2, JPG file, 0.5 MB.Detailed analysis of mutation sites in the PMV 3′ UTR identified from the Copyright © 2019 Pyle et al.2019Pyle et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Brachypodium and/or was a result of mechanical inoculation of T7-synthesized RNAs. To address this, we analyzed leaf tissues of naturally infected St. Augustinegrass (Stenotaphrum secundatum), a C4 warm-season turfgrass that is phylogenetically divergent from Brachypodium, a temperate C3 grass. St. Augustinegrass is a very prevalent host for PMV and SPMV in the Gulf Coast region of the United States , another well-established alternate host of PMV and SPMV  RNAs were detected in purified PMV and SPMV virions via amplification of oligo(dT)-primed cDNAs . In summPanicum mosaic virus and its two distinct satellite agents are modified in a previously unknown way, challenging our understanding of tombusvirus replication events and viral population dynamics within the infected hosts. For PMV, these alterations are defined by de novo polyadenylation and extensive editing  events within the 3′ UTR. Furthermore, we found that the modifications occur in both laboratory and natural infections of multiple hosts , underscoring its relevance beyond a single host-virus pathosystem.The principle finding of this study is that the RNAs of Our initial hypothesis for the polyadenylation involved virus-mediated acquisition of 3′ poly(A) tails in order to self-protect from host RNA degradation or surveillance mechanisms, an inherently proviral mechanism. This “tail-snatching” strategy was hypothesized to be analogous to cap-snatching, a process mediated by the RdRPs of segmented negative-sense RNA viruses for acquisition of capped primers for synthesis of viral mRNAs –51. Cap-–de novo polyadenylated RNAs and low-frequency viral genome variants on quasispecies population dynamics  by using a single subgenomic RNA originating from a prematurely terminated minus-sense replication intermediate , 23. Sin54–57–le virus , 61, thile virus , the prode novo added A/U-rich sequences were indeed identified in the genomes of diverse plant and animal RNA viruses that are known to lack genomic poly(A) tails, suggesting that this mechanism is conserved among several eukaryotic hosts  polymerases (PAPs) and polynucleotide phosphorylases (PNPases) are associated with the addition of poly(A) and A-rich tails to the 3′ ends of cellular RNAs –66. Thes–65–71–ic hosts –79. The c hosts – and B. Rc hosts –, 81. CytA third possibility involves trafficking of the PMV, SPMV, and satellite RNAs into the host nucleus for polyadenylation. The capsid proteins of PMV and SPMV localize to the nucleus and nucleolus/Cajal-like bodies, respectively , 82. Thein vivo modifications to the RNAs of PMV and its satellites. The mutation and deletion signatures within the PMV 3′ UTR implicate a host poly(A)-mediated RNA degradation strategy with antiviral potential. We hypothesize that this phenomenon is not unique to tombusviruses and may represent a host-directed RNA degradation strategy with antiviral implications. Similar observations have been sporadically reported in the literature for additional RNA viruses from diverse eukaryotic hosts, indicating that this phenomenon extends beyond the plant immune response against invasive nucleic acids. This RNA modification strategy deserves further attention in the broad context of known antiviral host responses and viral evasion mechanisms.In summary, we have defined previously underappreciated Brachypodium distachyon (accession Bd21-3) and proso millet (Panicum miliaceum cv. Sunup) were grown under previously described conditions (2/s) and 10 h of darkness at 21°C and 18°C, respectively. The St. Augustinegrass (Stenotaphrum secundatum) leaf samples were collected from a 10-m2 lawn area on the Texas A&M University campus , as reported previously , as described previously  with TRI reagent (Ambion) (Taq DNA polymerase (NEB) with standard reaction conditions and appropriate primers  , 35. RNABrachypodium samples analyzed  primer for cDNA synthesis containing an overhanging adapter sequence for subsequence PCR amplification (OligodT-Adapter)  . PCRs weAdapter)  and a reAdapter) . PCR proBrachypodium in the SRA . The full-length reference genomes of PMV (NC_002598.1) and SPMV (NC_003847.1) were used as search queries. Search parameters were optimized for blastn, megablast, and discontiguous megablast to identify the maximum number of reads for each query and data set (Brachypodium data sets using the tblastn suite with the capsid protein amino acid sequences of PMV (NP_068346.1) and SPMV (NP_620827.1).The blastn suite at the NCBI SRA was used to