C. elegans, but our understanding of their conservation and contributions in other nematodes is limited. We analyzed small RNA pathways in the divergent parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that associate with secondary 5’-triphosphate 22-24G-RNAs. These small RNAs target repetitive sequences or mature mRNAs and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Even in the absence of a piRNA pathway, Ascaris CSR-1 may still function to “license” as well as fine-tune or repress gene expression. Ascaris ALG-4 and its associated 26G-RNAs target and likely repress specific mRNAs during testis meiosis. Ascaris WAGO small RNAs demonstrate target plasticity changing their targets between repeats and mRNAs during development. We provide a unique and comprehensive view of mRNA and small RNA expression throughout spermatogenesis. Overall, our study illustrates the conservation, divergence, dynamics, and flexibility of small RNA pathways in nematodes.Small RNA pathways play key and diverse regulatory roles in  Ascaris lacks piRNAs. Here the authors compare Argonaute proteins and small RNAs from C. elegans and Ascaris, expanding our understanding of the conservation, divergence, and flexibility of Argonautes and small RNA pathways in nematodes.The parasitic nematode  One of the first discoveries of small RNAs and their role in gene regulation was in the free-living nematode Caenorhabditis elegans7. Subsequent studies in C. elegans have revealed a diverse and complex set of small RNAs, pathways, and associated Argonautes11.Small RNAs contribute to many regulatory processes. They are associated with diverse functions including repressing foreign invaders and mobile elements, transcriptional regulation, mRNA translation and degradation, DNA repair, chromatin regulation and epigenetic inheritance, and ciliate genome rearrangementsC. elegans small RNAs include miRNAs, 21U-RNAs (piRNAs) and small-interfering RNAs (siRNAs). C. elegans miRNAs are involved in post-transcriptional gene regulation through regulation of mRNA translation and degradation12 but have also been associated with transcriptional activation13. C. elegans can generate siRNAs against foreign elements and also has a large repertoire of endogenous siRNAs11. These C. elegans small RNAs are named based on their length and the predominant first nucleotide of the RNA, e.g., 21U-, 22G-, and 26G-RNAs. 21U-RNAs have a 5’-monophosphate and 3’ 2’-O-methylation16. They are primarily thought to identify foreign (non-self) RNA elements. The identification of these foreign elements through RNA base-pairing leads to the subsequent synthesis of secondary siRNAs that repress their targets. 22G-RNAs have a 5’-triphosphate20. These are secondary siRNAs as they are generated in response to other small RNAs 21. Thus, primary siRNAs base-pair with transcript targets, marking or identifying them for the synthesis of the more abundant antisense, secondary 22G-RNAs by RNA-dependent RNA polymerases (RdRPs)23. 22G-RNAs silence mobile elements, pseudogenes, non-annotated loci, and select endogenous, germline genes18. They also serve to “activate or license”, “tune”, or repress gene expression27. 26G-RNAs have a 5’ monophosphate29. There are two classes of 26G-RNAs in C. elegans, one is testis-specific and associated with the Argonautes ALG-3/432, the other is expressed in early embryos and associated with the Argonaute ERGO-133. Like 21U-RNAs, 26G-RNAs trigger or act to prime the synthesis of secondary siRNAs (22G-RNAs) through base-pairing with targets11. Overall, C. elegans secondary 22G-RNAs are amplified responses to targets identified by 21U- and 26G-RNAs and other primary siRNAs.C. elegans Argonautes have undergone significant expansion and diversification. Twenty-seven Argonaute genes were originally described23; 19 are expressed . These include members of the AGO, PIWI, and WAGO (Worm-specific Argonautes) Argonaute clades34. The C. elegans AGO-clade Argonautes (5 AGOs) interact with miRNAs and 26G-RNAs, the PIWI clade (1 AGO) with 21U-RNAs (the worm piRNA ortholog), and the WAGO-clade (13 AGOs) with 22G-RNAs. Several of the WAGO clade Argonautes function in the nucleus regulating transcription and heterochromatin formation36. The largest expansion of C. elegans Argonautes is in the WAGO clade23. Many of these WAGOs are thought to have redundant functions in C. elegans.Small RNAs are bound by effector Argonaute proteins. 39. While extensive analyses of C. elegans Argonautes, small RNAs, and pathways have been carried out, relatively little is known regarding the conservation, divergence, or function of these pathways in other nematodes41. Nematodes have been divided into three major classes and five clades with C. elegans and its relatives as members of Clade V45. The nematode Ascaris is a parasite of humans (and pigs) infecting upwards of 800,000 people48. Ascaris is a Clade III nematode estimated to have diverged from C. elegans ~365–400 million years ago50. Previous studies in Ascaris indicated that piRNAs and PIWI Argonautes are absent in Ascaris51. PIWI Argonautes and piRNAs play a key role in repressing mobile elements in the germline. C. elegans piRNAs have been proposed to serve in identifying and defining foreign genetic elements acting upstream of secondary siRNA pathways56. Thus, they have been described as defining self vs. non-self57. The WAGO-associated 22 G secondary RNAs function in silencing and can maintain silencing of the foreign elements over generations in C. elegans11. To counteract silencing, it has been proposed that 22G-RNAs associated with the C. elegans CSR-1 function to ‘license”, identify self, or protect germline genes from repression and allow for their expression27. Given the key role of piRNAs in many organisms, and their role in C. elegans, the absence of piRNAs and PIWI in Ascaris raises the question of how Ascaris small RNA pathways have adapted to the absence of piRNAs and PIWI . Does Ascaris still need small RNA pathways to “license” gene expression without the presence of piRNAs? How is self vs non-self elements determined?Nematodes are an extremely diverse and abundant phylum adapted to a wide variety of lifestylesAscaris Argonaute proteins and carried out Argonaute IP and small RNA sequencing to characterize small RNA pathways. Two Ascaris Argonautes are highly specific for binding miRNAs (AsALG-1) and 26G-RNAs (AsALG-4). AsWAGO Argonautes bind 5’-triphosphate small RNAs (22–24G-RNAs). These small RNAs target repetitive sequences including mobile elements, but they also target mRNAs and likely “license”, “tune”, and/or repress gene expression. Two Ascaris WAGOs change their genomic targets (mRNAs vs repetitive sequences) in different developmental stages. We exploited the long ~1 m Ascaris male germline to obtain discrete regions of the testis and analyzed Ascaris Argonautes and their small RNAs throughout spermatogenesis. These analyses provide a unique and comprehensive timeline for the expression of Argonautes, their bound small RNAs and targets, and changes in expression of their corresponding genomic or mRNA targets throughout nematode spermatogenesis. Our study suggests that in the absence of piRNAs and with extensive evolutionary divergence from C. elegans, several Ascaris small RNA pathways and Argonautes appear to bind similar small RNAs, have similar targets, and in many cases appear to serve similar roles in both nematodes. Therefore, the potential “licensing” or fine-tuning of gene expression by Ascaris CSR-1 does not appear to be a consequence or counter-response to the presence of piRNAs. Overall, our studies illustrate the conservation and divergence of small RNA pathways that illustrate the flexibility and adaptability of Argonautes and small RNA pathways in nematodes.Here, we generated antibodies to Ascaris Argonautes51. Alignment and phylogenetic analyses of these Argonautes indicates the presence of 5 AGO-clade and 5 WAGO (Worm-specific Argonautes) Clade Argonautes, but the absence of PIWI Argonautes  cluster with C. elegans miRNA associated Argonautes and one (AsALG-4) with C. elegans ALG-3/4 Argonautes. The two additional Argonautes (AsALG-5 and AsALG-7) cluster with 26G-RNA-like Argonautes or RDE-1 (Note: AsALG-5 is not orthologous to C. elegans AsALG-5)   and NRDE-3 (soma)59. Our phylogenetic analysis suggests one Ascaris