Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed ‘ClickSeq’ due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies’s (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these ‘intermediate’ species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs. Defective-Interfering RNA’ (DI-RNA). As a result, DI-RNAs may promote the establishment of chronic viral infections, may prolong the period during which the host is infectious, and may even be exploited as antiviral therapies or vaccines. Therefore, understanding the reasons and mechanisms of how DI-RNAs are formed and subsequently evolve is important. Here, we sought to characterize these processes by passaging Flock House virus (a highly tractable and well-characterized model RNA virus) in cell-culture for approximately one month and sequencing the viral genomes every three days using a combination of ‘ClickSeq’ (to resolve recombination events with nucleotide resolution) and the Oxford Nanopore Technologies MinION (to characterize full-length and defective genomes). This provides a highly detailed characterization of the pathways of DI-RNA emergence and their progression to dominance over the wild-type viral genome.Defective RNAs are versions of a viral genome that arise naturally during viral infections but have been truncated or rearranged by non-homologous recombination. While not encoding for functional viruses, they can be amplified and co-passaged with the wild-type virus, effectively parasitizing the normal viral machinery. Some defective RNAs can replicate so successfully so as to subdue the replication of the wild-type virus, forming a ‘ RNA viruses are extremely diverse and rapidly evolving. Their RNA-dependent RNA polymerases (RdRps) readily generate single-nucleotide variants whilst lacking proof-reading capabilities. RdRps aDI-RNAs can attenuate the viral infection via a variety of proposed mechanisms such as the saturation of the viral replicative machinery, sequestration of essential cellular cofactors, and/or induction of innate immune responses–14. DI-Rin vivo, DI-RNAs were considered to be a curious epiphenomenon of cell-culturing practices. As a result, our appreciation of the diversity of DI-RNAs and the range of situations in which they could play a role was greatly limited. Increasingly, due to the use and sensitivity of Next-Generation Sequencing (NGS) technologies, DI-RNAs have been observed in a multitude of viral systems under laboratory conditions  and in ronavirus, influenronavirus). DespitAlphanodavirus from the Nodaviridae family. FHV infects Drosophila flies and cells in culture as well as medically important genera of insects including mosquitos, (Anopheles gambiae), the tsetse fly (Glossina morsitans morsitans Westwood), and the Chagas vector [Drosophila melanogaster[Flock House virus (FHV) is a positive-sense single-stranded RNA (+ssRNA) virus originally isolated from grass grubs in New Zealand and is pxus Stal). Infectixus Stal). Moreovexus Stal). FHV hasxus Stal), 29–32 aanogaster. Many ofanogaster. DespiteIn order to resolve the potential mechanisms of DI-RNA emergence and elucidate the evolutionary pathways that lead to the formation of ‘mature’ DI-RNAs, we performed high-titer serial passaging of FHV in cell culture and characterized the encapsidated RNA using RNAseq. We used Illumina HiSeq sequencing of ClickSeq generated libraries to provide a high-resolution and high-confidence quantification of individual recombination events. We combined this information with long-read Oxford Nanopore Technologies’s (ONT) MinION sequencing to resolve the topology of full-length and defective RNA genomes. By combining these data, we aimed to determine the correlation of recombination events within single RNA virus genomes, characterize the distribution of defective RNA genomes, and determine the exact make-up of DI-RNAs during serial passaging of FHV in cell culture.We recently developed the ‘ClickSeq’ method for RNAseq that uses copper-catalyzed alkyne-azide cycloaddition (CuAAC), a click-chemistry reaction, for RNAseq library synthesis. ClickSeThe Oxford Nanopore Technologies’s (ONT) MinION is a small handheld sequencing device poised tThis study provides a comprehensive analysis of the steps and pathways governing DI-RNA emergence and evolution starting from a plasmid-driven inoculum through to a highly-passaged sample. By combining short-read and long-read sequencing technologies, we determine both the exact identity of RNA recombination sites and their correlation within the viral quasispecies. We find little evidence for the accumulation of intermediate defective RNA species that contain either only one, or smaller, deletions during the course of passaging. Rather, fully formed ‘mature’ DI-RNAs that are characterized by two to three deletions between a limited number of positions in each of the FHV genomic RNAs appear after approximately 9 days of viral passaging and accumulate rapidly. The accumulation of DI-RNAs corresponds with a reduction in the specific infectivity of the viral samples in each passage. This implies that partially formed DI-RNA species are not competitive and cannot accumulate in the manner that mature DI-RNA species do, perhaps due to the epistatic interaction of multiple recombination events. Alternatively, the formation of mature DI-RNAs may occur in a single step involving multiple simultaneous genome rearrangements.D. melanogaster (S2) cells were grown at 28°C in Schneider’s Drosophila Media supplemented with 10% fetal bovine serum and 1X Penicillin-Streptomycin using standard laboratory procedures. To generate the initial Flock House virus inoculum, S2 cells were plated at 50–70% confluency in a six well plate and were transfected with 2.5μg of pMT plasmid containing FHV RNA1 (NC_004146) and 2.5μg of pMT plasmid containing FHV RNA2 (NC_004144) using Lipofectamine 3000 Transfection Reagent as per the manufacturer’s protocol. Plasmid transcription was induced 24 hours post transfection with the addition of 50mM CuSO4. Virus was then allowed to propagate for 3 days post induction. For successive passages (Passages 1–9), S2 cells were grown in T-25 flasks to 70–90% confluency (~1 x 107 cells), then infected with 1mL of viral inoculum from the previous passage. Virus was grown for 3 days, then fractions were harvested for viral purification or inoculation of the next passage.To purify virus from each consecutive serial passage, cells and supernatant were subjected to a freeze-thaw cycle in the presence of 1% Triton X-100 to release viral particles from infected cells. Virus particles were then purified on a 30% sucrose cushion by spinning the cell lysate at 40,000 RPM for 2.5 hours. The viral pellet was resuspended in 10mM Tris (pH 7.4). Virus was further purified by applying resuspended virus atop a 10–40% sucrose gradient and spun at 40,000 RPM for 1.5 hours. The viral band was collected and subsequently treated with 1 Unit DNase and 1 Unit RNase and incubated at room temperature for at least one hour to remove any cellular nucleic acids not protected by the viral capsid. The virus sample was concentrated on a 100,000 NMWL centrifugal filter column and washed with at least 2 volumes of 10mM Tris pH 7.4. Finally, encapsidated viral RNA was extracted using a QIAGEN RNeasy Mini Kit as per the manufacturer’s protocol.S2 Fig were sequenced on a MiSeq platform with v3 chemistry for 600 cycles (2x300). Reads were trimmed to 150nts prior to analysis to emulate the libraries sequenced on the HiSeq.Next generation sequencing (NGS) libraries were generated using 100ng of RNA using the ‘ClickSeq’ protocol as previously described by Routh et al. , 44, 45.Cutadapt [FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). The remaining reads were aligned end-to-end with Bowtie (v1.0.1) [-v 3 –-best) first to the FHV genome (NC_004414 and NC_004146) and next to host D. melanogaster genome (fb5_22). The remaining unmapped reads were processed to identify recombination events using the python script ‘ViReMa’ [--N 1 --X 5 --Seed 25 --Host_Seed 30 --Defuzz 0 --MicroInDel 5). The frequency of a specific recombination event is approximated by dividing the number of reads mapping to this recombination (N) by N plus the average of the number of reads mapping to the wild-type genome at each of the recombination coordinates.Raw reads were processed by first removing the Illumina TruSeq adaptor using Cutadapt  with def(v1.0.1)  (commandn Mapper) (commandporetools[The Oxford Nanopore Technologies’s (ONT) MinION and flowcells were acquired as part of the ONT early-access program. To prepare sequencing libraries for the MinION, 50ng of RNA was reverse transcribed using RNA specific primers that were complimentary to the 3’ end of the respective genome (RNA1_RP: ACCTCTGCCCTTTCGGGCTA or RNA2_RP: ACCTTAGTCTGTTGACTTAA). cDNA was then amplified using the standard Phusion (NEB) PCR protocol using genome specific primers (RNA1_FP: GTTTTCGAAACAAATAAAAC or RNA2_FP: GTAAACAATTCCAAGTTCCA) for 19 cycles. Excess primers were removed from the PCR product using AMPure XP beads (Beckman Coulter) at a ratio of 1:1 AMPure bead:PCR product. Samples were then barcoded and prepared following the manufacture’s protocol (R9 Native Barcoding Kit I and Nanopore Sequencing Kit) with adjustments to tailor input cDNA quantities. A target of 1ug of fragmented DNA at approximately 8’000 nts is considered optimal for library generation using this kit. The input amounts for RNA1 (3107 bp) and RNA2 (1400 bp) were thus adjusted to 192ng and 88ng respectively and combined in 46uL water to maintain optimal DNA end molarity. After ligation of barcodes, equal amount of each DNA library  were pooled and loaded onto a MinION MkIB device equipped with an R9 flow cell. The MinKNOW control software was used to select a 48-hour sequencing protocol and was allowed to proceed for at least 36 hours, until high-quality data accumulation ceased. Raw data was uploaded automatically by Metrichor software for cloud-based base-calling using default settings and quality filtering for 2-dimensional reads. Reads were extracted from HDF5 format files (fast5) using BBMAP v36 suite . Alignment SAM files were visualized using the Tablet sequence viewer [samtools mpileup command and error rates at each position were calculated by dividing this value by the read depth at this position . Insertion and deletion events longer that 25 nts were extracted using the CIGAR string of the SAM files using simple in-house scripts. For recombination sites containing ‘fuzz’, where nucleotides surrounding the putative recombination events are the same for both the acceptor and donor sites, the recombination event was reported as occurring in the middle of the ‘fuzzy’ region, or at the 5’ side of the middle two nucleotides in the orientation of the reference if the fuzzy site contained an even number of residues. This is the same methodology as employed in the ViReMa script[Full-length ONT reads were mapped to the Flock House virus genome using the pacbio wrapper from the e viewer . SAM filMa script used to 1_317^1091_1242^2301_3107” indicates an authentic mapping from nt 1 to 317, then a deletion event removing nts 318 through 1090, then another authentic mapping from 1091 to 1242, followed by another deletion removing nts 1243 through 2300, and finally an authentic mapping from nt 2301 to 3107.To annotate the defective genomes detected by MinION nanopore sequencing or recombination events detected by ClickSeq, we use underscores ‘_’ to denote continued mapping, and carets ‘^’ to denote a recombination events. For example, “The Shannon Entropy Index is given by: 5 cells (S2) per well were plated in 96 well format. Virus samples were quantified by measuring the OD260nm. An OD of 4.15 corresponds to 1mg virus [13 virus particles assuming a virion mass of 9.4MDa [10 virus particles per 10μL. These quantities were chosen as particle-to-PFU ratios for rescued FHV has previously been reported to be 300–400 particles [10 particles per 105 cells corresponds to approximately 1000 PFUs per cell. Eight serial 10-fold dilutions were subsequently made and added to each column of the 96-well plate (8 replicate wells per dilution), as per standard TCID50 protocols. Virus was allowed to grow for 4 days after which the number of positive wells exhibiting cytopathic effect (CPE) were counted. The TCID50 values and effective MOI were calculated using the Reed and Muench Calculator [S7 Fig).Tissue culture infective dose 50 (TCID50) analyses of the supernatants from each passage were performed using standard protocols . For purmg virus , which if 9.4MDa . Purifiearticles . Therefolculator . We furtAll raw Illumina data and demultiplexed MinION nanopore data passing quality filters  associated with this manuscript are available on the SRA NCBI archive with study number SRP094723 and BioProject number PRJNA352872.D. melanogaster (S2) cells in culture were transfected with cDNA plasmids containing each of the Flock House virus genomic RNAs followed by a hepatitis D virus (HDV) ribozyme sequence. After induction, the HDV ribozyme regenerates the authentic 3’ end of the positive sense viral RNA, which is thus successfully recognized by the FHV RdRp allowing the initiation of viral replication[Fig 1. For each passage and replicate, including the original inoculum, viral particles were purified over a sucrose cushion and non-encapsidated genetic material was degraded to ensure that the genetic material subsequently analyzed was packaged within the viral capsid. RNA was extracted from the purified viral particles using standard silica-based spin columns.plication. We chooTable 1and . 1 million reads corresponds to an average coverage of greater than 33’000X across the FHV genome. Reads were aligned to the FHV genome and the host genome  using Bowtie end-to-end mapping [.ClickSeq libraries were syn mapping . As expe mapping , 55, 0.3ViReMa (“Virus Recombination Mapper”) [ViReMa is a computational pipeline optimized for mapping virus recombination junctions in NGS data with nucleotide resolution by dynamically generating moving read segments. ViReMa is sensitive to many types of RNA recombination events. This includes micro-insertions and deletions (InDels comprising 5 or fewer nucleotides), duplications, deletions, inter-RNA recombination (denoting recombination between FHV RNA1 and RNA2) and virus-to-host recombination events, and reports both the identity and frequency of recombination events. Recombination events that cannot be unambiguously identified due to unmapped read segments, mismatches occurring near to putative recombination events, or reads containing fragments of sequencing adaptors are flagged as ‘other’ [S1 Datafile). Broadly, we see an increase in the total number of recombination events during serial passaging of FHV and a corresponding increase in the Shannon diversity index .Subsequently, we further characterized the unmapped reads with the Python script Mapper”) . ViReMa ‘other’ . In eachTable 1and S1 Table) remained uncharacterized. As in previous studies, these were found to be derived from incorrect demultiplexing of neighboring samples on the HiSeq flow-cell[Following these mapping procedures, few reads  of RNA1 population and 0.044%  of the total RNA2 population when obtaining ~1M sequence reads.To demonstrate the reproducibility of the ClickSeq approach and to assess the limit in terms of our ability to successfully detect rare recombination events, we generated three replicate ClickSeq libraries from the RNA sample P7R2, obtained 1x150bp reads and performed the same computational analyses as described above. We mapped 0.83M, 1.45M and 1.18M reads per library, giving an approximate coverage of ~42,000–139,000x coverage over FHV RNA1 and ~18,000–67,000x over FHV RNA2 . When comparing the frequency of unique recombination events in either RNA1 or RNA2 between any pair of the three replicates, we find excellent correlation (Pearson >0.99) even for very infrequent recombination events, as illustrated in the scatter plots in Fig 2A). Inspection of these events reveals that they are dispersed throughout each of the genomic RNAs. RNA1 recombination events are the least frequent, with only 22 unique events detected represented by 2920 reads and without an apparent bias toward any specific location. The three most common RNA1 events in the inoculum are 2325^1241, 1484^1944, and 2199^393 with 514, 229, and 177 mapped reads respectively (S1 Datafile). Read depth for the wild-type genome at these loci ranges from 400K to 1.9M reads, therefore these recombination events make-up at less than 0.1% of the total viral population. These are not the events that have been previously reported as forming FHV DI-RNAs, moreover none of these events are observed again in subsequent passages, perhaps due to approaching the sensitivity of discovery limit as described above. However, due to the low-rate of artifactual recombination of the ClickSeq approach (>3 events per millions reads [In the inoculum (P0), less than 0.2% of the all the reads mapped to recombination events (ns reads ), we canns reads . TherefoS1 Datafile). Coverage in the wild-type RNA2 mapping in these regions ranges between 2.6M and 4.8M mapped reads, therefore these recombinant species make-up approximately 0.2% of the viral RNA2 population. The majority of other recombination events are found to delete a similar region of RNA2. This region is important as it has previously been reported to be deleted in FHV DI-RNAs. However, in the inoculum we do not observe deletions upstream in the RNA2 gene (for example 250^513), also reported to be deleted in previously characterized FHV DI-RNAs. Therefore, this dataset suggests that intermediate DI-RNAs with only a single region deleted between ~740–1220 are formed very early during virus passaging (within 3 days).For RNA2 in the inoculum, the three most frequently observed events were 738^1219, 738^1222 and 1024^1190, with 9849, 5237, and 3673 mapped reads respectively . Again, these occur throughout each of the genomic RNAs. However, we do begin to see events that have previously been characterized as forming DI-RNAs, such as 311^1104 and 301^1100, although these events are present at low levels  (S1 Datafile). In subsequent passages there was a rapid increase in the total proportion of mapped recombination events (peaking at 11.9% in P8R1) . In these later passages for each replicate, it can be seen that the most common recombination events are deletions that span two regions in each genomic RNA including: nts 300–940 and nts 1240–2300 of RNA1; and nts 250–510 and nts 740–1220 of RNA2, consistent with previous observations of FHV DI-RNAs[S1 Datafile). However, the exact sites of the recombination events, while repeatedly observed over time in each replicate, varied between replicates and each had distinct ‘most popular’ species in the final passages (Table 2). In some instances we are able to find that a specific event is predominant in one replicate while at low levels in another. Overall, these trends in the frequency of recombination events throughout passaging reveal that once defective RNA species emerge during viral passaging they rapidly accumulate.In passages P1-P2, less than 1.3% of the all the reads mapped to recombination events , unlike the DI-RNAs. Therefore we extracted all recombination events occurring with fewer than 10 mapped reads throughout all FHV passages and replicates  and cou pipeline). This r pipeline. HoweverFig 2D), although this was not the case for all replicates. Specifically, while initially showing an increase in ORF conservation, replicate 2 of RNA2 showed a decrease in the conservation of the ORF after passage 4, in contrast to the other two replicates, and in fact dips below 33%. Closer inspection of individual recombination events shows that this trend is driven by three of the four most common recombination events in replicate 2 passages 4 to 9: 249^517, 736^1219, 250^513 and 249^519 (the latter three events in bold do not maintain the ORF). These events are observed in the other replicates, but at a much reduced frequency  (S1 Datafile). This indicates that DI-RNAs can indeed accumulate without a strict requirement for a functional ORF. However, this analysis only takes a single recombination event into consideration and the Illumina reads are not long enough (150bp) to resolve multiple deletions at the same time. Nevertheless, neither compensatory recombination events including small InDels that might restore the ORF, nor single nucleotide variants at putative stop-codons, were found.Since many recombination events resulted in deletions of the viral genome, we were curious to see if the open reading frame (ORF) was conserved, as conservation of an ORF has frequently been observed to be a property of defective and defective-interfering RNAs. MoreoveViReMa, we were able to calculate the frequency with which each nucleotide was deleted, revealing areas of the viral genome that are conserved during serial passaging and required for DI-RNA replication. We plotted these data to generate recombination profiling maps for each RNA of FHV throughout passaging . In the first passage, there is a relatively even distribution of nucleotide deletions along the whole length of the genome with the exception of two frequently excised regions in the 3’ end in RNA1 due to two common recombination events in each of the replicates: 2545^2685 and 2277^2435. By passage 3 the deletions along the genomic landscape begin to be ‘sculpted’ whereby certain regions are deleted with a greater frequency than others. Passages 5, 7, and 9 were sculpted further revealing three major regions that were deleted in RNA1 and two in RNA2. Interestingly, for both RNA segments, while a range of deletions and rearrangements are generated during early passages, only the deletions that maintain regulatory and control elements are amplified during continued passaging, as previously observed in FHV [Fig 3), which correlates to the findings that these regions are important and required for RNA replication and encapsidation [Using d in FHV . These isidation , 60–64.Fig 4and S4 Fig). We reverse transcribed and amplified both RNA genes using primers specific to the 5’ and 3’ UTRs of RNA1 and RNA2 from all passages of replicate 2 to obtain cDNA that could be barcoded and analyzed using protocols for 2D sequencing on the ONT MinION. The ClickSeq data shows that these regions were highly conserved during passaging , therefore we were confident using template-specific primers to these regions would capture both the full-length wild-type virus genomes as well as any defective RNAs. After PCR amplification, we analyzed the MinION cDNA libraries using agarose gel electrophoresis to observe the distribution of cDNA fragments and ratios of full-length to defective RNA genomes . While the cDNAs from the early passages are predominantly of the expected size for full-length RNA genomes, later passages contain an array of band sizes. This shows that in the early passages the full-length genome is the predominant species while in later passages the truncated version becomes predominant. We also observe species appearing to be larger than RNA2. It is possible that some of these species correspond to RNA2 homodimers or other complex rearrangements that have previously been observed[S1 Datafile).The short-read ClickSeq data provide in-depth and high resolution details of individual recombination events. However, in order to determine the correlation of these events over time, we used long-read ONT nanopore sequencing, which can characterize full-length wild-type and defective genomes . This pipeline tolerates large insertions and deletions in the long-reads, thus allowing us to characterize the overall topology of the defective RNAs. We mapped between 94 and 97.5% of the MinION reads from each passage to the FHV reference genome (Table 3). An example of reads aligned to FHV RNA2 is shown in S4 Fig. The error rate of aligned reads, including single nucleotide mismatches and small InDels, was determined from alignment pileup files. Consistent with recent reports [S5A Fig. Due to the large number of small InDels generated during nanopore sequencing [S5B and S5C Fig). This shows that small InDels are frequent, but fall quickly in abundance with increasing length. 