mine the transcriptome data sets for mock-, PMV-, and PMV-SPMV-infected data set . SearcheBrachypodium mRNA-enriched transcriptome data set (accession no. SRX747740) yielded >20,000 reads with sequence similarity to the PMV genome (NC_002598.1). These reads were assembled into two preliminary contigs using the CodonCode Aligner desktop sequence assembly software . Contig1 had a length of 2,384 bases and was assembled from 10,847 reads, while contig2 had a length of 1,905 bases and was assembled from 9,153 reads. Contig1 was used as query for megablast in NCBI. Hits included the 3′ end of the PMV genome (plus/plus orientation), the 3′ ends of Thin paspalum asymptomatic virus (TPAV) isolates, and the 3′ ends of the PMV chimeric satellite RNAs. Contig1 had >99% sequence identity with the PMV reference genome, with one mutation at position 3752 (U3752C). Contig2 was used as query for megablast in NCBI. Three hits were identified, including the 5′ end of the PMV reference genome (plus/minus orientation) and two TPAV isolates. Two additional mutations were identified within contig2 (G848U and U1799G). The terminal 9 bases at the 5′ end of the PMV reference genome were not identified in contig2.A search of the PMV-infected Brachypodium mRNA-enriched transcriptome data set (accession no. SRX747746) yielded 19,891 reads with sequence similarity to the PMV genome (A search of the PMV-SPMV-infected V genome . These rBrachypodium poly(A)-selected transcriptome data set (accession no. SRX747746) was performed using the SPMV reference genome as the query (NC_003847.1). This search yielded 763 reads  for 10 min. Virions were concentrated by high-speed centrifugation  on a 20% sucrose cushion for 2 h. Virus pellets were resuspended in 50 mM PPB and subjected to a second low-speed centrifugation  for 10 min. The clarified supernatant was loaded onto 10% to 40% sucrose density gradients in 50 mM PPB, and the virions were separated by high-speed centrifugation  for 2 h. Following the density gradient separation, the light scattering bands for PMV and SPMV were removed and concentrated by a third high-speed centrifugation step  for 3 h in 50 mM PPB. The pelleted purified virions were resuspended in 1 ml of 50 mM PPB and stored at 4°C for further RT-PCR analysis.PMV and SPMV virions were purified from transcript-inoculated proso millet tissues as described previously, with minor modifications , 84. Inf


L-glutamate is the major excitatory amino acid in the mammalian central nervous system (CNS). This neurotransmitter is essential for higher brain functions such as learning, cognition and memory. A tight regulation of extra-synaptic glutamate levels is needed to prevent a neurotoxic insult. Glutamate removal from the synaptic cleft is carried out by a family of sodium-dependent high-affinity transporters, collectively known as excitatory amino acid transporters. Dysfunction of glutamate transporters is generally involved in acute neuronal injury and neurodegenerative diseases, so characterizing and understanding the mechanisms that lead to the development of these disorders is an important goal in the design of novel treatments for the neurodegenerative diseases. Increasing evidence indicates glutamate transporters are controlled by the circadian system in direct and indirect manners, so in this contribution we focus on the mechanisms of circadian regulation  of glutamate transport in neuronal and glial cells, and their consequence in brain function. Thi the SCN –22, alth the SCN .Glutamate (Glu), the main excitatory neurotransmitter in the mammalian central nervous system (CNS), activates two subtypes of Glu receptors: ionotropic (iGluRs) and metabotropic (mGluRs) –25. The +-dependent high affinity Glu transporters carries out the Glu removal from the synaptic space. The excitatory amino acid transporters (EAATs) comprise five different Glu transporters: Glu/aspartate transporter (GLAST), Glu transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4), and excitatory amino acid transporter 5 (EAAT5) or EAAT 1-5 according to rodent and human nomenclature, respectively , as with AD, there is also a decrease in Glu uptake; in PD, Glu transporters have an unusual trafficking between membrane and cytoplasm leading a decrease in Glu transporters at the plasma membrane . This phLikewise, Glu transporters have a critical role in Huntington's disease (HD), in which the expression of these transporters is diminished, the symptoms of HD worsen . In thisIt is well-known that Glu transporters are regulated at different levels, at the transcriptional translational and post translational levels through modifications of transporter protein, as