WAGO, named AsNRDE-3, is related to these C. elegans nuclear WAGOs. It is expressed in zygotes and early embryos and presents both in the cytoplasm and nuclei of embryos. We note that with only one Ascaris nuclear WAGO, AsNRDE-3 may also serve functions similar to C. elegans HRDE-1. Only AsCSR-1 and AsWAGO-3 have the RNase H, catalytic tetrad residues that confer slicer activity.The 5 Ascaris has three RdRPs  and is expressed in the germline, oocytes, zygotes, and during early development. In the male germline, RdRP-3 RNA expression is highest at M5 when the initiation of 26G-RNAs occurs and likely plays a major role in their biogenesis. The other two Ascaris RdRPs are likely orthologous to either C. elegans EGO-1 or RRF-1 and are expressed in the germlines . Their targets and functions in these tissues remain to be determined.ues Fig. . The mosR-1 Fig. , which iAscaris Argonautes, the miRNA Argonautes (AsALG-1 and AsALG-6) are expressed in both the male and female germline, early development, and larvae but are present at much lower levels in somatic tissues including the muscle, intestine, and carcass. AsALG-6 is eliminated during Ascaris programmed DNA elimination67. The 26G-RNA Argonaute, AsALG-4, is predominantly expressed in the male germline and highest during meiosis. AsALG-5 and AsALG-7 expression is in general low in the male and female germlines and early embryos through the comma stage, except for AsALG-7 in early spermatogenesis. The phylogenetic analysis in Fig. C. elegans ERGO-1 or RDE-1. However, C. elegans ERGO-1 binds 26G-RNAs that are 2’-O-methylated by HENN-1 but Ascaris lacks HENN-151.For AGO-clade Ascaris Argonautes, we generated polyclonal antibodies  to AsALG-1 (miRNA), AsALG-4 (26G-RNA), and all five WAGO Argonautes . These Argonautes were chosen as they represent the diversity of small RNA pathways in Ascaris, are the most highly expressed, and appeared orthologous to major C. elegans Argonautes. Western blot analyses show these antibodies specifically recognize the corresponding Argonaute proteins  support the specificity of the antibodies.To identify small RNAs associated with the Ascaris Argonautes indicates that AsCSR-1, AsWAGO-1, AsWAGO-2, AsWAGO-3, AsNRDE-3, AsALG-4, and AsALG-1 are present in both the cytoplasm and nucleus in early embryos  in the germline and early embryo68. We carried out Ascaris Argonaute antibody histochemistry on the male and female germlines and early embryo. Using a variety of conditions, we have not been able to observe germ granules in the germline or early embryo. Similarly, other antibodies to Ascaris proteins known to localize in germ granules in the early C. elegans embryo  also do not identify discrete granules in Ascaris early embryos. While it is possible that optimal fixation and other conditions may not have been identified for Ascaris germ granules, we have carried out successful immunohistochemistry in Ascaris embryos69. It is possible that such granules and functional complexes are present, but do not form large enough complexes to be visualized. Interestingly, CSR-1 antibodies did not identify P-granules in early embryos of C. briggsae64 and P-granules have not been examined in other nematodes.Ascaris Argonaute IP and small RNA sequencing demonstrate that the Argonautes associate with distinct small RNA classes that are antisense to different elements in the genome . However, AsALG-1 and AsALG-4 associated small RNA libraries were also constructed using 5’-dependent libraries to enrich for 5’-monophospahte RNAs , AsALG-4 binds 5’-monophosphate 26G-RNAs, and the WAGOs typically bind 5’-triphosphate 22G-RNAs (22–24G-RNAs). Neither Ascaris miRNAs nor 26G-RNAs are represented at high abundance compared to the predominant 22G-RNAs (22–24G-RNAs) present in the total small RNA population  and in the middle of a few chromosomes. The eliminated sequences include 30 Mb of a 120 bp tandem repeat that is present in the internal and terminal regions of chromosomes. Based on a liberal prediction, we estimate that repetitive and mobile sequences constitute ~41% or 126 Mb of the Ascaris germline genome .Ascaris small RNAs (80–90%) are antisense to repetitive sequences and/or mobile elements in the genome  and cover ~9.5 Mb or 3.4% of the genome  of defined repetitive sequences compared to C. elegans. However, the AsWAGO-1/2 targeting of repeats is not a consequence of the increased repeat content as less than 8% of these repeats (3% of the genome) are high-confidence AsWAGO-1/2 small RNAs targets.The majority of the ome Fig. . HoweverAscaris genes that are potentially targeted by small RNAs, we normalized the small RNA reads to mRNAs based on reads-per-kb-per-million (rpkm). We considered mRNAs with antisense small RNAs over 10 rpkm in a stage as high-confidence targets (Source Data file). Small RNAs that target mRNAs are generally antisense and fully complementary to mature mRNAs and associated with AsCSR-1, AsWAGO-3, AsNRDE-3 (in the testis), and AsALG-4. The distribution of these small RNAs across mRNAs is typically uniform across transcripts or with a 5’ bias , we compared the levels of small RNAs in these IPs matching mRNAs and repeats using principal component analysis (PCA) , many of these targeted mRNAs overlap  Fig. . Small RCA) Fig. . Small RCA) Fig. . These dC. elegans female germline. In contrast, analysis of C. elegans small RNAs and pathways are more limited in the male germline74. 21U-RNAs, 26G-RNAs, and 22G-RNAs are all known to play an important role in the C. elegans male germline. Our PCA analysis of small RNAs suggested a greater diversity of small RNAs in the testis 76. These regions include the mitotic region, transition zone, early meiosis (pachytene), late meiosis I and II, and spermatids  during spermatogenesis. Many of these targets overlap with enriched H3K9me2/3 histone marks  in the male and female germline (but not the early embryo) for one of these, a non-LTR R4 retrotransposon that inserts into the ribosomal locus. R4 was previously described as mobile based on its presence in different genomic locations among individuals82. In contrast to the expression of R4, our RNA-seq analyses suggest that most other predicted mobile elements (previous work and current predictions) are not highly expressed. However, PRO-seq suggests many of these mobile element loci are bidirectionally transcribed at low levels. This bi-directional transcription could lead to dsRNA substrates for Dicer to generate primary siRNAs, leading to subsequent secondary siRNA generation to further silence the loci. Consistent with this, high levels of AsWAGO-1, AsWAGO-2 and AsNRDE-3 associated 22G-RNAs are observed to target many of these elements.Several previous studies defined 67). Figure To define the timing and expression of Argonaute associated small RNAs and their mRNA targets during spermatogenesis, we first identified 5526 genes targeted by at least one Argonaute  with antisense small RNAs over 10 rpkm in one or more of the spermatogenesis stages (see Source Data file). These genes can be categorized into two broad groups based on their expression profiles. One group includes genes expressed throughout male germline development  whereas the other group are genes specifically expressed during meiosis  is in general later than their targeted mRNAs (M5–7), including AsALG-4 mRNA itself  during spermatogenesis and suggest an important role for AsNRDE-3 during the later stages of spermatogenesis. This plasticity of small RNA targets is also observed in AsWAGO-3  in these divergent nematodes88. Here, we characterize Argonautes and their small RNAs in the parasitic nematode Ascaris. Ascaris is a Clade III nematode that diverged from C. elegans (Clade V) ~365–400 million years ago89. Ascaris has a reduced set of expressed Argonautes compared to C. elegans (10 vs. 19) including 5 AGO-clade and 5 WAGO-clade Argonautes  lacks piRNAs (21U-RNAs) and PIWI Argonautes88. We generated specific polyclonal antibodies to seven Ascaris Argonautes and analyzed small RNAs associated with AsALG-1, AsALG-4, AsWAGO-1, AsWAGO-2, AsWAGO-3, AsCSR-1, and AsNRDE-3 in early embryos, the female germline, and throughout the male germline. Overall, several small RNA pathways and their functions appear conserved between Ascaris and C. elegans while others have diverged in function and targets or been lost.Our