99.8% and 99.9% of all MinION deletions and insertions respectively were shorter than 25nts. Therefore, we considered only deletions and insertions of at least 25nts to be likely to be bona fide InDels present in the original viral RNA and corresponding to recombination events comprising defective RNA species rather than a sequencing error.The long-read nanopore sequencing data were aligned to the full-length FHV genome using the BBMAP suite . The full table of characterized defective RNAs and their frequencies in each passage is detailed in S2 Datafile. In total, we found 6030 and 3639 unique defective RNAs of RNA1 and RNA2 respectively throughout all passages.The nanopore data reveals the presence and frequency of large deletions and insertions within defective RNA genomes. From these, we can reconstruct the population of either full-length or defective RNA genomes present in each of the viral passages (annotated as described in the Table 3). The correlation in the earliest passages was poor, due to the low abundance of events in both datasets. However, later passages correlate well with Pearson coefficients reaching 0.85. This is important as it demonstrates that the frequency of recombination events was not biased during cDNA amplification of the full-length or defective viral genomes. Similarly, the Shannon Entropy indices increase during passaging , consistent with those from the ClickSeq data.The frequency of individual recombination events found in both the ClickSeq data and the MinION data were compared and correlation coefficients calculated  are easily identifiable as early as passage 2 (S2 Datafile) and match well with the expected identities based on our ClickSeq results and previous studies[The number of deletions in each passage in RNA1 and RNA2 are given in S6 Fig and S2 Datafile). Importantly, most of these single events do not delete the expected regions common to FHV DI-RNAs. Therefore, they may either correspond to sequencing artifacts, or transient defective RNA species generated due to stochastic RNA recombination, similar to the low-frequency events observed in the ClickSeq datasets. In later passages (beginning at passage 3), we do begin to see the presumptive intermediates (e.g. 1_317^1091_3107 or 1_1242^2301_3107) of mature DI-RNAs (e.g. 1_317^1091_1242^2301_3107). However, this is after the mature DI-RNAs were first observed, and after the point at which DI-RNAs have begun to accumulate. Indeed, in passages 2 and 3 respectively, mature DI-RNAs make up 1% and 30% of the viral population while the singly-deleted intermediates make up 0% (unobserved) and 3%. Together with the observation of rare DI-RNAs in the inoculum with the ClickSeq recombination analysis, these data indicate that single-deletion species do occur early during passaging, but remain poorly abundant and do not accumulate. In contrast, mature DI-RNAs are observed to rapidly accumulate between passages, indicating that they possess a replicative advantage above both wild-type viral genomes and intermediate defective RNA species.While we can readily identify mature DI-RNAs containing two or more deletions, few single-reads contain just one deletion (<6%) in all of the passages. Moreover, individual species are rarely observed again in subsequent passages  of the Proximal Sub-genomic RNA Control Element (PSCE) previously identified as being essential for FHV RNA replication and conserved in DI-RNAs species . The mosS1 Datafile). This matches precisely the 3’ junction site of the insertion event detected in the MinION data. However, the event 20^2300 corresponding to the 5’ junction site was not detected in our initial ViReMa analysis of the ClickSeq data as this would have required a search seed length of less than 20 nts. Repeating the ViReMa analysis using a seed length of 17 does indeed reveal the presence of the 20^2300 recombination event. This event is rarely observed in either of the other two replicate ClickSeq data . These data indicate that in a number of defective RNA genomes, the intRE element has been deleted and subsequently re-inserted at the 5’ end of the defective RNA genome. As the intRE element is required for regulation of RNA replication, presumably this maintains the ability for this highly-rearranged defective RNA to replicate.The ClickSeq data also shows a frequent recombination event, 2513^21, which appears first in passage 4 and is among the 10 most common events in the final 5 passages  and frequencies of these species vary substantially over time. We found that illustrating these data as a stacked area plot gave the most informative summary of the changes of the many different type of DI-RNA species over time. Due to the moderate error-rate of the nanopore read data, the exact identification of a recombination event and thus annotation of that genome may be incorrect. This would result in an over-estimation in the potential number of unique structural variants. Therefore we filtered datasets by requiring genomes to be represented by three or more reads. While removing a lot of noise, this has the drawback that we might be losing rare defective RNAs. Stacked area plots for genomes represented by three or more reads are shown in Fig 6A) shows that the composition of DI-RNAs in the viral population changes over time and new species appear at each passage. For example, the most abundant defective RNA1 species in passage 5 is ‘1_317^1091_1242^2301_3107’ but reduces in relative frequency in later passages. The most abundant species in the final passage 9 is ‘1_313^941_1241^2325_3107’, which appears at low levels as early as passage 2, but does not begin to accumulate until passage 6 (S2 Datafile). Why this DI-RNA only begins to accumulate at later passages despite being present in the early passages is not clear. The ‘complex DI-RNA’ that deletes the PSCE in RNA1 referred to in the previous section (‘1_342^1083_1245^2514_3107’) is also observed  first appearing at passage 5.The stacked area plot for RNA1 , the general composition of DI-RNA species is established at passages 4–5. Subsequently, the relative frequencies of the DI-RNA fluctuate but the overall diversity changes little with few new species appearing after passage 4. This is also observed when calculating the Shannon Diversity index  whereby entropy reaches a maximum at passage 5 and decreases thereafter. Interestingly, this range of fluctuations resemble the sinusoidal patterns of DI-RNA abundance that have been observed in other studies of RNA viruses where the ratio of the frequency of DI-RNAs to wild-type genome has been measured through longitudinal studies[As can be seen in the stacked area plot for RNA2 /ml) drops considerably during passaging by over four orders of magnitude. The corresponding effective MOI (PFUs per cell) also drops from 38.5 to 0.0003 during passaging .The reduction in full-length infectious viral genomes and the accumulation of defective RNAs during passaging is likely to correspond to a decrease in the specific infectivity of the virus samples. To determine the effect of defective RNAs characterized by combined ClickSeq and nanopore sequencing of replicate 2 upon specific infectivity, we performed 50% tissue culture infectious dose (TCID50) assays for each passage 1–9 for both the original inoculum used to infect each sample and for the particles purified from each passage . The TCImethods). This allowed us to determine and normalize the number of virus particles delivered per cell between each passage. As the particle-per-PFU ratio has previously been estimated at 300–400 particles-per-PFU [Fig 7A) with a trend very similar to that for the unpurified inocula. Together, these data indicate virus specific infectivity drops with a corresponding increase in the defective RNA population. There was an exception at passage 7 where TCID50 actually increased ~5 fold from the previous passage. This could be correlated to our observation of a decrease in the amount of defective RNA2 species in the MinION analysis .To determine whether the drop in effective MOI was driven by reduced total particle yield or from reduced specific infectivity (i.e. virus particles per PFU), we also performed TCID50 analysis of our purified and quantified virus stocks . We observed a reduced overall CPE in later passages at the highest virus dose as well as an increase in the number of viral particles require to induce the same amount of CPE  than for cells infected with the same inoculum (and therefore same ratio of full-length to defective RNAs) but at a lower dose . This observation indicates the protection of cells from infection and/or CPE when supplied with a large dose of viral particles that contain a large proportion of DI-RNAs.We further characterized each well of the TCID50 assay of our purified particles using flow-cytometry to give a quantitative assessment of cell survival and death in response to virus dose. We calculated the number of live cells that remained after infection at each dilution and for each passage (vice versa), which are always low. Furthermore, the majority of the detected inter-RNA recombination events correspond to genomic RNA hetero- and homo-dimers, which have previously been characterized as replication intermediates [In this manuscript, we sought to provide a thorough and comprehensive analysis of the frequency and identity of recombination events present during the serial passaging of Flock House virus in cell culture in order to elucidate the pathways and mechanism of DI-RNA emergence and evolution. We began with a homogenous inoculum derived from plasmid cDNAs of each of the FHV genomes. In the inoculum and in the early passages, we find a wide range of low-frequency recombination events corresponding to deletions and duplications that are dispersed through-out the viral genomic RNAs. We can be confident that these species do not constitute sequencing artifacts as we made our RNAseq libraries using ‘ClickSeq’ that hasmediates .Within only 2–3 passages, however, deletion events similar to those previously observed in DI-RNAs appear in all three passaging replicates. In subsequent passages, these recombination events begin to accumulate rapidly so as to predominate over full-length viral RNAs. This observation of the emergence