well as by the transporter targeting and trafficking –64. NeveGlast mRNA and protein levels present a diurnal rhythm in 12/12 h light-dark conditions  to ZT18  are a good example of a system that can rapidly respond to external stimuli since it is activated without changes in transcription and/or translation . In thiscis- and trans-elements involved in its transcriptional regulation. Specifically, the circadian regulation of Glu transporters is an emerging theme that promises to be an indispensable tool in the preventing and/or treatment of diseases related to alterations in glutamatergic system. Future research should be directed to study of molecular mechanisms involved in circadian modulation of these transporters.In the last two decades, several research groups have examined the different signaling pathways that modulate glial Glu transporters expression (GLAST and GLT1). Scarce information about EAAC1, EAAT4, and EAAT5 transporters is available. Particularly, EAAC1 has a much less evolutionarily conserved sequence in the 5′ noncoding region compared to GLAST and GLT-1, hindering the identification of Optimal functioning and precise regulation of Glu removal from the synaptic cleft is critical to prevent an excitotoxic insult and thus avoid several neurodegenerative pathologies. To date, compelling evidence suggests that Glu transporters could be regulated in a circadian fashion Figure . It is cDC-C gathered the relevant information, wrote the manuscript, as well as elaborated the figure. AO revised and edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Other methods create artificial chimeric transcripts coupled with bacteriophage-derived coat proteins  to tag molecules in live cells. In other approaches, endogenous RNAs are recognized by complementary RNAs complexed with noncatalytic Cas proteins. Each technique has its own set of strengths and limitations that must be considered when planning an experiment. Here, we discuss the mechanisms, advantages, and weaknesses of in situ hybridization, molecular beacons, MS2 tagging and Cas-derived systems, as well as how RNA tracking can be employed to study various aspects of molecular biology.RNA tracking allows researchers to visualize RNA molecules in cells and tissues, providing important spatio-temporal information regarding RNA dynamics and function. Methods such as fluorescent  Coding RNAs (messenger (m)RNAs) serve as templates for the translation of proteins, while noncoding RNAs (including microRNAs (miRNAs) and long non coding RNAs (lncRNAs)) regulate gene expression programmes on many levels ,2. RNAs en masse. Microarrays are fast and cost-effective methods for quantifying RNA levels in distinct populations, and RNA sequencing (RNA-seq) can further elucidate the identity of the RNAs at the cell or tissue levels [Recent advances in RNA biotechnology have allowed researchers to interrogate the transcriptome e levels –7. Immune levels . These mAdvances in RNA localization at a subcellular level have improved our understanding of viral and neurodegenerative diseases through characterization of RNA-mediated pathological mechanisms. For example, analysis of the trafficking of hepatitis C virus (HCV) RNA revealed that host cells increase the production of type-I interferons by packaging viral RNAs into exosomes; these are then transported to nearby dendritic cells, activating the TLR-7-mediated antiviral response . Other sDrosophila oocytes, wherein regulation of bicoid mRNA localization establishes head and thorax regions in the egg [Xenopus and zebrafish development [RNA localization is also important for establishing cell polarity and asymmetry during development. Developmental patterning is achieved by restricting mRNA translation to one side of the cell, effectively localizing the respective protein products to subcellular compartments. This is evident in  the egg . Similarelopment ,14.In adult mammals, one notable example of long-range mRNA transport is found in neurons in the form of messenger ribonucleoprotein (mRNP) granules . These min situ hybridization (FISH)) or beacons, to track individual RNAs by making chimeric RNAs bearing tractable elements , and to identify endogenous RNAs through a complementary RNA complexed with noncatalytic Cas9/Cas13 , FISH was soon adapted to detect various types of RNA molecules [figure 1b) [FISH was introduced in 1980 by the van Duijin lab , and wasfigure 1olecules –25. A kegure 1b) . This teWhile RNA FISH allows tracking of RNA molecules in cells, it is also a valuable tool for quantifying the spatial distribution and copy number of a target RNA. RNA-seq and reverse transcription (RT) followed by real-time