understanding of nematode small RNA pathways comes from detailed studies in tes Fig. . AscarisAscaris are 22G-RNA secondary siRNAs. The majority are antisense to repetitive sequences and are associated with Ascaris WAGOs, particularly AsWAGO-1 and AsWAGO-2. However, other Ascaris WAGOs, including CSR-1, WAGO-3, and NRDE-3, also associate with 22G-24G-RNAs that are antisense to mature mRNAs. Detailed analysis of AsCSR-1 and AsWAGO-3 small RNAs indicates they are primarily 24G-RNAs and 23G-RNAs, respectively  that target mRNAs expressed primarily in the male germline during meiosis. AsALG-4 function may differ in Ascaris compared to C. elegans, whereas the AsCSR-1 pathways appear to function similarly as in C. elegans91 even in the absence of an Ascaris piRNA pathway.The most abundant small RNAs in C. elegans is to define “self” vs “non-self” (C. elegans genes vs foreign elements)56. C. elegans piRNAs have limited, miRNA-like complementarity to their targets , thus providing the potential for targeting a vast array of sequences93. It has been proposed that a role of the C. elegans CSR-1 pathway is for “licensing” gene expression, thus enabling expression of genes, to counteract promiscuous piRNA silencing of germline transcripts27. Given the absence of piRNAs and PIWI in Ascaris, we sought to determine the targets of AsCSR-1 small RNAs and thus the potential function of the Ascaris CSR-1 pathway. AsCSR-1 represents the most abundant Argonaute mRNA in the Ascaris germline and early embryos. Its associated 24G-RNAs are antisense to almost all transcribed mature mRNAs. Why does AsCSR-1 target so many mRNAs? Does this indicate it still plays a role in “licensing” gene expression in Ascaris? In the absence of piRNAs, perhaps the Ascaris WAGOs or other genome regulatory processes involved in gene silencing (including programmed DNA elimination94) must be counteracted. Alternatively, the AsCSR-1 pathway may not be involved in “licensing gene expression”. Instead, it functions in fine-tuning gene expression90 or reducing noise to prevent epimutations95.It has been suggested that a central role of piRNAs in C. elegans CSR-1 has been proposed to “fine tune” gene expression of mRNAs loaded into oocytes90, clear maternal mRNAs25, and degrade mRNAs with non-optimal codons96. AsCSR-1 is present in oocytes and at its highest level during the maternal to zygotic transition which initiates at the one-cell stage in Ascaris prior to pronuclear fusion60. AsCSR-1 disappears early in Ascaris development at the 32–64 cell stage. These data are consistent with AsCSR-1 also playing a similar role as observed in C. elegans in fine-tuning oocyte transcripts and clearing mRNAs during the maternal to zygotic transition. The balance among these functions of AsCSR-1  may be developmental stage and/or mRNA dependent and remains to be determined even in C. elegans.Ascaris is sexually dimorphic. We used the ~1 m length of the male Ascaris germline to comprehensively examine mRNA, Argonautes and their associated small RNAs, and the small RNA targets throughout nematode spermatogenesis. A striking feature of Ascaris small RNA pathways is the unique expression of AsALG-4 and associated 26G-RNAs during the later stages of spermatogenesis (M6–M7). This is followed by a significant decrease in the mRNAs targeted by the 26G-RNAs . Thus, independently evolved small RNA pathways may have convergent functions and play key roles during pachytene of spermatogenesis.Small RNA pathways and targets throughout the developmental stages of nematode spermatogenesis have not been analyzed. C. elegans, it has been proposed that 26G-RNAs likely identify spermatogenesis mRNA targets for generation of secondary 22G-RNAs72. This does not appear to be the case in Ascaris as our current data further refine our previous finding51 and indicate that AsALG-4 and 26G-RNA expression does not precede the appearance of secondary 22G-RNAs to the same mRNA targets. In contrast, our data suggest that 26G-RNAs appear much later than most of the small RNAs targeting mRNAs during spermatogenesis and 26G-RNAs are associated with the loss of mRNAs in late spermatogenesis and the formation of spermatids . This contrasts with the 5’ and 3’ end targeting of transcripts of C. elegans ALG-3/4 26G-RNAs during spermatogenesis72. Thus, AsALG-4 transcript targeting differs from C. elegans ALG-3/4 indicating flexibility and differences in targeting between nematodes. The AsALG-4 transcript targeting appears more similar to C. elegans ERGO-1 26G-RNAs that uniformly target across transcripts72.The current C. elegans NRDE-3 is involved in the nuclear RNAi pathway and contributes to co-transcriptional repression (inhibiting elongation)101. AsNRDE-3 small RNAs target the 5’ ends of transcript in M6–M7. This may be associated with co-transcriptional repression of targets. The decrease in AsCSR-1 levels during the later stages of spermatogenesis may lead to loss of licensing or protection from repression also resulting in a decrease in AsCSR-1 target RNAs. AsCSR-1 could also play a repressive role as recently described96. Notably, targets of AsCSR-1, AsALG-4, and AsNRDE-3 in M6–M7 largely overlap. Ascaris spermatids exhibit very low levels of mRNAs and small RNAs. We suggest that during the later stages of Ascaris meiosis these Argonautes, their changes in expression, and small RNAs act in concert to clear mRNAs in the formation of spermatids. Overall, complex and diverse small RNA pathways contribute to key patterns of gene regulation likely involving licensing during early spermatogenesis and transcriptional and post-transcriptional repression during the later stages of Ascaris spermatogenesis.Strikingly, AsNRDE-3 22G-RNA targets are largely repetitive sequences during early spermatogenesis but during the later stages (M6–M7) their targets are predominantly mRNAs. Ascaris WAGO-1 and WAGO-2 associated small RNAs only target repetitive sequences. However, AsWAGO-3 and AsNRDE-3 small RNAs, depending on the stage and tissue, can target either repetitive sequences or mature mRNAs. AsWAGO-3 small RNAs in early embryos predominantly target mature mRNAs that overlap with AsCSR-1 (AsWAGO-3 RNA is expressed at much lower levels than AsCSR-1) whereas in the male gonad they largely target repetitive sequences. Sequence alignment and phylogenetic analyses suggest AsWAGO-3 appears similar to C. elegans C04F12.1 and clusters with CSR-1. C. elegans NRDE-3 IP and 22G-RNA analysis indicate the small RNAs target intergenic repetitive loci102. In Ascaris, NRDE-3 also targets primarily repetitive sequences during early spermatogenesis, the female germline, and in 4-cell embryos. However, during the later stages of spermatogenesis, a large percentage of NRDE-3 small RNAs target mRNAs. Secondary 22G-siRNAs in C. elegans are generated by RNA-dependent RNA polymerase following identification of target RNAs by primary siRNAs. The change in small RNA targets in AsWAGO-3 and AsNRDE-3 raises questions regarding how distinct types of targets are identified as substrates for the biogenesis of small RNAs, the RdRPs used to generate the 22G-RNAs, and the loading of these RNAs into distinct Argonautes in different stages78.Ascaris embryos , larvae104, the isolated and perfused adult intestine105, and muscle106 through injection of dsRNA into the pseudocoelom of adults. The key C. elegans RDE-1 Argonaute binds exogenous siRNAs derived from dsRNA. Ascaris appears to lack an RDE-1 Argonaute. What Ascaris Argonaute then acts on introduced dsRNA or endogenous dsRNA to generate primary siRNAs that induce the generation of secondary siRNAs? Most of our phylogenetic analyses suggest that AsALG-5 and AsALG-7 appear related C. elegans ALG-3/4, while in a few cases, they cluster with either RDE-1 or ERGO-1. AsALG-7 expression is highest in the early stages of spermatogenesis. However, we observe very low levels of 26G-RNAs in early spermatogenesis suggesting AsALG-7 may not bind 26G-RNAs. The lack of HENN-1 and 26G-RNAs in other stages suggests that AsALG-5 and AsALG-7 are not likely orthologs of ERGO-1. It is possible that the widely expressed AsALG-7 might serve functions similar to C. elegans RDE-1. Overall, with no Ascaris piRNAs and 26G-RNAs not likely serving as primary siRNAs to initiate secondary siRNA generation, the origin of endogenous primary siRNAs in Ascaris that