of DI-RNA species, followed by their rapid accumulation is consistent with existing theories on the evolution of DI-RNAs that postulate that a wide range of potential DI-RNA species are generated by non-programmed RNA recombination and that only a handful are successfully replicated and thus accumulate. While tTable 2). Few of the RNA1 recombination events observed in the inoculum are observed again in subsequent passages. Additionally, even though we found the event ‘738^1219’ in RNA2 the inoculum, this was not the final predominant species in any replicate. Therefore, the evolution of DI-RNAs was not pre-determined by the presence of rare DI-RNA species in the common inoculum (a founder-effect), but rather by the selection of well-replicating DI-RNAs that arose later during serial passaging. Nonetheless, the final recombination events are highly similar between replicates and to previous reports from different laboratories. Therefore, this indicates that either the DI-RNAs have emerged due to a common mechanism of formation, the presence of a common selectivity filter, or both.The variation in recombination boundaries of the DI-RNAs suggests that a range of deletions can be tolerated. However, it is important to note that while each replicate is its own distinct lineage, each replicate passaging experiment was derived from the same initial inoculum. We chose to design the experiment this way to determine if the same DI-RNAs would be generated independently or if completely different deletions would arise and be selected for even though the environmental conditions are practically the same. Here, we observe the latter  may arise through any of these potential mechanisms. Alternatively, it is possible that multiple reassembly/deletion events occur in a single step, in a manner reminiscent of chromosome shattering (chromothripsis)[virothripsis’. Within the confined invaginations of the mitochondrial membranes that form the replication factories of RNA viruses such as FHV[An alternative reason for the rarity of precursor/intermediate defective RNAs is that the mature DI-RNAs are generated in one single event. We are yet to determine the molecular mechanism of recombination that leads to DI-RNA formation. Both template-switching, secondary-structure jumping, and non-replicative mechanisms have been proposed, and indeed these mechanisms need not be mutually exclusive. Our observation of nucleotide preferences at recombination junctions (thripsis); or ‘virch as FHV, the frainterfering RNA is a defective RNA that has the ability to compete with or otherwise attenuate the replication and proliferation of the wild-type helper virus. In our study we demonstrate that the viral swarm, even after only a few passages, is replete with many varieties of defective RNAs. With a single sequencing experiment, we would not be able to determine whether these defective RNAs are accumulating, diminishing or make-up a static component of the viral quasi-species. However, as we perform serial passaging with sequential sequencing experiments, we can determine which defective RNAs are accumulating (for example the ‘mature’ DI-RNAs) and which are not . It would be impractical to validate each of the many hundreds of detected defective RNA species with molecular virological experiments to determine whether they truly can attenuate or interfere with wild-type virus replication and to therefore categorize that species as a defective-interfering RNA. Indeed, we cannot exclude the possibility that multiple DI-RNAs act co-operatively within the viral quasi-species and are mutually dependent upon one another. However, the demonstration here of an accumulation during serial passaging is strong evidence that these species are interfering, as their accumulation essentially dilutes the pool of wild-type functional virus. With this in mind, we believe it would be suitable to describe the mature defective RNAs as defective-interfering RNAs (DI-RNAs), and the ‘immature’ only as defective RNAs.A defective-in vivo viral passaging scenarios. However, in this experiment we were actually passaging a large number of virus particles between cells, but with a low specific infectivity. It is also interesting to observe that for the final passages there appears to be a protective property of the DI-RNAs as determined by flow-cytometry, but only when administered at the highest doses corresponding to over ~30,000 particles per cell. However, the role that DI-RNAs might play in vivo is not clear, as these very high doses may not be physiologically relevant. DI-RNAs for FHV similar to the ones described here have been observed to arise in experimental fruit fly infections [in vivo. However, whether RNA viruses such as FHV have evolved to favor the spontaneous formation of DI-RNAs and if so whether these DI-RNAs play an important role in modifying the life-cycle of the virus, is yet to be determined.It is remarkable that FHV is able to maintain a viable infection despite being burdened with such a gross excess of DI-RNAs in the final passages presented here. By performing TCID50 assays of the original inocula used between each of the passages and of the particles purified from each passage, we show that there is a dramatic reduction in specific infectivity during passaging corresponding with the increase in the DI-RNA content. DI-RNAs have generally been demonstrated to arise at high MOIs, as was the scenario with our first passages. However, our calculated MOI drops rapidly after DI-RNAs have formed to levels that might be expected during typical fections  and so tS1 FigPercent mapping is calculated by the frequency of reads that mapped to the host genome compared to all processed reads.(PDF)Click here for additional data file.S2 FigThree replicate ClickSeq libraries were generated from the same RNA sample to validate the reproducibility of ClickSeq and to determine the cut-off for sensitivity of discovery. Each point represents an individual recombination event and the x- and y- axes is the number of reads mapping to that specific event for each data set. The size of the point indicates the number of different events that share the same coordinates, as indicated by the key. These data illustrate the reproducibility with which recombination events are found when multiple libraries are generated side-by-side. Pearson correlation coefficients exceed 0.99 when comparing RNA1 or RNA2 recombination between each pair of replicates.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 FigA composite snapshot of the TABLET sequence viewer alignment of RNA2 from the reads generated by the MinION (P4R2).(PDF)Click here for additional data file.S5 Fig(A) A histogram of the frequency of error rates over every mapped position across all Nanopore datasets is shown. Errors include substitutions, insertions and deletions shorter than 25 nts. The proportion of correct base matches to mismatches is shown on the x-axis. The y-axis indicates the number of nucleotide coordinates with the corresponding error rate. This reveals a mode and mean error rate of 5.0% and 6.3% respectively. Frequency of (B) deletions and (C) insertions within the MinION dataset.(PDF)Click here for additional data file.S6 FigStacked-area plot of showing the pathways of FHV DI-RNA evolution for (A) RNA1 and (B) RNA2. Similar to Fig 6, except all species are included, including genomes represented by only one MinION nanopore read. The passage number is indicated on the x-axis and the stacked frequencies of each detected defective RNA is shown in the y-axis. Each non-contiguous color represents a specific genome characterized by MinION nanopore sequencing. Wild-type genomes are colored green, genomes with one deletion are colored in shades of blue, and genomes with two or more deletions are colored in shades of oranges .(PDF)Click here for additional data file.S7 Fig(A) Uninfected S2 cells. (B) S2 cells four days post-infection with FHV exhibiting cytopathic effect.Screenshots of the InCyte software (of the Guava easyCyte HT flow cytometer) indicating gating used to count live cells. (PDF)Click here for additional data file.S1 TableTable 1.Each passage in each replicate as well as the inoculum is shown, similar to (PDF)Click here for additional data file.S1 DatafileDonorCoord_to_AcceptorCoord_#_Counts”. This is the raw data used to populate Table 1and S1 Table.Each passage in each replicate as well as the inoculum is shown. Output format, as described in Routh et al. , is give(ZIP)Click here for additional data file.S2 DatafileFig 6and S6 Fig.Table reports the annotated genomes (wild-type or defective) and the number of mapped reads in each passage. This the raw data used to populate the stacked-area plots in (XLSX)Click here for additional data file.


The World Health Organization estimates that the yearly number of dengue cases averages 390 million. This mosquito-borne virus disease is endemic in over 100 countries and will probably continue spreading, given the observed trend in global warming. So far, there is no antiviral drug available against dengue, but a vaccine has been recently marketed. Dengue virus also serves as a prototype for the study of other pathogenic flaviviruses that are emerging, like West Nile virus and Zika virus. Upon viral entry into the host cell and fusion of the viral lipid membrane with the endosomal membrane, the viral RNA is released and expressed as a polyprotein, that is then matured into three structural and seven non-structural (NS) proteins. The envelope, membrane and capsid proteins form the viral particle while NS1-NS2A-NS2B-NS3-NS4A-NS4B and NS5 assemble inside a cellular replication complex, which is embedded in endoplasmic reticulum (ER)-derived vesicles. In addition to their roles in RNA replication within the infected cell, NS proteins help the virus escape the host innate immunity and reshape the host-cell inner structure. This review focuses on recent progress in characterizing the structure and functions of NS5, a protein responsible for the replication and capping of viral RNA that represents a promising drug target. Aedes genus . These mosquito vectors are commonly found in inter-tropical regions but now also in more temperate areas to due global warming, thus raising the possibility of further expansions of the areas affected by dengue. Most infections are asymptomatic or cause mild symptoms  lasting for a few days. However, in some severe cases, consequences can be more dramatic  causing about 20,000 deaths per year. The dengue virus belongs to the flavivirus family along with West Nile virus (WNV), yellow fever virus and the recently emerging Zika virus (ZIKV). Dengue virus comprises four serotypes labelled DENV 1 to 4. The viral particle encapsulates a positive strand RNA of 11 kb that constitutes the viral genome, with 5’ and 3’ untranslated regions (UTR) and a 5’ cap. Upon infection, the RNA is translated into a single polypeptide chain embedded in endoplasmic reticulum (ER) membranes that is processed in ten proteins following proteolytic maturation by viral and host cell proteases. The envelope, the precursor-membrane and the capsid form the structural proteins encasing the viral RNA. NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 are non-structural (NS) proteins expressed in the host cell that are essentially not incorporated in the viral particle. With the help of host proteins, NS proteins reshape the inner organization of the cell, mature the polyprotein, replicate the viral RNA and help the virus evade the immune