quantitative polymerase chain reaction (qPCR) assays can directly quantify transcripts in tissues or single cells ,27,28, bβ-actin (ACTB) mRNAs revealed that their localization in the leading lamellae in fibroblasts contributes to cell motility [β-actin mRNA transport via zipcode-binding protein 1 in neural growth cones regulates the direction of axonal growth, as demonstrated by FISH coupled with immunostaining [FISH has been used since its advent to study cellular RNA trafficking. FISH analysis of individual motility . Similarstaining . The expstaining  and nuclstaining  were alsDrosophila embryos. The main limitation of this approach is the number of available microscope filters, as the emission spectrum of each fluorophore is unique. Although FISH methods are ideal for in situ analysis of fixed samples, they are not amenable to visualization of RNA in live cells. The need to fix the samples for analysis makes it impossible to study subcellular RNA dynamics in real time. In addition, the fixation and preparation of samples with strong chemicals like paraformaldehyde or hydrochloric acid can lead to biochemical alterations within the cells, disrupting cellular structures or denaturing proteins and organelles [RNA FISH can be multiplexed to identify different transcripts in the same sample. Kosman and colleagues  used thiganelles . To alle2.2.a) [figure 2b) [The basic principle of molecular beacons is the same as FISH: to employ fluorescently tagged oligos that bind complementary transcripts of interest in the cell. The main difference lies in the coupling of the fluorophore to a quencher . Quenchefigure 2a) . The tara) . When thgure 2b) . The absgure 2b) . Beaconsgure 2b) ,37.Figu2.3.While RNA visualization is commonly used in basic biomedical research, these techniques are not widely used in diagnostics, where RT-qPCR analysis is preferred for the detection of oncogenic or viral transcripts . HoweverInstead of using antisense oligomers directly conjugated to a fluorophore, RNAscope amplifies fluorescence by first assembling a scaffold on the RNA of interest, the foundation of which consists of Z-shaped probes . These pZ probes must bind directly adjacent to the target transcript to fully assemble the RNAscope scaffold, reducing much of the background fluorescence and improving the signal-to-noise ratio substantially. In addition, one set of Z probes can contain up to 400 dyes, and therefore designing multiple Z probes to tile the RNA of interest dramatically increases the ability to detect single RNA molecules. When detecting rare or isolated transcripts in large tissue sections, one set of RNAscope probes will provide a strong signal for detection; in contrast, standard RNA FISH would require tiling of oligomers, which entails designing dozens of oligomer sequences for a single target RNA. Depending on the length of the RNA of interest, this may not even be possible, making RNAscope a more suitable approach. The short length of the Z-probe targeting sequences (two 18–25 bp sequences) makes them suitable for targeting miRNAs, and perhaps circular RNAs at splice junctions as well. RNAscope can be multiplexed to up to four independent targets in a single sample , limited3.trans-acting factors, including RBPs, miRNAs and lncRNAs [The methods described so far rely on complementary base-pairing of oligomers to the target transcript. One drawback of these approaches is that if the target sequence is unavailable, either because it is embedded in a hairpin, bound to another nucleic acid, or associated with an RNA-binding protein (RBP), then hybridization cannot occur . In addi lncRNAs ,43.3.1.The MS2 tagging system is based on the coat protein of the MS2 bacteriophage, which contains an RNA-binding site with high binding affinity for RNA stem-loop structures found only in the bacteriophage RNA . BacteriDrosophila oocytes expressing ectopic nos-MS2 and MS2-GFP were used to demonstrate the timing of mRNA transport in developing oocytes and the role of the cytoskeleton in transcript trafficking [Pkp4 3′UTR constructs were used to demonstrate the preferential recruitment of RNAs found in adenomatous polyposis coli-containing ribonucleoprotein (APC-RNP) complexes to granules that contained mutant forms of the protein FUS and have been linked to amyotrophic lateral sclerosis [in vivo. mRNAs were tracked with MS2, and the nascent FLAG-tagged polypeptides were tagged with fluorescently labelled anti-FLAG Fab fragments. Importantly, neither the tags nor the fluorescent proteins disrupted normal protein or transcript distribution.The MS2 system has been employed to study RNA localization in a multitude of biological