lead to the abundant 22G-RNAs is unclear. Recent data suggest that in some C. elegans stages or compartments the CSR-1 slicer activity22 may initially function to target and cleave mRNAs thereby serving to identify RNAs for RdRPs to initiate the generation of secondary siRNAs for CSR-196. A similar mechanism may occur in Ascaris.Several studies suggest that the introduction of dsRNA leads to RNA interference in Ascaris and these are the most abundant small RNAs. Notably, Ascaris Argonautes that associate with small RNAs targeting repetitive sequences  do not have the catalytic tetrad for slicing activity.A diverse array of repetitive sequences are targeted by 22G-RNAs in Ascaris are transcribed bidirectionally at low levels and may generate dsRNAs for primary siRNA generation that may then lead to secondary, amplified 22G-RNAs and suppression of mobile element expression. High levels of these 22G-RNAs target mobile elements in most developmental stages. Many, but not all of these loci exhibit repressive histone marks including H3K9me2/3  are present in many Caenorhabditids and some Clade III nematodes, but they appear absent in other Clades113. Here, we have shown general conservation of Caenorhabditid CSR-1, ALG-3/4, and WAGO-associated small RNAs and their targeting in Ascaris (Clade III). Secondary siRNAs (22G-RNAs or longer) are present in Clades III–V, but they are absent in Clades I–II113. Strongyloididae (Clade IV) appear to have 27GA-RNAs instead of 22G-RNAs and the secondary siRNAs of Globodera pallida (Clade IV) are 22–26 nt in length88. Differences in the sizes of small RNAs is also observed in Ascaris with discrete small RNAs (22–24 G) associated with different WAGOs. Although orthologs of ALG-3/4 appear present in most nematodes, 26G-RNAs appear generally absent; Ascaris appears to be an exception with abundant 26G-RNAs during spermatogenesis meiosis. If 26G-RNAs in other nematodes are restricted to meiotic regions of the male germline as seen in Ascaris, they may not have been enriched in samples used in previous analyses of those nematodes.Previous analysis of the evolutionary conservation of Argonautes and different types of small RNAs in nematodes suggest that the complexity of Argonautes and small RNA pathways in C. elegans is associated with piRNAs, 22G-RNAs, and histone H3K9me3 and H3K27me3 marks114. C. elegans HRDE-1 is important for the maintenance of inheritance over several generations whereas NRDE-3 appears limited to one generation. An interesting question is whether Argonautes and small RNA pathways are associated with transgenerational inheritance in Ascaris and other nematodes. Transgenerational inheritance in C. elegans has been associated with environmental information , behavior, lifespan, and viral and bacterial pathogens120. In parasitic nematodes, transgenerational inheritance of information like these and particularly host immune responses would be clearly beneficial. For example, exosomes from the parasitic nematode, Heligmosomoides polygyrus (bakeri), contain 22G-RNAs and a WAGO Argonaute, and can suppress Type 2 innate responses and eosinophilia121. Nematode Argonautes and small RNA pathways may play key roles in host-parasite interactions and communication among nematodes. As many of these nematode Argonautes and small RNA pathways are worm-specific and thus differ from their hosts, they could be potential targets for new therapies against parasitic nematodes.Transgenerational inheritance in Ascaris lacks piRNAs but maintains a CSR-1 pathway with small RNAs that target most expressed mRNAs. Thus, AsCSR-1 may act in a “licensing” pathway as observed in C. elegans in the absence of a piRNA pathway as well as serving other tuning or repressive functions. Ascaris ALG-4 associated 26G-RNAs target male meiosis-specific genes commensurate with their mRNA degradation and do not appear to act as primary siRNAs for targeting and generating secondary 22G-RNAs during spermatogenesis. Several Ascaris Argonautes, including AsNRDE-3 and AsWAGO-3 targets, are stage dependent altering their targets between mRNAs and repeats during spermatogenesis and development. Our data significantly expand our understanding of the conservation, divergence, and flexibility of nematode Argonautes and small RNA pathways. Nematode Argonautes and small RNA pathways may have initially evolved for specific types of gene regulation leading to better fitness, but over time may have been co-opted for other functions in diverse nematodes.In summary, nematodes are a diverse phylum including free-living and parasitic species. Ascaris tissues, sperm, and embryonation was as previously described60. Male and female germline tissues were washed in PBS, frozen whole or following dissection in liquid nitrogen, and stored at −80 °C , 200 U of NxGen RNase Inhibitor (Lucigen), and protease inhibitor cocktail (Roche)122. Fresh Ascaris 4-cell embryos were decoated and directly homogenized in a metal dounce and nuclei lysed as described above. Lysates were then used for total RNA isolation or immunoprecipitation.Collection of Ascaris fusion proteins for AsALG-1, AsALG-4, AsCSR-1, AsWAGO-1, AsWAGO-2, AsWAGO-3, and AsNRDE-3 and also to peptides for AsWAGO-2 and AsWAGO-3. AsALG-1 amino acids 1-165 (HQ611964), AsALG-4 amino acids 1–75 (HQ611965), AsCSR-1 amino acids 1–162 (HQ611969), AsWAGO-1 amino acids 1–113 (HQ611970) AsWAGO-2 amino acids 1–141 (HQ611971), AsWAGO-3 amino acids 49–202 (HQ611972), and AsNRDE-3 amino acids 45–246 (HQ611973) were fused in frame with GST using the Bam H1 site of pGEX-6P-1 , the proteins were expressed in E. coli, purified using Glutathione Sepharose 4B columns, and used as the immunogens for the initial boost to generate polyclonal rabbit antibodies (Covance). For additional boosts, we used the Ascaris proteins cleaved from GST-fusions bound to Glutathione Sepharose 4B by treatment with PreScission protease . AsWAGO-2 peptides (YASRRMFELTKQSRDDYRA and RNGGQETSSSSSGEAGHLDI) and AsWAGO-3 peptides (VISYGRGKARRKEFPKQAGT and RENDLPRNGESVDWNRITD) were fused to KLH and used as immunogens to generate polyclonal rabbit antibodies . Antibodies were affinity-purified using either the peptides linked to SulfoLink beads (Pierce) or the Ascaris proteins (without GST) linked to a mix of Affigel-10/15 (BioRad).We generated polyclonal antibodies to Ascaris embryo immunohistochemistry was carried out as described60 using a modified freeze-crack method to permeabilize and fix embryos. Briefly, decoated embryos were suspended in 50% methanol and 2% formaldehyde solution and were frozen and thawed three times using a dry ice/ethanol bath. The embryos were re-hydrated with 25% methanol in PBS pH 7.4 for 1 min. After washing twice with PBS pH 7.4, the embryos were incubated in signal enhancer solution (Invitrogen I36933) for 30 min at RT. The embryos were then resuspended in blocking solution (0.5% BSA in PBS pH7.4) for 30 min at RT, followed by overnight incubation in primary antibodies at 4 °C, and then a 2 h incubation in secondary antibodies (Invitrogen) at room temperature. Nuclei were stained with DAPI.Regions of 1–5 cm were collected from 1% formaldehyde-fixed dissected gonads at defined distances from the distal tip. Germline fragments were placed on a glass slide with PBS pH 7.4 and gently rolled over with a glass rod to release germline tissue from the outer somatic sheath. Samples were next stained with DAPI and the slides mounted in Prolong anti-fade medium (Invitrogen). Regions were characterized and defined based on nuclear morphology and presence or absence of mitotic/meiotic structures as follows: (1) Mitotic: round-shape interphase nuclei with evenly distributed chromatin and defined nucleolus and the presence of mitotic metaphases and anaphase. (2) Transition zone: more compact chromatin with punctate aspects and irregular shape, no mitoses observed. (3) Meiosis I—Pachytene: Chromatin aspects similar to a “bowl of spaghetti”, increasing nuclear size from early (closer to TZ) to late (closer to meiotic progression). (4) Meiosis 1 and 2: increased chromatin compaction (diplotene), visualization of individual bivalents (diakinesis), further localization to the metaphase plate (metaphase I) and segregation to opposite poles with lagging material in the middle corresponding to sex chromosomes (anaphase I). While no clear observations