system. Infection by dengue virus is followed by noticeable changes in the inner cell substructures, including formation of ER vesicles, double membrane particles formation and elongation of mitochondria [Dengue virus is a mosquito-borne human pathogen affecting mostly inter-tropical regions, where 3.9 billion persons live, with nearly 100 million infections provoking clinical symptoms every year [chondria . In the chondria . NS4A acchondria ,9,10,11.chondria  whilst tchondria ,14 alongchondria . Here, we summarize recently published research on the NS5 protein, an important enzyme drug target. NS5 is the largest (102 kDa) and the most conserved protein  sensor proteins [The MTase is responsible for capping the nascent genomic RNA sequentially using S-adenosylmethionine as the methyl donor, via sequential methylation on the N7 atom of the cap guanine and the 2’O atom of the ribose of the first strictly conserved adenine of the genome ,24. Viraproteins O-ribose methylation [Crystal structures of the MTase domain (residues 1–263) showed that it shares the characteristic α/β fold  as seen hylation . The thihylation . A magneGuanylyltransferase activity was proposed for NS5 based on in vitro experiments, using a triphosphorylated RNA strand in the presence of the NS5 and NS3 proteins and GTP . The putO-ribose methylation of genomic viral RNA [O-ribose of the adenine base [O-methylation is proposed to occur after the N7-methylation [O-ribose methylation) and the SAM pocket, it is likely that the three reactions involve dissociation followed by repositioning of the RNA for the following enzymatic step , the partially capped RNA (at the N7 position) and S-adenosyl-L-homocysteine (the by-product of the reaction) allowed the authors to propose a detailed mechanism for 2’-iral RNA : The catiral RNA ,24,34. Tiral RNA . Of the ine base ), 2’-O-mhylation ,35. Onlyhylation , capturitic step .Recently, Gokhale et al. reported that the flavivirus genomic RNA is also internally methylated on adenosines . SurprisUpon infection, the positive sense viral RNA is released in the cytoplasm. NS5 protein first transcribes it as a negative sense strand before using the negative strand (in the context of a dsRNA intermediate) to synthesize a large excess of + RNA. The viral mRNA is then used to express the polyprotein by host cell ribosomes and also for encapsulation into new viral particles.Flavivirus genus  consistently shows a closed conformation of the RdRP domain. However, a major difference resides in the various relative orientations between the RdRp and the MTase domains, leading essentially to two sets of interfaces, with the NS5FL from DENV being in one group with the NS5FL from JEV and ZIKV belonging to another related evolutionary group.The first structure of the DENV RdRp domain (residues 270–900) published 10 years ago revealed a right-hand architecture found in many DNA and RNA polymerases, with a palm, a thumb and a fingers subdomain. Conserved motifs A–G each with a precise function are present in the flavivirus RdRp : motif AO-ribose on the α-phosphate of the incorporated nucleotide. The first published structure of the RdRp domain in complex with a chain terminator nucleoside analogue 3’dGTP mimicking the positioning of the first base of the newly synthetized RNA showed the importance of residues R729 and R737 in coordinating the phosphate groups. Moreover, a role for the indole group of W795 from the priming loop for stacking the base of the nucleotide was proposed, to position the first bases for efficient polymerization [Flavivirus RdRps use RNA as a template and do not require a primer to elongate nascent RNA . The RNA synthesis mechanism was proposed based on structures of RdRp from various viruses ,51,52,53rization . This warization  and laterization .Once the first ribose-phosphate bond is formed with the production of pyrophosphate as a by-product, the dsRNA is then translocated into the exit tunnel to allow the positioning of the next nucleotide. Thus, the mechanism of RdRp can be broken down into four steps: (1) the protein is in a resting mode in a preinitiation form, most probably in the closed conformation seen in the crystal structures with the priming loop in an extended conformation; (2) in the initiation state, the 3’ end of the ssRNA template is bound along with the A and G ribonucleotides until the initial phosphodiester bond is formed. Here the priming loop acts as a stabilizing platform maintaining the adenosine in position to allow synthesis of the initial dinucleotide primer; and (3) the NS5 protein undergoes a large conformational change with a concomitant retraction of the priming loop leading to an opening of the dsRNA exit tunnel. This conformational change is thought to be the rate-limiting step for the RNA polymerase activity . Finally2+ or Mn2+, whilst ZIKV NS5 can only use Mn2+ and is inhibited by Mg2+. However, the in vivo metal dependency of the enzymatic activity is not clear yet. Until recently, NS5 was thought to solely use RNA as a template. In the case of the Zika virus, a recent study suggested that ab-initio polymerization is also possible using ssDNA as template. Moreover, the elongation reaction can occur with a heteroduplex formed by a DNA template and an RNA primer . InteresInterestingly, the effect of the presence of the MTase domain stimulates the RdRp activity; moreover, in the presence of residues from the linker region, both the activity and stability of the RdRp domain is significantly enhanced ,58,59. Tcis in a sequential manner but it is easier to conceive that several molecules cooperate in trans, each one adding a modification to the RNA substrate. Comparison of the overall structures of DENV, ZIKV and JEV NS5 proteins shows that although the folding of the distinct domains is conserved (root mean square deviation (rmsd) ~1Å for the MTase and the RdRp domains respectively, see PDB access codes 4V0R, 5TFR and 4K6M), their relative orientation changes by a rotation of ≈100°. The linker domain has been proposed to allow the MTase and the RdRp domains to adopt different relative conformations upon binding to RNA, NS3 or host partners. The linker region of NS5 (residues 266–271) connects the MTase and the RdRp domains. In the crystal structures of the full length DENV3 NS5; the linker comprises a short 310 helix of four residues [Detailed information on the cooperation between NS5 and NS3 proteins and how the helicase and RdRp activities and the triphosphatase and capping activities are subjected to a cross-talk between the various domains is still lacking. Thus, it is not clear how a newly synthetized RNA strand is separated from the template, undergoes removal of the 5’ pyrophosphate, and becomes guanylylated before methylation. The various enzyme activities are handled by different proteins and the cognate domains would need drastic conformation changes that are not easily visualized based on the available crystal structures of the NS3 and NS5 protein, and on what we know from their interaction. We cannot exclude the possibility that synthesis and capping are performed in residues . This reresidues . The linresidues . The linresidues . NS5 protein has been considered to be active as a monomer. A recent study reported a crystal structure of the full length NS5 also from DENV , where tIn addition to its central role in genome replication, NS5 proteins from flaviviruses play a crucial part in evading the host immune system. Amino acids between residues 202 and 306 from the N-terminal region of the protein from the dengue 2 serotype have been shown to interact directly with STAT2  21,68,6,70 and pSurprisingly, in infected cells, NS5 protein is mainly localized in the nucleus rather than in ER vesicles, especially in the DENV 2, 3, 4 serotypes ,74. ThisA comprehensive review of the antiviral efforts directed against DENV was recently published . InhibitThe second class of compounds are the non-nucleoside inhibitors NNIs, . They arMethylation of the RNA cap is essential for virus replication and represents an important drug target ,27,28,99Recently, an alternative approach to design NNIs targeting disordered regions of NS5 has been proposed . The ide


The discovery of RNA interference (RNAi) has been a major scientificbreakthrough. This RNA-guided RNA interference system plays a crucial role in awide range of regulatory and defense mechanisms in eukaryotes. The key enzyme ofthe RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNAguide molecule to specifically target a complementary RNA transcript. Twofunctional classes of eukaryotic Ago have been described: catalytically activeAgo that cleaves RNA targets complementary to its guide, and inactive Ago thatuses its guide to bind target RNA to down-regulate translation efficiency. Arecent comparative genomics study has revealed that Argonaute-like proteins arealso encoded by prokaryotic genomes. Interestingly, there is a lot of variationamong these prokaryotic Argonaute (pAgo) proteins with respect to domainarchitecture: some resemble the eukaryotic Ago (long pAgo) containing a completeor disrupted catalytic site, while others are truncated versions (short pAgo)that generally contain an incomplete catalytic site. Prokaryotic Agos with anincomplete catalytic site often co-occur with (predicted) nucleases. Based onthis diversity, and on the fact that homologs of other RNAi-related proteincomponents (such as Dicer nucleases) have never been identified in prokaryotes,it has been predicted that variations on the eukaryotic RNAi theme may occur inprokaryotes. TtAgo fromThermus thermophilus and RsAgo fromRhodobacter sphaeroides. Like eukaryotic Argonautes, bothTtAgo and RsAgo are long pAgos that areco-purified with oligonucleotide guides , themajority of which are complementary to plasmids. Together with the observation thatTtAgo  target DNA,this has led to the conclusion that both pAgos play a role in host defense .However, apart from these similarities there are important functional differencesbetween the two bacterial Argonaute proteins. The guides acquired byRsAgo are mRNA-derived RNA oligonucleotides that target the templatestrand of plasmid genes. As RsAgo lacks a catalytic site, it mostlikely requires a partner nuclease for target cleavage. In contrast,TtAgo acquires DNA guides that allow targeting of AT-rich sequences ofdouble-stranded plasmid DNA. Whereas the functional nuclease site ofTtAgo catalyzes nicking of a single targeted DNA strand, twoTtAgos loaded with overlapping complementary guides can generatedouble-stranded DNA breaks. Recent studies by us and the working group of Alexei Aravin have described molecularanalyses of two distinct bacterial Argonautes, These studies have revealed interesting variations on the eukaryotic RNAi theme. Severalbasic features of these two variant pAgos remain elusive, concerning mechanistic detailsof both guide acquisition and target interference. Moreover, apart from the twocharacterized prokaryotic Argonautes, many more pAgo variants exist, that may differ infunctionality with respect to (i) guide preference (RNA/DNA), (ii) target specificity(RNA/DNA), and (iii) catalytic mechanism (nuclease activity). Apart from providinginsights in the evolution of the prokaryotic Ago variants and their eukaryoticcounterparts, future research will aim at revealing the molecular basis for the distinctfunctionality of these different pAgo variants. Moreover, gained insights will result inan interesting set of novel nucleases that may allow for dedicated geneticengineering.