contexts. Sheth and Parker  used it fficking . MS2-tagclerosis ,51. Receclerosis  used MS2In each of these studies, RNA localization relative to subcellular structures was tracked by its association with fluorescently tagged proteins and/or immunostaining of protein markers. The MS2 system enables studies of association of an RNA of interest with an endogenous protein more easily than FISH, since fixation is often not required.3.2.MS2 tagging is best suited to track RNA in live cells, a virtually impossible task with FISH. An important advantage of the MS2 system is that, in theory, the MS2 RNA tag should not impact upon the natural function of the endogenous RNA. By contrast, when a transcript of interest is detected via associated (tagged) antisense oligomers, there is a chance that the oligomers may cover an RBP binding site, disrupting normal processes such as trafficking or loading into an RNP complex. Conversely, in live cells, antisense oligomers may directly interfere with mRNA translation or other RNA functions. In addition, in fixed cells, antisense oligomers recognizing the same site as an RBP may be masked when the RBP is bound, thereby preventing visualization. While careful oligomer design can avoid some of this interference, unknown interactions between oligomers and RBPs can create further artefacts. By contrast, since MS2 hairpins are generally added to the distal end of the 3′UTR, and the coat protein will bind these hairpin repeats exclusively, there is less concern of accidentally interrupting cellular processes during experimentation.Despite these advantages, there are limitations to this technique as well. While cloning extra hairpin repeats may enhance detection, replication and transcription of long sequences of palindromic DNA repeats can be unstable and lead to slips in the DNA polymerase, which may result in loss or extra insertions of these hairpins . In addi4.CRISPR/Cas-based technologies are well known for their immense potential in genome editing and genetic engineering . The tec4.1.Streptococcus pyogenes [The most widely studied Cas protein is Cas9 from pyogenes ,57. Recepyogenes ,59. Beinpyogenes ); since pyogenes  developepyogenes . When nopyogenes .Figure a). Exposing cells to this volume of foreign nucleic acids could be stressful [Limitations of this method include the necessity of developing PAMmers, which increases the complexity of implementation of this method, requiring the transfection of (i) a plasmid expressing dCas9 , (ii) the sgRNA, and (iii) the PAMmer oligomers a. Expositressful . More cotressful .4.2.Leptotrichia wadei (LwaCas13a) [Another Cas system, Cas13a, isolated from aCas13a) , has recaCas13a) . This teaCas13a) . HoweverThe use of dLwaCas13a technique has several shortcomings. The first is that several sgRNAs need to be tested to optimize sequence recognition. With the catalytically active LwaCas13a, the guide position had a dramatic impact on the level of knockdown, and shifting the guide position a few bases reduced gene knockdown . It is pS. aureus (SauCas9) can bind and cleave ssRNA, requiring only the sgRNA, not a PAMmer. While the study only demonstrates the cleavage capacity of SauCas9, an inactive variant with a fluorescent tag would likely be capable of binding and tracking RNA in live cells. In this case, the tagged variant would function in a similar manner to the dLwaCas13a system. Given how well characterized Cas9 systems are, this may potentially become a next robust method of Cas-derived RNA tracking.A recent study  demonstr5.We have described several popular RNA visualization methods that rely either on fluorescent RNAs  or fluorescent proteins recognizing unique RNA tags .5.1.As discussed above, FISH and molecular beacons employ fluorophore-labelled antisense oligos that bind target RNAs based on complementary sequences ,26. The cis  or trans (gRNA). When using bacteriophage RNAs , one plasmid expressing the fluorescently tagged MS2 or λN proteins and another plasmid expressing the RNA of interest tagged with MS2 or boxB hairpin repeats are introduced into cells. The fluorescent proteins then bind the RNA tags, permitting the localization of exogenous transcripts in live cells. Since this assay does not depend on complementary oligomers, there is less risk of RNA structure or binding proteins decreasing the sensitivity and lower concern with background. The sensitivity for a single transcript can be increased by cloning additional MS2/boxB tags into the chimeric RNA, creating more RBP recognition sites. Bacteriophage-derived RNA tracking methods require transfection of two plasmids (the phage-tagged RNA and the tag-binding protein), and