of meiosis II prophase to anaphase were obtained, groups of 4 nuclei in very close proximity to each other indicated completion of the second meiotic division and formation of haploid nuclei. (5) Spermatids: dispersed, very small and compact nuclei.Ascaris germline was carried out on the same samples as described above. Tissue fragments were kept on the slide throughout the whole immunohistochemistry procedure, with careful pipetting of the solutions to avoid sample loss. Samples were first permeabilized in 0.2% Triton X-100/PBS for 15 min at room temperature, followed by two washes in PBS. Next, samples were suspended in blocking solution (1% BSA in PBS pH 7.4), covered with parafilm and incubated for 1 h at room temperature in a wet chamber. Primary and secondary antibody (Invitrogen) incubations were performed overnight at 4 C and for 2 h at RT, respectively, with slides covered with parafilm and placed in a wet chamber. After staining of nuclei with DAPI, slides were mounted in an anti-fade medium (Invitrogen) and kept in the dark at room temperature for 24 h before imaging.Immunohistochemistry of the Ascaris germline immunohistochemistry and DAPI-stained preparations were imaged on an Applied Precision DeltaVision microscope, using a ×60 immersion objective and FITC/DAPI excitation filter set. Images were deconvolved with Applied Precision’s Softworx software and analyzed using Fiji software. Ascaris embryos immunohistochemistry images were captured on a Leica DM6B fluorescence microscope, equipped with a Leica DFC 7000 T camera, using a ×40 objective, FITC/DAPI excitation filter set and LASX software.123. Germline or embryo whole-cell lysates were mixed with Protein A Dynabeads and rotated overnight at 4 °C. Protein A Dynabeads were recovered and washed with high-salt buffer  three times. The beads were resuspended in 250 ul of Proteinase K buffer containing 200 µg/ml Proteinase K and incubated for 1 h at 37 °C. RNA was extracted using Trizol LS (Invitrogen) adapted for small RNA recovery by precipitation using 2 volumes of isopropanol, 15 µg of GlycoBlue (Invitrogen), and 30 min incubation at −80 °C followed by centrifugation at 18,500 × g for 35 min at 4 °C. The RNA was treated with RppH  in Thermopol buffer for 5’ independent cloning124. Both untreated and RppH-treated samples were done for AsALG-1 and AsALG-4 samples. Following RppH treatment, samples were repurified with Trizol LS (Invitrogen)  and stored at −80 °C.Argonaute IPs were carried out using affinity-purified rabbit antibodies pre-bound to Protein A Dynabeads (Fisher Scientific) (5–10 ug of antibody per 100 ul of beads)124 were prepared. Libraries from 4-cell embryos were also prepared using 18–30 nt gel-purified RNA using the SMARTer smRNA-Seq Kit (Clontech) and NEXTflex-Small-RNA-Seq (New England Biolabs) with similar results. RNA > 200 nt was treated with TURBO DNase (Ambion) and rRNA depletion carried out using RiboCop rRNA Depletion Kit for Human/Mouse/Rat (Lexogen). Long RNA (>200 nt) libraries were made using CORALL Total RNA-Seq Library Prep Kit (Lexogen). Both small and long RNA libraries were sequenced 150 nt from both ends using the Illumina NovaSeq 6000 System.RNA was isolated using Trizol or LS Trizol (Invitrogen). Total RNA samples were fractionated into small RNA (<200 nt) and long RNA (>200 nt) using the Monarch RNA cleaner (New England Biolabs). Small RNA libraries were prepared using the Small RNA-Seq Library Prep Kit (Lexogen). Both 5’-phosphate and 5’-phosphate independent libraries (RppH-treated)51. Briefly, adaptor sequences were trimmed and then the reads collapsed to non-redundant datasets. The reads were then mapped to the Ascaris genome and other various datasets using bowtie2125 to determine targets and their expression levels. To define antisense regions in the genome corresponding to AsWAGO-1 and AsWAGO-2 small RNAs, we first mapped reads from each Argonaute IP-small RNA library to the genome using bowtie126. We normalized the coverage for each library to 30 million reads, a number close to the average number of raw input reads for the libraries. With an average length of ~22 nt for the small RNAs, this corresponds to ~2.5× coverage of the 280 Mb genome. As AsWAGO-1 and AsWAGO-2 small RNA targets are very similar, we merged genomic regions in close proximity  if they have > = 20 fold average coverage (50×) from any of the 20 AsWAGO-1 and AsWAGO-2 libraries (using bedtools -mergeBed -d 2000). This merge is necessary as a locus is often not fully covered with high-levels of siRNAs. This approach defined an initial set of enriched loci (9164 with length ≥ 50 bp). We then used the normalized reads (rpkm) mapped to these loci and a 10-fold enrichment in at least one library to define these genome regions as enriched for AsWAGO-1 and AsWAGO-2 small RNAs. These regions were further filtered to remove loci related to rRNA, tRNAs, miRNAs or mitochondrial DNA. Overall, we defined 3948 AsWAGO-1 and AsWAGO-2 targeted (10× enriched) genomic regions that constitute 8.1 Mb of sequence and named them WAGO-repeats. Using the same approach, we defined 2912 (6.2 Mb) AsNRDE-3 targets. The majority (~77%) of the sequences defined as AsNRDE-3 targets overlap with AsWAGO-1/2 targets. See Source Data file for the defined WAGO-repeats and mRNAs and their WAGO-associated small RNAs. PCA analysis on the small RNA libraries was done using DESeq2 plotPCA127. Heatmaps were generated using Treeview 3128.Bioinformatic processing of small RNA sequencing data was carried out as previously described129, LTRharvest130, RepeatScout131, RepeatExplorer132, dnaPipeTE133, MGEScan-non-LTR134, Helsearch135, MITE-Hunter136, SINEfinder137, TEdenovo138, and RepARK139. The final collection of repetitive sequences was filtered for redundancy with CD-hit and are available within the repeat track at http://genome.ucsc.edu/s/jianbinwang/Ascaris_small_RNAs.Repetitive sequences were identified using a combination of homology-based and de novo approaches, including RepeatMaskerFurther information on research design is available in the Suppementary InformationPeer Review FileReporting Summary


Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease. MDA5 is the primary immune sensor for SARS-CoV-2 and many other viruses. Mutations in MDA5 can cause disease. Here the authors employ CryoEM and biochemical methods to show how steric constraints cause MDA5 to misrecognize endogenous RNA as viral RNA. MDA5 cooperatively assembles into helical filaments on uninterrupted RNA duplexes longer than a few hundred base pairs (bp)6. LGP2 promotes and stabilizes MDA5-RNA complex formation8. RIG-I, MDA5, and LGP2 bind dsRNA with a modified DExD/H-box helicase core and a C-terminal domain (CTD)14. The helicase consists of two RecA-like domains, Hel1 and Hel2, and an insert domain, Hel2i, all of which form contacts with phosphate and ribose moieties of both RNA strands. The helicase and CTD, linked by a pair of α-helices referred to as the pincer domain, form a ring around the RNA. Upon binding RNA, RIG-I-RNA oligomers and MDA5-RNA filaments nucleate the assembly of MAVS into microfibrils16, which then recruit proteins from the TRAF and TRIM families to activate potent type I interferon and NF-κB inflammatory responses15.Viruses deliver or generate RNA in the cytosol of the host cell to have their genes expressed and replicated by cellular machinery. Recognition of cytosolic viral double-stranded RNA (dsRNA) is one of the most important and conserved mechanisms for sensing infection. dsRNA is recognized in the cytosol by the innate immune receptors RIG-I, MDA5, and LGP2. RIG-I binds dsRNA blunt ends with di- or triphosphorylated 5′-ends2, including SARS-CoV-218, but viral dsRNA can be difficult to distinguish from endogenous dsRNAs. Retrotransposon transcripts, including inverted Alu repeats within mRNA and bidirectionally transcribed LINE-1 elements can anneal into RNA duplexes that are sensed by MDA5 as viral dsRNA22. Retrotransposons constitute one-third of the human genome. A-to-I deamination by ADAR1 destabilizes Alu:Alu duplexes sufficiently to prevent MDA5 filament formation20. Nevertheless, efficient proofreading and fine-tuned signal transduction are necessary for cells to respond appropriately to infection without triggering autoimmune responses. Cryogenic electron microscopy (cryoEM) structures