The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3′ UTR and a 7-methylguanosine cap on the 5′ end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5′ mRNA cap and the 4ESE element in the 3′ UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E–4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. Here, edoes not , 2006. Fdoes not , 2006. Rdoes not , 2006.Despite the clear biological relevance of eIF4E activity, there is no biochemical framework to provide an understanding of the mechanics of this mRNA export pathway. Some important features have been characterized  and thesTo define biochemical factors in this export pathway, we carried out proteomic studies and found that the leucine-rich pentatricopeptide repeat protein (LRPPRC) immunoprecipitates with both eIF4E and 4ESE RNAs in the nucleus . ImportaIn this study, biochemical, biophysical, and NMR methods have been used to characterize the eIF4E export RNP and to determine essential components for RNA recognition and for association with the nuclear pore. We show that LRPPRC is a central component of this RNA export pathway. Specifically, LRPPRC directly binds eIF4E, the 4ESE RNA element, and CRM1. Thus, LRPPRC is the first factor shown to act in 4ESE RNA selection. Further, we reconstituted the eIF4E–LRPPRC–4ESE RNA–CRM1 complex in vitro suggesting that this could be the basic export mRNP in the nucleus. Finally, we show that RNA-free LRPPRC, like cap-free eIF4E, is reimported into the nucleus via Importin 8, suggesting coordinated recycling of these export components. Our studies provide a molecular basis for essential steps in the eIF4E-dependent mRNA export pathway as well as a molecular understanding of how cap- and RNA-binding status underlie critical control points in this process.We examined whether LRPPRC plays a direct role in eIF4E-dependent mRNA export by testing if it directly interacts with eIF4E in pull-down assays. Recombinant GST-tagged eIF4E and the mature form of LRPPRC  were expressed and purified from bacterial cells. LRPΔMTS directly bound eIF4E in this assay B. Furthe15N-labeled eIF4E upon addition of the unlabeled LRP10A  reported previously . The 4ESll-downs B. Then, ll-downs C. Thus, FL) and truncated (4ESETR) forms where the unfolded 5′ and 3′ ends were deleted (TR and c4ESETR were at least as active in eIF4E-dependent mRNA export as full-length constructs using standard export assays and qPCR (mut)  D based oCR (mut) . Our CD CR (mut) G. Strikinstructs E. Finallnstructs D. Intereucts  had no RNA binding activity on its own C. In con and SL2 C,E. ThesFL and m7GDP cap analog, respectively, both complexes were combined and subjected to gel filtration. We observed a peak corresponding to a size of ∼200 kDa, using globular proteins to calibrate the column. This value is only slightly larger than the expected 158 kDa molecular weight for an equimolar complex of LRPPRC–eIF4E–4ESE RNA. Importantly, the elongated shape that these PPR-containing proteins like LRPPRC typically adopt, likely affect migration on the column. To determine that all the components were indeed present, column fractions were subjected to gel electrophoresis and visualized by Coomassie blue staining to detect LRPΔMTS and eIF4E, and on denaturing polyacrylamide stained with silver stain for the c4ESEFL RNA. All three factors were found in the ∼200 kDa fraction consistent with the formation of the equimolar trimeric complex in solution.Finally, we examined whether LRPΔMTS could simultaneously bind eIF4E and 4ESE RNA using gel filtration chromatography A. After Our above studies indicated that LRPPRC binds both eIF4E and the cargo 4ESE RNA. In order to be exported through the nuclear pore, RNA and protein complexes must interact with an export receptor. Given our previous findings that eIF4E-dependent mRNA export is abrogated by inhibition of CRM1 with Leptomycin B , we examAll our previous work suggested a model whereby eIF4E associates with the cap of 4ESE containing RNAs in the nucleus. This is based on previous cap competition studies and experiments with eIF4E mutated in the cap-binding site (eIF4E W56A), which indicated that transcripts required capping for eIF4E interactions , 2006. H7G cap analog (m7GDP) to the preformed 15N-labeled eIF4E-unlabeled 4ESE RNA complex. Cap addition led to dissociation of the eIF4E–4ESE RNA complex as evidenced by the reappearance of resonances but now in cap-bound positions  our NMR results and (ii) the observation that this region has a net positive charge B. First,ositions C,D. Thus studies E using w studies . Specifi studies F, as it  studies D. ConsisThe final step of every export cycle is the reentry of the machinery into the nucleus to support future export rounds. Our previous studies indicated that Importin 8 imports eIF4E . Thus, wTo determine whether these interactions were functional, we carried out in vitro nuclear import assays. Import of LRPCt was monitored in permeabilized U2Os cells using immunofluorescence in conjunction with confocal microscopy. We used GST antibodies to specifically detect exogenous LRPCt. Import assays were performed using a buffer with a mixture of cofactors required for karyopherin-mediated import. In the presence of this buffer, LRPCt remained in the cytoplasm, whereas Importin 8 addition in the presence of Ran led to its nuclear entry D, consisBased on these studies, we propose the first biochemical model for eIF4E-dependent mRNA export. We identify LRPPRC as the RNA specificity component of the complex, directly binding 4ESE RNAs and also as the factor that interacts with the export receptor CRM1. Further, our studies show that LRPPRC binds eIF4E and 4ESE RNA simultaneously through domains in the N and C termini, respectively. LRPPRC also directly binds CRM1, allowing the eIF4E-LRPPRC-4ESE RNA complex to engage the export machinery and transit through the nuclear pore to the cytoplasm .Once RNA cargoes are released, the export machinery must be reimported into the nucleus. In our model, after the dissociation of 4ESE RNA cargoes, eIF4E and LRPPRC return to the nucleus via Importin 8  and RNA-An important consideration is how these findings translate from in vitro biochemistry to the cell. Our previous studies in cells support the proposed model in Our model provides many testable features that will be the subject of future investigations. For instance, it would be interesting to isolate eIF4E–LRPPRC–4ESE–CRM1 complexes from cells. In this way, we could determine whether our biochemical models fully recapitulate the in vivo situation. We previously showed that eIF4E's ability to bind 4ESE-containing RNAs is linked to its cap-binding activity , 2006. TE. coli. When bacterial cultures reached OD600 nm of 0.8, expression of recombinant LRPPRC fragments were induced with 0.5 mM isopropyl-β-D-thiogalatopyranoside (IPTG) and allowed to grow at 20°C overnight. The cells were harvested and resuspended in TB-LRP buffer  supplemented with protease inhibitors (Roche), DNase, and RNase. The cells were lysed by sonication and supernatant of the lysate added to Glutathione Sepharose 4B  for affinity purification. After extensive washing with buffer TB-LRP, followed by WB-LRP buffer  to reduce the salt concentration, the GST-tagged LRPPRC fragments were eluted with WB-LRP buffer containing 50 mM reduced glutathione, and loaded onto a Mono Q HP  column, followed by gel filtration chromatography  in 50 mM sodium phosphate pH 7.5, 100 mM NaCl, 10% glycerol, and 1 mM DTT.Human LRPPRC  was expressed and purified as described by 15N-labeled human eIF4E (pET-15b) used in the NMR experiments; the mouse GST-tagged eIF4E (pGEX-20T), human GST-CRM1 and GST-Importin 8 (modified pGEX-4T-3 with TEV site), and human Ran (pET-15b) used in the import assays and the different pull-downs. The eIF4E mutants were generated by performing site-directed mutagenesis and verified by DNA sequencing.Overexpression and purification of the following proteins were as described in 7GDP was obtained from Sigma. The synthetic peptide LRP10A (N-Ac-EGFPIRPHYFWPLLVGRRKEK-NH2), in >95% purity was purchased from Biomatic Corporation and stored as a lyophilized powder. The RNA was purchased from Dharmacon, Inc. Prior to use, each nucleotide was heated at 85°C for 5 min, followed by a ramp to 60°C at 0.1°C/sec, 37°C for 5 min, then 25°C for 20 min.Antibodies used were rabbit polyclonal anti-LRP130 , rabbit polyclonal anti-Importin 8 (LifeSpan BioSciences), mouse monoclonal anti-eIF4E (BD Transduction Laboratories), goat polyclonal anti-GST , and anti-goat Cy3 (Jackson ImmunoResearch). The cap analog m1H–15N HSQCs were recorded at 600 MHz on a Bruker Avance III HD spectrometer equipped with a cryoprobe. Solution conditions were ∼40 µM uniformly 15N-labeled eIF4E at 20°C in 50 mM sodium phosphate pH 7.3, 100 mM NaCl, 1 mM DTT, 7% D2O. Data were processed using NMRPipe (obs = [(ΔδHN2 + ΔδN2/25)/2]1/2 /2]1/2 .GST and GST-tagged (wild-type and mutant) proteins (75 pmol) were mixed by rotation with its prey (15 pmol) for 1 h at 4°C in 500 μL of binding buffer , supplemented with either 0.001% or 0.005% NP40 for the CRM1/LRPPRC or Importin 8/LRPPRC pull-downs, respectively. Then, 20 µL of pre-equilibrated Glutathione Sepharose 4B beads  were added and mixed for another 2 h. Unbound protein was removed by washing four times with their corresponding binding buffer and bound proteins were eluted with hot Laemmli sample buffer. The eluted protein complexes were then separated by SDS–PAGE and revealed using specific antibodies and Western blot analysis. In additional competition experiments, Ran-GTP (at fivefold molar excess) was added individually to the binding buffer along with Importin 8 or CRM1 during the incubation step. All experiments were carried out at least three independent times.2, 1 mM DTT, 0.05% NP40) for 1 h at 4°C. After the beads were pelleted by centrifugation , the supernatant was transferred to another Eppendorf tube and 0.1 nmol of biotinylated RNA was added and mixed for another hour. Then, 30 µL of Streptavidin-agarose beads was added, and after incubation for an additional 2 h, beads were washed five times with buffer RNA-X. Pulled-down materials were eluted by boiling with 30 µL Laemmli buffer. Protein content was monitored by Western blot analysis, and RNA loading was visualized on a 20% acrylamide–8 M urea denaturing gel stained with silver nitrate. All experiments were carried out at least three independent times.To remove nonspecific binding, the beads were precleared as follows: 0.01 nmol of protein was mixed by rotation with 20 µL of Streptavidin-agarose beads equilibrated in buffer RNA-X  Superdex 200 10/300 GL column (Amersham Biosciences). The separation was performed at a flow rate of 0.25 mL/min. The amounts of LRPPRC and eIF4E were 0.4 and 1.2 nmol, respectively. The c4ESE2, to achieve 20 µM sample concentration. Measurements were taken with a 1-nm data interval, and spectra, established as an average of three CD scans, were subtracted