transfection ratios must be optimized to achieve an acceptable signal : noise ratio. The plasmids are typically introduced through transient transfection without stable incorporation into the genome, so this method is mainly useful for cultured cells and single-cell organisms, such as yeast. Following transfection and assay execution, samples may be fixed for static imaging as well.RNA tagging using chimeric fluorescent RBPs relies on the addition of RNA sequences in bona fide transcript signals [Similarly, fluorescent, nuclease-deficient Cas proteins (e.g. dCas9-GFP) can be directed by sgRNAs to target RNAs in live cells in the presence of an additional oligomer, a PAMmer ,63. A va signals .5.2.RNA aptamers are remarkably specific RNA structures that are capable of binding myriad targets . AptamerThese aptamers are chosen by rounds of artificial selection for RNAs that bind dyes such as the synthetic GFP mimic DFHBI (Spinach) or thiazole orange (TO) derivatives. When these dyes are not bound to aptamers, excited energy is released through molecular movement, such as bond rotation . Howeverin vitro and pre-incubated with dye [in vivo with minimal background signal.Application of these aptamers is remarkably flexible. The aptamer sequence can be cloned into the 3′ region of an RNA of interest, similar to an MS2 hairpin, but the sequence is much shorter, generally in the range 20–80 nts . Once clwith dye . FollowiDespite the introduction of fluorescent Spinach RNA aptamers in 2011 , there h5.3.in situ sequencing (FISSEQ) of RNAs in fixed cells and tissues [in situ reverse transcription. During circularization, amines incorporated into the cDNA are used to cross-link and prevent diffusion. The cDNA is circularized and amplified by rolling-circle amplification; cross-linking of amines produces cDNA libraries in situ. The cDNA is then subjected to sequencing through SOLiD sequencing, a sequence-by-ligation approach . Recent work from the Church lab has introduced novel methods for fluorescent  tissues . Samplesiewed in ).Drosophila embryos, and organoids derived from induced pluripotent stem cells [This technique preserves the cytoskeleton and overall sample structure, and it can detect biologically active RNAs, so researchers can detect functional differences between cell types or regions of tissue. It can be used in cells, formalin-fixed tissues, em cells . The entem cells . At the em cells ; no tech5.4.Given their functional diversity, RNA studies have taken centre stage in cellular biology. mRNAs encode proteins needed for cellular processes, while non coding RNAs can perform structural and regulatory functions, such as chromatin organization, ribosome assembly, and transcriptional control, post-transcriptional RNA regulation and post-translational control of protein levels and function –78. The MALAT1) [One area that may benefit from RNA tracking is the field of extracellular vesicles  ,82. ThesMALAT1) ,87, but MALAT1) . Trackin5.5.MALAT1 and HOTAIR are metastasis markers in cancer such as lung, breast and nasopharyngeal carcinomas [RNAs of many types  are known to play key roles in cancers ,80, and rcinomas –91. Althrcinomas . It is drcinomas ,94. Ther5.6.Outside of biomedical applications, there are many mysteries surrounding RNA, particularly non coding RNAs. The project ENCODE (Encyclopedia of DNA Elements) aims to identify all functional regions in the human genome and characterize their purpose in the cell . This prDespite important advances in the field, however, there are still some restrictions to the application of RNA tracking methods. The systems described here generally require a known RNA sequence, either by creating complementary nucleic acids that seek out the transcript of interest, or by developing vectors that encode chimeric (tractable) versions of the RNA. Therefore, most of the current tracking technologies are not adequate for discovering new RNAs and are limited to studying the function of known RNAs.There is rising recognition that coding and noncoding RNAs play pivotal functions in all cellular processes, including chromatin organization, transcriptional control, regulation of post-transcriptional events , and post-translational processes like protein stability and multiprotein assembly. Along with these expanding functions, evidence is also accumulating that RNA dysregulation can lead to major disease categories including cancer, neurodegeneration, cardiovascular disease and metabolic syndrome. As our ability to track RNAs improves, we will gain insight into their mechanisms of action, expanding our functional understanding of RNAs and enabling the development of therapeutic venues.