of MDA5-dsRNA filaments at different stages of the ATPase cycle—bound to ATP, transition-state analog ADP-AlF4, or no nucleotide—showed that ATP hydrolysis by MDA5 is coupled to conformational changes in MDA5-dsRNA filaments14. We proposed the ATPase cycle performs a mechanical proofreading function by testing the interactions with the bound RNA, promoting dissociation of MDA5 from loosely-bound endogenous RΝΑs while allowing it to remain bound to viral RNAs long enough to activate signaling14.MDA5 is the primary innate immune sensor for many viruses24. Approximately 30 MDA5 point mutations have been linked to severe autoinflammatory disorders in heterozygous human patients, including Aicardi–Goutières syndrome (AGS), Singleton–Merten syndrome (SMS), spastic-dystonic syndrome, and neuroregression27. Pathogenic MDA5 mutations are associated with increased expression of interferon-stimulated genes in patients27 and increased IFN-β signaling in cultured cells, particularly without stimulation with exogenous dsRNA26. MDA5 mutations are known or predicted to be a pathogenic map to the RNA and ATP binding sites, with a few exceptions , found in patients from two unrelated families, is associated with symptoms of AGS and SMS caused by elevated type I interferon signaling29. Residue 854 is located on the first pincer helix, distant from the ATP and RNA binding sites. Therefore, the molecular mechanism of disease pathogenesis from the M854K mutation remains unknown.Mutations that alter the parameters governing MDA5-dsRNA filament dynamics can cause inappropriate activation of the antiviral innate response. MDA5 gain-of-function mutations and ADAR1 loss-of-function can cause PKR-mediated translational shutdown and severe autoinflammatory disordersHere, we examine the consequences of the M854K disease variant on the structure and activities of MDA5. We show that the M854K mutation abrogates ATPase activity, stabilizes MDA5-dsRNA complexes, increases IFN-β transcription, and dampens the cooperativity of signal transduction. CryoEM structures of WT and M854K MDA5-dsRNA filaments at different stages of ATP hydrolysis, determined at resolutions of up to 2.8 Å, reveal in detail how the M854K mutation constrains conformational changes in MDA5 necessary for ATP hydrolysis and ATPase-dependent dissociation from endogenous RNAs, ultimately leading to constitutive signaling activation.26. To determine the functional consequences of the M854K mutation, we measured its activity in a cell signaling assay. We compared the M854K variant to WT MDA5 and to variants with point mutations at the filament-forming interface engineered to increase or decrease signaling activity  RNA, a dsRNA analog, to induce MDA5 signaling. IFN-β-dependent gene expression was measured as the ratio of firefly to Renilla luciferase luminescence14. The expression level of each MDA5 variant was assessed by Western blotting  stimulation, and 10–30% higher than WT with poly(I:C) stimulation  stimulation and 50% of WT activity with stimulation. The D848K/F849A/R850E mutations, which completely inhibit filament formation but not ATPase activity14, abolished signaling with and without poly(I:C) stimulation   cells. The mutant with a stabilized filament interface, H871A/E875A, was constitutively active in ADAR1-KO cells, with poly(I:C) stimulation providing no further increase in signaling  Fig. . This suing Fig. . The muting Fig. . TogetheThe constitutive signaling activity of variant M854K in the absence of exogenous long dsRNAs implies that the M854K mutation disrupts the ATPase-dependent structural selectivity of MDA5 for long uninterrupted RNA duplexes. Cellular dsRNAs are relatively short. Alu:Alu RNA duplexes span up to 300 bp but contain a mismatch or bulge every 13 bp on average. We hypothesized MDA5 signaling from cellular RNAs to be less sensitive and cooperative than signaling from long viral dsRNA. To test this, we measured the cell signaling activity of the WT, M854K, and ∆C12 variants with different expression levels of each variant. As expected, WT MDA5 had no significant signaling activity without poly(I:C) stimulation, regardless of MDA5 expression level, and high signaling with poly(I:C) stimulation. Signaling of WT with poly(I:C) increased slightly, but not proportionally with increasing MDA5 expression levels Fig. . With thWe also measured signaling activity in cells stimulated with linear 100-bp, 300-bp, and 1-kbp in vitro-transcribed dsRNA. The signaling activity of M854K MDA5 with each of these dsRNAs or with no stimuli was similar as when transfected with poly(I:C)  Alu(+):Alu(−) dsRNA hybrid from the NICN1 3′UTR known to support IRF3 dimerization in vitro in the context of ADAR1 deficiency or MDA5 gain-of-function mutations19. At the lowest M854K MDA5 protein concentration tested of 200 nM, we found that the dsRNA migrated primarily as a saturated complex with M854K MDA5 whereas with WT MDA5 the dsRNA migrated as a smeared band  dsRNAs present in the cytosol such as Alu:Alu hybrids. Our cell signaling data imply that, like other gain-of-function disease-associated MDA5 mutationsand Fig. . This inand Fig. . At highand Fig. . The binNegative-stain electron microscopy showed M854K MDA5 formed filaments on Alu(+):Alu(−) dsRNA and 100-bp dsRNA in the presence of ATP and failed to form filaments without dsRNA Fig. . WT MDA531 produced maps sufficiently detailed for atomic models to be built and refined (Table 12) and the helical twist of 75˚ is low (as defined previously14). With a helical rise of 44 Å, the asymmetric unit spans 14 bp of dsRNA with a protein–RNA interface area of 2168 Å2 similar to WT ATP-bound filaments. The M854K variant binds ATP in the same manner as WT MDA5, with a magnesium ion coordinating the β- and γ-phosphate groups. The most notable difference in the ATP-bound M854K structure is that the mutant Lys854 side chain forms a salt bridge with Glu813 in Hel2, and a hydrogen bond with Ser491 in Hel1, within the same MDA5 protomer , closed helicase motifs, and intermediate helical twists (87–88˚), like WT-ADP-AlF4-bound filaments14. However, the commonly detected structure, Class 167k, with 166,554 segments collected, contained only 14 bp of RNA per MDA5 molecule, the same number as ATP-bound filaments but one base pair less than WT-ADP-AlF4-bound filaments. In the less common structure, Class 62k , the footprint was 15 bp . We obtained two similarly populated structural classes, one with an intermediate helical twist (88˚) and the other with a high twist (92˚), with overall resolutions of 3.9 and 3.35 Å, respectively . However, we noticed that density in the map was weak for residues 810–827, to the extent that it was uninterpretable in the (higher-resolution) high-twist structure , may constrain motifs Vc-VI and hence reduce dissociation of dsRNA and nucleotide, in association with the hydrogen bond between Lys854 and Ser491 . The twist distribution was narrower than for ATP-bound WT MDA5 filament segments, of which 30% had an intermediate twist (81˚–91˚)   RNA duplexes. In the M854K variant, however, the salt bridge between Lys854 and Glu813, located in motif Vc, may constrain motifs Vc-VI, and hence prevent dissociation of certain endogenous dsRNAs and of ADP/Pi.The structure of ADP-bound WT MDA5 filaments completes our structural picture of the MDA5 ATPase cycle Fig. . In contThe structural data in this study provide insights into the role of ATP hydrolysis in RNA recognition by MDA5. In the ATP and transition states, MDA5 has a tight grip on the RNA, as evidenced by the larger protein-RNA interface, narrow twist distribution, and ordered motif Vc-VI structure in these two states. Upon ATP hydrolysis, a reduction in the RNA binding surface, broadening of the twist distribution, and disordering of motif Vc-VI cause MDA5 to relax its grip on the RNA. This in turn provides an opportunity for dissociation of RNAs with lower binding affinities, such as endogenous RNAs. In the M854K variant, polar contacts between the Lys854 sidechain and the Hel1 and Hel2 domains constrain some of the conformational changes necessary for ATP hydrolysis, including those required for RNA footprint expansion. This interferes with ATP-dependent RNA discrimination or proofreading and ultimately leads to constitutive signaling from endogenous RNAs.Our study uncovers the