with the spectrum of the buffer. All experiments were carried out at least three independent times and representative data are shown.CD spectra were recorded on a Jasco-810 spectropolarimeter using a 0.1 cm path length quartz cuvette (Hellma). The temperature was maintained at 20°C with a Peltier thermostated cell holder. The oligonucleotides were dissolved in 10 mM sodium phosphate, 50 mM NaCl, 2.5 mM MgClU2Os cells were grown on coverslips to 80%–90% confluence. Cells were washed two times in PBS and one time in Transport buffer  and permeabilized with digitonin (50 µg/mL) for 5 min at RT. The permeabilized cells were washed six times with ice cold Transport buffer. The cells were incubated with import mix at 37°C for 20 min in a humid chamber. Import mixtures contained an energy-regenerating system consisting of the following components: 1 mM ATP, 0.2 mM GDP, 0.2 mM GTP, 5 µM creatine phosphate, and 20 U/mL creatine-phospho kinase (Sigma). The final concentrations for each protein in the mix were 1.5 µM GST-LRPCt, 1.5 µM Importin 8, and 2 µM Ran. After washing three times with Transport buffer, cells were fixed with 4% paraformaldehyde 10 min at RT, then washed three times with PBS to remove paraformaldyde and permeabilized with 0.2% (v/v) Triton X-100 for 10 min at RT. Upon permeabilization, cells were washed four times with PBS, blocked with Blocking buffer (10% FBS and 0.1% Tween 20 in PBS) for 1 h, and incubated with anti-GST antibody (1:1000 dilution) overnight at 4°C in wet chamber, followed by three washes in PBS. Cells were then incubated with Cy3 conjugated anti-goat antibody (1:200 dilution) for 1 h at RT. The cells were then washed four times with 1×PBS and mounted in mounting media with DAPI (Vector Laboratories). Analysis was carried out using a laser-scanning confocal microscope  exciting 405 and 543 nm with a 63× oil objective and 1× digital zoom. Channels were detected separately, with no crosstalk observed. Confocal micrographs represent single sections through the plane of the cell. All experiments were carried out three independent times and representative data are shown.The 4ESE-LacZ RNA export assays were followed as described in


RMRP, a component of the mitochondrial RNase MRP, is important for mitochondrial DNA replication and RNA processing, and the steroid receptor RNA activator, SRA, is a key modulator of hormone signaling and is present in both the nucleus and mitochondria. Some RNA-binding proteins maybe play a role in the lncRNAs transport system, such as HuR, GRSF1, SHARP, SLIRP, PPR, and PNPASE. Furthermore, a series of nuclear DNA-encoded lncRNAs were implicated in mitochondria-mediated apoptosis, mitochondrial bioenergetics and biosynthesis, and glutamine metabolism. The mitochondrial genome can also encode a set of lncRNAs, and they are divided into three categories: (1) lncND5, lncND6, and lncCyt b RNA; (2) chimeric mitochondrial DNA-encoded lncRNAs; and (3) putative mitochondrial DNA-encoded lncRNAs. It has been reported that the mitochondrial DNA-encoded lncRNAs appear to operate in the nucleus. The molecular mechanisms underlying trafficking of the mitochondrial DNA-encoded lncRNAs to the nucleus in mammals are only now beginning to emerge. In conclusion, both nuclear- and mitochondrial DNA-encoded lncRNAs mediate an intense intercompartmental cross-talk, which opens a rich field for investigation of the mechanism underlying the intercompartmental coordination and the maintenance of whole cell homeostasis.In animal cells, mitochondria are the primary powerhouses and metabolic factories. They also contain genomes and can produce mitochondrial-specific nucleic acids and proteins. To maintain homeostasis of the entire cell, an intense cross-talk between mitochondria and the nucleus, mediated by encoded noncoding RNAs (ncRNAs), as well as proteins, is required. Long ncRNAs (lncRNAs) contain characteristic structures, and they are involved in the regulation of almost every stage of gene expression, as well as being implicated in a variety of disease states, such as cancer. In the coordinated signaling system, several lncRNAs, transcribed in the nucleus but residing in mitochondria, play a key role in regulating mitochondrial functions or dynamics. For example,  It has been reported that mitochondrial DNA-encoded lncRNAs seem to operate in the nucleus [In animal cells, mitochondria also contain genomes, and they can produce mitochondrial-specific nucleic acids and proteins. Because the mitochondria are the primary powerhouses and metabolic factories in the cell, they need to be fully integrated into the whole cell regulation and coordination mechanisms to ensure that cellular needs are met. Thus, the mitochondria must continuously communicate with the nucleus . It is nMitochondria are the only organelles in animal cells that possess their own genomes . It is nmtRNA) transcription [mtRNAs are processed according to the “transfer RNA (tRNA) punctuation model,” whereby 22 interspersed tRNAs are excised to concomitantly release individual rRNAs and mRNAs [The human mitochondrial DNA (mtDNA) genome is a compact, circular, double-stranded DNA encoding only 13 proteins, which are all subunits of the electron transport chain, as well as two rRNAs 12S and 16S) and 22 tRNAs required for their translation . In addiS and 22 cription . mtRNAs 2O and CO2 [2, the TCA cycle generates intermediates that can funnel into multiple biosynthetic metabolic pathways to produce glucose, amino acids, lipids, heme, and nucleotides. Thus, the mitochondria operate as a central hub of both catabolic (the breakdown of large macromolecules to produce energy) and anabolic  metabolism.In classical biochemistry textbooks, the major tasks of the mitochondria are the production of ATP and the metabolites necessary to fulfill the bioenergetic and biosynthetic demands of the cell . Mitocho and CO2 . In addiIn fact, mitochondrial biological activities have progressively expanded to include not only various bioenergetic processes and important biosynthetic pathways but also calcium homeostasis and thermogenesis, cell death by apoptosis, and several different signal transduction pathways that are primarily related to redox control of gene expression . OncogenThe human mtDNA encodes only 13 proteins of the respiratory chain and ATP synthase complexes; a large proportion of mitochondrial proteins necessary for maintaining mitochondrial structure and function are encoded by the nuclear genome. These nuclear DNA-encoded proteins must be synthesized in the rough endoplasmic reticulum and imported into mitochondria . ProteinAs mentioned above, the mitochondria have distinct genetic systems that need to be coordinated with the nucleus to ensure cellular demands. Consequently, mechanisms have evolved in which nuclear genes exert direct control of mitochondrial gene expression and posttranslational modifications . However, the mitochondrial genome allows individual mitochondria to respond to changes in membrane potential and maintain oxidative phosphorylation by expressing their own genes . This deAlthough biologists often speak of a tight coupling between “genes and their encoded protein products,” it is important to remember that thousands of human genes produce ncRNAs as their ultimate products . ncRNAs Based on the length of ncRNAs, regulatory ncRNAs can be divided into at least three groups: (1) short ncRNAs, including microRNA (miRNA) (22–23 nts) and piwi-interacting RNA (piRNA) (26–31 nts); (2) medium ncRNAs 50–200 nts); (3) lncRNAs (>200 nts) [0 nts;  transcript may generate a lncRNA that is the target for a range of RNA-binding nuclear receptor coregulator proteins, including p68, SHARP, and SLIRP, and is a key modulator of hormone signaling [Because of its long length, lncRNA has the potential to develop multiple intramolecular RNA–RNA interactions conferring on them complex three-dimensional structures. It is possible that all lncRNAs consist of multiple structural or functional domains. In addition, in comparison to other classes of RNA, a feature of lncRNA is its propensity to contain a huge number of protein-binding sites. Thus, lncRNA can provide a platform for the multimerization of proteins  or can pignaling .RBPs are collectively referred to as heterogeneous ribonucleoproteins that associate with certain transcripts and profoundly influence all steps of posttranscriptional regulation of RNAs, including pre-mRNA splicing and mRNA polyadenylation, stability, localization, translation, and turnover –29. A seHuman antigen R (HuR), a ubiquitous RBP, recognizes specific RNA signature sequences that are typically U- or AU-rich and are usually found in the 3′-untranslated region of short-lived mRNAs , 32. HuRmtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, the lncCyt b and lncND5 RNAs, all of which contain the multiple-consensus binding sequence AGGGD. GRSF1 appears to be crucial for posttranscriptional storage, handling, and translation of mtRNAs [G-rich RNA sequence-binding factor 1 (GRSF1) is a cytoplasmic RBP and bears three RRMs with high affinity for G-rich sequences . GRSF1 wf mtRNAs .SRA. As mentioned above, SRA, a nuclear DNA-encoded lncRNA, has been shown to potentiate steroid hormone receptor transcriptional activity. SHARP binds to the SRA via three conserved RRMs and suppresses SRA activity. Surprisingly, the expression of SHARP is estrogen inducible, providing a potential autoregulatory mechanism to attenuate the hormonal response [SMRT/HDAC1-associated repressor protein (SHARP) is an RNA-binding nuclear receptor coregulator protein that includes three RRMs. SHARP is a transcriptional repressor, which can interact with the response .SRA stem-loop interacting RNA-binding protein (SLIRP) contains an RNA-binding domain and associates with and represses the activity of SRA [mtRNAs by directly modulating the expression, processing or stability of the mitochondrial transcripts that encode oxidative phosphorylation protein subunits. Furthermore, SLIRP is consistently coexpressed with the nuclear oxidative phosphorylation machinery. These properties raise the interesting possibility that SLIRP is coregulated with mitochondrial and nuclear genes to coordinate the oxidative phosphorylation gene expression of these two genomes [y of SRA . Howevery of SRA , 39. SLI genomes .168, and PTCD3 is a translational regulator [lncND5 transcript, whereas impairing PTCD2 decreased the levels of both lncND5 and lncND6 RNA [lncND5, lncND6, and lncCyt b RNA; Thus, MRPP3 is necessary for the accumulation of mitochondrial lncRNAs [The pentatricopeptide repeat (PPR) protein family is characterized by the presence of a 35-amino-acid structural motif that is tandemly repeated 2–26 times per protein. The PPR domain is involved in RNA–protein interaction, and PPR proteins play various roles in RNA editing, stability, and translation activation . In mammegulator . KnockdocND6 RNA . Knockdo lncRNAs .LRPPRC, an RNA-binding protein, is involved with mtDNA transcript