Mitochondria harbor their own genetic system, yet critically depend on the import of a number of nuclear-encoded macromolecules to ensure their expression. In all eukaryotes, selected non-coding RNAs produced from the nuclear genome are partially redirected into the mitochondria, where they participate in gene expression. Therefore, the mitochondrial RNome represents an intricate mixture of the intrinsic transcriptome and the extrinsic RNA importome. In this review, we summarize and critically analyze data on the nuclear-encoded transcripts detected in human mitochondria and outline the proposed molecular mechanisms of their mitochondrial import. Special attention is given to the various experimental approaches used to study the mitochondrial RNome, including some recently developed genome-wide and in situ techniques. Trypanosoma brucei . PN. PN19].  and IMS ,102. In solution ,106,107.solution , and maysolution . Mutatiosolution ,112,113.Since PNPase is a nuclease, the mechanism by which it could be involved in RNA import remains puzzling, although bacterial PNPase homologues are known to eventually act as simple RNA-binding proteins without degrading their associated transcripts (reviewed in ). It wasBesides the RNA species described above, many other types of small non-coding RNA have been detected in human mitochondria, including snRNAs, snoRNAs, piRNAs, and tRNA fragments ,28. To vIdentification of RNA in isolated mitochondria has been the first approach used to discover imported RNAs . It reliIsolation of mitochondria requires an initial step of cell homogenization in conditions that maintain mitochondrial integrity, usually followed by differential centrifugations . Low-speIn either case, the isolated crude mitochondria remain contaminated with other cellular components such as the endoplasmic reticulum ; Golgi; This RNase treatment step is critical as the choice of RNase, and the conditions of digestion have a direct and decisive impact on the interpretation of the results of the analysis. For instance, different enzymes have varying specificities and unequal abilities to degrade RNAs, and some types of transcripts  are significantly more resistant to RNase-mediated degradation  or extenIn a hypothesis-driven analysis, when the identities of candidate RNAs are known, northern blotting and RT-qPCR can be performed to evaluate their presence in the isolated mitoplasts. This is normally accompanied by quantification of several negative controls (bona fide cytosolic contaminant RNAs) that should be absent or strongly depleted from the mitoplast preparation.However, to analyze the mitochondrial RNAs in an unbiased and high-throughput way and detect potentially new imported transcripts, next-generation sequencing should be opted for. The experimental design of RNA-seq and the post-analysis pipeline vary greatly in function of the model system and the biological question. The sequencing of RNA itself may exploit different methodologies, depending on the library generation protocol and the commercial instrument used . The divLarge-scale analyses of mammalian mitochondrial RNA samples by RNA-seq have been attempted by many laboratories ,28,31. HObviously, the problem of robust identification of the imported RNAs with RNA-seq cannot be solved in a naïve “simple detection” mode. Instead, better-controlled enrichment-based approaches should be adopted. For instance, comparison of transcript levels between mitoplast- and crude mitochondria-derived RNA samples offer the first simple means to prioritize potentially imported RNA candidates and dismiss some typical abundant contaminants . Since RA simple and popular way to verify the ability of RNA to penetrate into the mitochondria and at the same time study the biochemical requirements for this process is the in vitro import test  116,117117. For Strong evidence of the specific localization of an RNA in mitochondria can be obtained by its direct observation in cells by fluorescence microscopy. Various approaches have been developed to this end. The first group of methods is based on the introduction of exogenous RNAs tagged with fluorophores, fluorogenic aptamers, or protein-binding domains. This can be achieved either by direct transfection of cells with RNA or by its expression from a plasmid or a stably integrated recombinant gene ,133. An Contrary to some proteins, RNAs cannot produce intrinsic fluorescent signal. Thus, fluorescence tools that had already been successfully used to study cellular proteins, such as the green fluorescent protein (GFP), had to be adapted to image RNAs in cells. MS2 tagging was the first system to provide GFP-mediated imaging of RNA localization . The bacImprovement of the technique was achieved with the development of a series of RNA aptamers that can capture specific fluorophores and become fluorescent. Unbound fluorophores are poorly fluorescent and light up only when bound by an RNA aptamer ,138. SevMT-ND6 mRNA, which reconstitute