structural basis and functional consequences of the M854K disease-associated MDA5 variant. The M854K mutation inhibits ATP hydrolysis through an allosteric mechanism, setting this variant apart from previously studied ATPase-deficient variants, which directly target nucleotide binding or catalysis in the active site pocket. By completing our picture of the MDA5 ATPase cycle, our structural data also provide insights on the structural changes driven by ATP hydrolysis that allow MDA5 to distinguish between self and non-self RNA. A clearer understanding of how genetic variants dysregulate these processes will help identify new immunotherapeutic strategies.MDA5 (IFIH1) were cloned into the pET28a vector with an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site as described5. MDA5 residues 646–663, in the flexible L2 surface loop of Hel2i, were deleted for solubility, resulting in a 114-kDa polypeptide chain. The ΔL2 loop deletion does not affect the dsRNA binding, ATPase, or interferon signaling activities of MDA514. The M854K and other mutations were introduced into the MDA5-ΔL2 constructs by overlap PCR.Genes encoding mouse Escherichia coli BL21(DE3) cells were transformed with an MDA5 construct and grown to OD600 0.4–0.6 at 37 ˚C. After cold shock on ice water for 30 min, protein expression was induced with 0.4–0.5 mM isopropyl-b-d-1-thiogalactopyranoside (IPTG) overnight at 22 ˚C. Harvested cells were resuspended in 50 mM HEPES 7.5, 0.15 M NaCl, 5% Glycerol, 20 mM imidazole, EDTA-free Complete Protease Inhibitor (Roche), and 8 mM 2-mercaptoethanol (β-ME), then lysed by ultrasonication on ice. The lysate was spun at 15,000×g for 1 h. The supernatant was loaded onto Ni-NTA agarose (QIAGEN) and MDA5 was eluted with 50 mM HEPES 7.5, 0.15 M NaCl, 5% Glycerol, 0.3 M imidazole, and 8 mM β-ME. MDA5 in the eluate was further purified on a Resource Q anion exchange column (Cytiva) ; buffer B: 20 mM HEPES 7.5, 0.5 M NaCl, 2 mM DTT), and a Superdex 200 Increase 10/300 GL size-exclusion column (Cytiva) in 20 mM HEPES 7.5, 0.1 M KCl, 5 mM MgCl2, and 2 mM DTT. For the ATP-bound M854K MDA5 dataset, the protein was expressed as above except for the following differences. The protein was expressed in E. coli Rosetta2(DE3) cells overnight at 17 ˚C. Cells were lysed in 30 mM HEPES pH 7.7, 0.15 M NaCl, 5% glycerol, 1 mM Tris(2-carboxyethyl)phosphine (TCEP), EDTA-free Complete Protease Inhibitor (Roche), and benzonase (Merck). The size-exclusion chromatography buffer was 30 mM HEPES pH 7.8, 0.15 M KCl, 1 mM DTT. Purified proteins were flash-frozen and stored at −80˚, except for M854K MDA5 EM grid preparation, for which fresh size-exclusion column elution fractions were used.−1 of firefly luciferase under the control of the IFN-β promoter , 40 ng ml−1 of Renilla luciferase under a constitutive promoter , and 40 ng ml−1 of pLEXm vector35 containing either no insert, WT human MDA5-∆L2 (pLEXm MDA5-Δ644-663), or mutant human MDA5-∆L2. Transfections were performed with polyethylenimine (Sigma-Aldrich). After expression for 6 h, cells were transfected with poly(I:C) (Tocris Bioscience). After 24 h, cell lysates were prepared, and luciferase activity was measured using Promega assay kits according to the manufacturer’s instructions. Firefly luciferase activity was normalized against the co-transfected Renilla luciferase14. For cell signaling assays with short dsRNAs, 96-well plates containing 4 × 104 HEK293T cells were transfected with 80 ng pIFN-Luc, 8 ng pRL-TK, and 8 ng of pLEXm vector containing either no insert, human WT MDA5-∆L2, or human M854K MDA5-∆L2 using Lipofectamine™ 3000 Transfection Reagent (ThermoFisher). After 6 h incubation at 37 °C and 5% CO2, cells were transfected with 100 ng of 100, 300 bp, or 1 kb in vitro-transcribed dsRNAs or with poly(I:C) using Lipofectamine™ MessengerMAX™ Transfection Reagent (ThermoFisher). For Western blots, the FLAG tag on MDA5 was detected with an anti-FLAG antibody  or an anti-MDA5 antibody . For the actin control, we used anti-actin antibody AC-40 .The ADAR1-knockout (KO) HEK293T cell line was a kind gift from Charles Rice (The Rockefeller University). WT or ADAR1-KO HEK293T cells in 12-well plates were transfected with 400 ng mlNICN1 gene19. The Alu(+) and Alu(−) sequences were synthesized as separate gene fragments, which were used as templates to transcribe Alu(+) and Alu(−) in separate reactions. The 1-kb dsRNA contained the first 1 kb of the IFIH1 gene flanked by 5′-GGGAGA and TCTCCC-3′. All transcripts were treated with DNase and purified by column-based purification (ThermoFisher PureLink RNA Mini Kit). To produce dsRNA or Alu(+):Alu(−) hybrids, transcripts were heated to 95 ˚C for 5 min then annealed by cooling to room temperature over at least 1 h.RNAs were transcribed in vitro using T7 RNA Polymerase (New England Biolabs) following the manufacturer’s instructions. The Alu(+):Alu(−) hybrid used was the inverted repeat from Alu retrotransposon in 3′-UTR of the −1 RNA was incubated on ice with purified MDA5 protein at the concentrations indicated in Fig. 2) for 30 min. ATP was added to a final concentration of 6 mM and the solutions were incubated at 20 ˚C for 20 min. The reactions were quenched with native loading dye containing EDTA . Samples were loaded on a 0.7% agarose gel and run at 120 V in 45 mM (0.5×) Tris-borate buffer on ice for 40–60 min. The gels were post-stained for RNA with 1× SYBR Gold stain (ThermoFisher). Gels were imaged with a G-BOX gel imager (Syngene).A total of 5 ng μl2, 1 mM DTT, and incubated at 37 ˚C. Samples of the reaction were extracted and quenched with 20 mM EDTA at 20-s intervals. The concentration of inorganic phosphate released by hydrolysis of ATP was measured by tracking absorbance at 620 nm of malachite green binding to phosphate ions using the ATPase/GTPase activity colorimetric assay (Sigma-Aldrich) as described in ref. 14. For the ATPase rescue assays ], and imaged with a 120 kV Technai 12 electron microscope (ThermoFisher). Images were taken at −2 to −4 µm defocus, 26,000× magnification, and with 4 Å per pixel.For the filament assembly assay, 26 µg ml−1 purified MDA5 protein was incubated with 0.05 g l−1 1-kb dsRNA in Buffer F  on ice for 2–3 min. For an optimal trade-off between filament quality and nucleotide-binding, 2 mM ADP or 1 mM ADP-AlF4  was incubated with preformed MDA5-dsRNA filaments for 6–7 min (ADP dataset) or 30 min (ADP-AlF4 dataset). Samples were diluted twofold with Buffer F and 3.5 µl of the sample was immediately applied onto a glow-discharged 300-mesh gold Quantifoil R1.2/1.3 grid (Quantifoil Micro Tools). Grids were blotted for 2–4 s and plunge-frozen in liquid ethane cooled by liquid nitrogen with a Vitrobot MarkIV (ThermoFisher) at 4 ˚C and 100% humidity. For ATP-bound MDA5 filaments, the sample was prepared as above except the protein was incubated with the dsRNA and 10 mM ATP in Buffer F2  on ice for 2–3 min. Samples were diluted twofold with Buffer F2 and applied to 300-mesh gold UltrAuFoil R1.2/1.3 grids, freshly glow-discharged for 60 s at 30 mA with an Edwards 12E6/531 glow discharger.A total of 1 g l−1) with a 12 s exposure time. Data were acquired with SerialEM with four shots per hole. ADP-AlF4-bound M854K MDA5 filaments were imaged at the Electron Bio-Imaging Centre  on a Titan Krios microscope with a Falcon3 detector in counting mode (ThermoFisher). Movies were recorded at a magnification of 75,000 with a 60 s exposure time. Data were acquired with EPU (ThermoFisher) with two shots per hole. ATP-bound M854K MDA5 filaments were imaged at the MRC-LMB on a Titan Krios microscope with a K3 detector in counting mode (ThermoFisher). Movies were recorded with a 1.4 s exposure time. Data were acquired with EPU with two shots per hole. See Table CryoEM imaging of ADP-bound WT MDA5-dsRNA filaments was performed at the CryoEM Service Platform at EMBL Heidelberg. Data were collected on a 300 kV Titan Krios microscope (ThermoFisher) with a K2 detector in counting mode. Movies were recorded at a magnification of 130,000  as the initial model. 