processing. A report found LRPPRC to be bound to polyadenylated mRNA in a shuttling complex with heteronuclear ribonuclear protein K , which hPolynucleotide phosphorylase (PNPASE) is a 3′- to 5′-exoribonuclease and poly-A polymerase, located in the mitochondrial intermembrane space –53. PNPAlncRNAs represent a new field of the molecular biology, as it is becoming increasingly apparent that they are involved in the regulation of almost every stage of gene expression, as well as being implicated in a variety of disease states . lncRNAsMitochondria have distinct genetic systems that need to be coordinated with the nuclear genetic system, to meet the cellular energy and metabolite needs . In the Mitochondria possess pathways that enable RNA uptake from the cytosol. Even though the RNA import is a universal process , 66, theRMRP, a nuclear DNA-encoded lncRNA, is important for mtDNA replication and RNA processing. The gene for RMRP is located on chromosome 9p; the full length of RMRP is 265 nucleotides in human [RMRP from the nucleus to mitochondria  and essential for enzymatic activity. RNase MRP has the capacity to cleave mitochondrial RNA complementary to the light strand of the D-loop at a unique site. This cleavage site is one of the transition sites of primer RNA synthesis to DNA synthesis at the leading-strand origin of mitochondrial DNA replication [RMRP is primarily located in the nucleolus, but they have also confirmed the presence of low amount of RMRP in mitochondria [RMRP even at the level of detectability would be sufficient to promote an RNase MRP activity [in human . Three Rria Fig. . It has ytoplasm . PNPASE,l matrix . GRSF1 he matrix . RMRP islication . Li and chondria . It has activity .Fig. 1CrSRA, a lncRNA, is the target for a range of RBPs, including SHARP and SLIRP, and it is a key modulator of hormone signaling [SRA. SRA acts as an RNA scaffold for other coregulators at the transcription initiation site. SHARP interacts with SRA through an RNA-binding domain comprised of three RRMs. These RRMs are required by SHARP to repress SRA-augmented estrogen-induced transactivation [SRA and SHARP and augments SHARP’s corepressor activity on estrogen signaling. Given SLIRP’s nuclear repressive activities and demonstrated presence in the nucleus, it was surprising that SLIRP is predominantly a mitochondrial protein [SRA and SLIRP are present in both the nucleus and mitochondria and may, therefore, be expected to perform tasks in both locations. The transport mechanism of SRA and SLIRP from the nucleus to mitochondria remains unknown.The steroid receptor RNA activator ignaling . Secondativation . SLIRP b protein . SRA andRNase P is a lncRNA subunit of ribonuclease P which processes the 5′ leader of precursor tRNA [RNase P was partially purified from mammalian mitochondria [RNase P is imported into the mitochondrial matrix [RNase P in the mitochondria. If the amount of a lncRNA is low, it is difficult to make a difference between the contamination threshold and a real mitochondrial localization. Furthermore, it cannot be excluded that a “passive” nonspecific process could permit the mitochondrial import of various nucleic acids at a low rate in vivo [sor tRNA . RNase Pchondria , and thel matrix , 4, 54. l matrix , 80. In  in vivo .ENSMUST00000136025 is a nearby enhancer-like lncRNA of its neighboring BIM gene; BIM is a pro-apoptotic protein and potentiates mitochondria-dependent apoptosis. After sevoflurane exposure, aberrantly upregulated ENSMUST00000136025 highly induced overexpression of BIM in sevoflurane-treated samples compared with control samples. BIM eventually promoted sevoflurane-induced hippocampal apoptosis [MEG3 induces renal cell carcinoma (RCC) cells apoptosis by activating the mitochondrial pathway. MEG3 was evidently downregulated in RCC tissues and RCC cell lines. The apoptosis rate of RCC cells was increased significantly after MEG3 overexpression. Furthermore, overexpression of MEG3 could reduce the expression of Bcl-2 and procaspase-9 proteins, enhance the expression of the cleaved caspase-9 protein, and promote the release of cytochrome c protein to the cytoplasm [CARL) can suppress mitochondrial fission and apoptosis by targeting miR-539 and PHB2 (a subunit of the prohibitin complex). PHB2 has a key role in maintaining the homeostasis of mitochondrial dynamics. PHB2 is negatively regulated by miR-539. CARL can directly bind to miR-539 and participates in the regulation of mitochondrial network and apoptosis through the miR-539/PHB2 pathway [HOTAIR) induces mitochondria-related apoptosis and inhibits tumor growth in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. The mitochondrial membrane potential was changed significantly by HOTAIR blockage. Mitochondrial calcium uptake 1-dependent cell death was induced by HOTAIR depletion. Mitochondrial-related cell death pathway  involved in HOTAIR-dependent apoptosis process. HOTAIR plays a vital role in cancer initiation and progression by affecting cell cycle progress, apoptosis, and invasion [Some nuclear DNA-encoded lncRNA could play a role in mitochondria-mediated apoptosis. poptosis . lncRNA ytoplasm . Cardiac pathway . Homeoboinvasion .Tug1), an evolutionarily conserved lncRNA, regulates mitochondrial bioenergetics in diabetic nephropathy (DN). The PGC-1α (encoded by Ppargc1a), whose expression is typically reduced in diabetes, is a key mediator of mitochondrial dysfunction and progression of DN. Tug1 is a regulator of PGC-1α transcription. Tug1 interacts with PGC-1α at its promoter, resulting in elevated Ppargc1a transcriptional output. A decrease in Tug1 in the diabetic milieu would decrease PGC-1α expression, resulting in decreased expression of downstream PGC-1α targets involved in regulating mitochondrial bioenergetics [SAMMSON, a recently annotated lncRNA gene, is located in the chromosome 3p melanoma-specific focal. SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Silencing of the lineage addiction oncogene SAMMSON caused mitochondrial protein synthesis defects [AK055347 may contribute to the pathogenesis of atrial fibrillation by dysregulating mitochondrial energy production via regulation of Cyp450, ATP synthase, and MSS51 [Evidence that several nuclear DNA-encoded lncRNAs may regulate mitochondrial bioenergetics and biosynthesis has also been reported. Taurine upregulated gene 1  formation [A series of lncRNAs was implicated in glutamine metabolism. Glutamine, one of the essential nutrients, is deaminated by glutaminase (GLS) to produce glutamate, which further serves as a substrate for the mitochondrial tricarboxylic cycle . lncRNA pecimens . GLS was [HOTTIP . The posc manner . Moreoveormation . ROS areormation .lncND5, lncND6, and lncCyt b RNA; (2) chimeric mitochondrial DNA-encoded lncRNAs; (3) putative mitochondrial DNA-encoded lncRNAs.In animals, after processing the polycistronic transcripts of mtDNA, a set of ncRNAs was found . The mitlncND5, lncND6, and lncCyt b RNA, have been identified in humans. The regions of the mitochondrial genome complementary to the genes that encode ND5, ND6, and Cyt b mRNAs were found to have high levels of lncRNAs, specifically, lncND5, lncND6, and lncCyt b RNA. These three lncRNAs are 58, 34, and 14% as abundant as their complementary coding ND5, ND6, and Cyt b mRNAs, respectively. These mitochondrial DNA-encoded lncRNAs primarily form intermolecular duplexes with their respective complementary mRNAs. This suggests that they may have a functional role in mitochondria to either stabilize their partner mRNAs or regulate their expression. lncND5 RNA was the most abundant of the three mitochondrial DNA-encoded lncRNAs. The expression of lncND5, lncND6, and lncCyt b RNA is regulated by nuclear-encoded mitochondrial processing proteins, such as the PPR protein family [Three abundant mitochondrial DNA-encoded lncRNAs, n family . It was SncmtRNA), was identified in proliferating human tumor cells [SncmtRNA, the mitochondrial 16S rRNA was associated with an 815 nucleotide 5′-leader fragment deriving from the complementary strand. The SncmtRNA was polyadenylated and formed an 820 bp, RNase-resistant double-stranded structure with a 40 nucleotide loop [ASncmtRNA-1 and ASncmtRNA-2, combining the antisense mitochondrial 16S rRNA with a 310 or 545 nucleotide 5′-leader sequence deriving from the sense strand of the gene encoding this rRNA [ASncmtRNAs formed distinct double-stranded structures of 310 or 545 bp with a 96 or 450 nucleotide loop, respectively.A set of chimeric mitochondrial DNA-encoded lncRNAs with surprising structures and behavior was identified in mice and humans. The first member (1685 nucleotides) includes the mitochondrial 16S rRNA, together with a 121 nucleotide 5′-leader sequence deriving from the complementary strand of the gene encoding this rRNA . A seconhis rRNA . These tSncmtRNA and ASncmtRNAs were reported to be present in both the mitochondria and the nucleus, suggesting a role in mitochondrial nuclear communication and retrograde signaling [ASncmtRNA-2 has been shown to be exported from the mitochondria into the nucleus [ASncmtRNA-2 might function as a precursor of two miRNAs (hsa-miR-4485 and hsa-miR-1973) [The ignaling . Further nucleus . It was iR-1973) , but itsASncmtRNAs might function as unique tumor suppressors [SncmtRNA and ASncmtRNAs, the ASncmtRNAs are downregulated in human tumor cell lines and tumor cells from biopsies of different types of cancer, and they may take part in the mitochondrial reprogramming of oncogenic pathways [ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis family. Downregulation of survivin is at the translational level and is probably mediated by miRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs [Recently, it has been postulated that the pressors . Althougpathways . KnockdoncmtRNAs .LIPCAR). LIPCAR, a chimeric lncRNA, contains 781 nucleotides. Mapping the complete LIPCAR sequence to the human mitochondrial genome showed that the 5′ half (nucleotides 1–392) maps to antisense of the mitochondrial CYTB gene, but the 3′ half (nucleotides 385–781) maps to antisense of the mitochondrial COX2 gene. Indeed, the 5′ half of LIPCAR is wholly contained within the previously described mitochondrial lncCyt b gene [LIPCAR was upregulated at the late stages of left ventricular remodeling and was elevated in patients with chronic heart failure, independently of the pathogenesis. Thus, this lncRNA behaved as a prognostic indicator for chronic heart failure and cardiovascular mortality [A series of putative mitochondrial DNA-encoded lncRNAs was identified in studies devoted to heart diseases , 99, 100t b gene . The levLncRNAs are emerging as new players in gene regulation, but how lncRNAs operate in the regulation of an intense cross-talk between nucleus and mitochondria is still unclear. Because mitochondrial biological activities include not only bioenergetic processes and biosynthetic pathways but also calcium homeostasis, thermogenesis, and cell apoptosis , the fun