a full-size fluorescent protein upon RNA binding [ND6 mRNA dynamics in living mammalian cells. However, using the Pumilio system requires extensive molecular design, and potential formation of protein dimers or oligomers may produce false-positive signal.An original protein-based probe for imaging of endogenous RNAs has been developed on the basis of Pumilio homology domain (PUM-HD) mutants that have the ability to recognize single-stranded RNAs in a programmable and modular manner . Ozawa e binding . This apMT-ND6 mRNA and 12S rRNA [In a simpler and more straightforward way, detection of specific nucleic acids in the cell can be performed by fluorescence in situ hybridization (FISH) . In its 12S rRNA . The sam12S rRNA . Applica12S rRNA .However, the canonical variant of FISH has limited sensitivity since a probe labeled with only one fluorophore provides a weak signal-over-background ratio. To bypass this limitation and additionally make the FISH technique truly quantitative, the so-called single-molecule FISH has been introduced . It is bE. coli [Despite significant advances in RNA labeling technology, studies of mitochondrial RNA import by fluorescence microscopy remain a challenging task because of the dual  localization of the imported molecules and generally low import efficiency. Furthermore, the very size of mitochondria borders the resolution limit of the conventional fluorescence microscopy . Thus, iE. coli . In contA conceptually different group of methods to assess the subcellular localisation of transcripts builds on in vivo chemical modification of RNAs confined in a membrane-bounded compartment, such as mitochondrion. This chemical “mark” permits one to specifically enrich and identify modified RNAs. In a recent embodiment of this principle a, it wasThe recent ClickIn approach exploits an in organello click reaction to verify the uptake of molecules, such as peptides and peptide nucleic acids, by isolated rat mitochondria . OrganelA methodologically similar group of approaches is based on compartment-specific crosslinking of RNAs to proteins for their subsequent isolation and identification. Two slightly different variations of this method have been published so-far: APEX-RIP  and ProxAlthough the history of research in mitochondrial RNA import counts now four decades, many nagging questions persist. How do the RNAs destined for mitochondria get selected from the cellular milieu and transported to the mitochondrial surface? Does this process differ between cell types and metabolic conditions? How do RNAs cross the inner mitochondrial membrane? Where are they located inside the organelles and, most importantly, what do they do there? With the recent development of microscopy and other in situ methods and interactomic and molecular biology approaches, it seems that the long-awaited methodology is coming of age to tackle these problems in a more conclusive way.Our knowledge of the spatial organization of the RNA metabolism in human cells has experienced a qualitative breakthrough over the last few years ,159. ChaThe question of which nuclear-encoded RNAs are imported in mammalian mitochondria is a matter of controversy, and some speculative arguments  have been forwarded . Taking


Self-rated health (SRH), an individual’s assessment of their own health status, is associated with older adults’ chronic and acute health conditions, as well as mortality. Assessments of SRH indicate individual’s global health is likely multifaceted. Level of education, particularly amount of post-secondary schooling, is associated with better SRH. Other indices of socioeconomic status (SES) such as income and wealth, have varying associations with SRH partly dependent on relative deprivation (e.g. Gini Index). The current study utilized data from 2,500 members of the Project Talent Twin and Sibling (PTTS) Study interviewed as adolescents in 1960 and followed up 54 years later. In 2014, participants were, on average, 70 years of age. Women comprised about 54% of the sample. We examined rearing family wealth, years of education, and functional independence as mediators of variance in SRH. Mean-level results indicated small positive associations between SES and SRH. Activities of Daily Living (ADL) accounted for about a quarter of variance in SRH, with higher functional independence predicting better SRH. Biometric analyses indicated that family wealth had small mediation effects on SRH via familial-environment (S) influences. Education mediated individual-specific (E) environmental influences. Functional independence (measured by ADL) mediated SRH via both additive genetic (A) and E influences. After adjusting for overall effects of sex, age, and specified mediators, a large portion of remaining variation in SRH was due to individual-specific (E) environmental influences. Current results suggest complex underlying genetic and environmental mechanisms contributing to an older adult’s assessment of their own health.