1,068,108 auto-picked segments were subjected to three rounds of 2D classification yielding 383,128 particles. Three independent 3D classifications of ATP-bound filament segments did not show any significant differences in a twist and helical rise distribution of the selected segments. The ATP-bound segments were 3D-refined, CTF-refined, Bayesian-polished, and post-processed following the workflow shown in Supplementary Fig. The ATP-bound M854K MDA5 dataset was processed in the same way as the ADP-bound dataset except that dose-weighted images were used for CTF estimation. A reference-free two-dimensional (2D) class was generated from manual picking as a template for auto-picking. Helical reconstruction was performed using the ATP-bound WT MDA5-dsRNA structure  refinement options were selected in PHENIX. Secondary structure restraints, non-crystallographic symmetry restraints between the protein subunits, sidechain rotamer restraints, and Ramachandran restraints were used in real space refinement42.Previously reported cryoEM structures of MDA5-dsRNA filaments12) using the secondary structure elements of Hel1 as the reference. The conformational state of the helicase domain was defined based on the rotation angles relating the Hel2 domains of the aligned structures as follows: closed state, 0˚–3˚ angle; semi-closed state, 7˚–13˚ angle12.To determine which conformational state the helicase modules were in, each model was superimposed onto the fully closed structure of LGP2 (PDB:5JAJNo statistical methods were used to predetermine sample size, experiments were not randomized, and the investigators were not blinded to experimental outcomes. Luciferase-reporter cell signaling data are represented as the mean ± standard error of the mean of three replicates conducted in a single independent experiment. Data were representative of at least three independent experiments. Scatter plots, histograms, and error bars were plotted with GraphPad Prism 9.2.0 and Microsoft Excel v.16.52.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Background: Genetic profiling of resected tumor or biopsy samples is increasingly used for cancer diagnosis and therapy selection for thyroid and other cancer types. Although mutations occur in cell DNA and are typically detected using DNA sequencing, recent attempts focused on detecting pathogenic variants from RNA. The aim of this study was to determine the completeness of capturing mutations using RNA sequencing (RNA-Seq) in thyroid tissue and fine-needle aspiration (FNA) samples.Methods: To compare the detection rate of mutations between DNA sequencing and RNA-Seq, 35 tissue samples were analyzed in parallel by whole-exome DNA sequencing (WES) and whole-transcriptome RNA-Seq at two study sites. Then, DNA and RNA from 44 thyroid FNA samples and 47 tissue samples were studied using both targeted DNA sequencing and RNA-Seq.Results: Of 162 genetic variants identified by WES of DNA in 35 tissue samples, 77 (48%) were captured by RNA-Seq, with a detection rate of 49% at site 1 and 46% at site 2 and no difference between thyroid and nonthyroid samples. Targeted DNA sequencing of 91 thyroid tissue and FNA samples detected 118 pathogenic variants, of which 57 (48%) were identified by RNA-Seq. For DNA variants present at >10% allelic frequency (AF), the detection rate of RNA-Seq was 62%, and for those at low (5–10%) AF, the detection rate of RNA-Seq was 7% (p < 0.0001). For common oncogenes (BRAF and RAS), 94% of mutations present at >10% AF and 11% of mutations present at 5–10% AF were captured by RNA-Seq. As expected, none of TERT promoter mutations were identified by RNA-Seq. The rate of mutation detection by RNA-Seq was lower in FNA samples than in tissue samples .Conclusions: In this study, RNA-Seq analysis detected only 46–49% of pathogenic variants identifiable by sequencing of tumor DNA. Detection of mutations by RNA-Seq was more successful for mutations present at a high allelic frequency. Mutations were more often missed by RNA-Seq when present at low frequency or when tested on FNA samples. All TERT mutations were missed by RNA-Seq. These data suggest that RNA-Seq does not detect a significant proportion of clinically relevant mutations and should be used with caution in clinical practice for detecting DNA mutations. TERT promoter mutations, have emerged as important prognostic markers that define the most aggressive class of thyroid cancers  and the University of Pittsburgh Medical Center, Pittsburgh, PA  with the approval by respective institutional review boards. In addition, DNA and RNA from 44 thyroid FNA samples and 47 thyroid tissues were studied by targeted DNA sequencing (112-gene panel) and RNA-Seq at the University of Pittsburgh Medical Center. The samples were pre-selected to represent all mutations most commonly occurring in thyroid cancer and mutations present at various levels including low level. The summary of the study design is given in De-identified DNA and RNA samples isolated from 35 randomly selected tissue specimens were studied using whole-exome DNA sequencing (WES) and whole-transcriptome (RNA-Seq) analysis at two study sites, the Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY  and FNA samples  was calculated  package with R (v3.3.2) and ggplot2 (2.2.1). Comparison between the detection rate of DNA variants by different sequencing approaches was performed using lculated . To complculated .p < 0.0001). There was no difference in the detection rate of variants by RNA-Seq between the two study sites (p = 0.89).To evaluate the rate of detection of cancer-related genetic variants that occur in DNA using tumor RNA, 35 tumor tissue samples were analyzed using whole-exome DNA sequencing and whole-transcriptome RNA-Seq at two participating study sites. Each study site performed detection of variants independently using their own clinical sequencing protocols and bioinformatics pipelines. Only cancer-related genetic variants were used for comparison between sequencing of DNA and RNA at each site. The results of the analysis are summarized in Next, we determined the rate of detection of pathogenic genetic variants, that is, mutations, by RNA-Seq compared with targeted DNA sequencing (ThyroSeq v3 GC) in thyroid samples. Forty-seven tissue samples and 44 thyroid FNA samples were used. Among the FNA samples, 21 were diagnosed as Bethesda III, 8 as Bethesda IV, 13 as Bethesda V, and 2 as Bethesda VI. Overall, targeted DNA sequencing detected 118 gene mutations in 91 thyroid samples , of whicBRAF (n = 27) and RAS (n = 25), of which RNA-Seq detected 13 (48%) of BRAF and 19 (75%) of RAS mutations  were detected using RNA-Seq. If TERT promoter mutations were excluded, the overall detection rate of expressed DNA variants by RNA-Seq would increase from 48% to 68%.We further analyzed the sequencing data to examine whether the rate of detection of mutations by RNA-Seq varies between different mutations. The most common mutations detected by targeted DNA sequencing in thyroid samples were utations . For tumfor TSHR . FinallyNext, we determined whether the rate of detection of mutations in these samples varied for mutations present at different allelic frequencies. Allelic frequency of a mutation is calculated as a percentage of sequencing reads containing the mutation divided by total number of reads covering the locus. It allows estimating the proportion of clonal tumor cells to all (neoplastic and nonneoplastic) cells collected from each sample . This is important for clinical samples that may contain low proportion of cancer cells, such as thyroid FNA samples. This is because thyroid tumors not infrequently have significant infiltration by lymphocytes and other inflammatory cells, stromal fibroblasts, or other nonneoplastic cells that “dilute” tumor cells collected by FNA.p < 0.0001). These detection rates without TERT promoter mutation in these data sets will be 78% and 8% for “high” and “low” AF, respectively. Among the most common mutations that affect BRAF and RAS oncogenes, 94% of those present at a high (>10%) allelic frequency and 11% of those present at a low (5–10%) allelic frequency were captured by RNA-Seq. None of the TP53, PIK3CA, and EIF1AX mutations present at low allelic frequency were detected using the RNA-Seq. None of the TERT mutations were captured by RNA-Seq regardless of allelic frequency. These results are summarized in In our group of samples, among 118 DNA mutations detected by targeted DNA sequencing, 89 had allelic frequency of >10% , which was designated as “high.” The remaining 29 variants had a “low” allelic frequency of 5–10% . When the rate of mutations detected by RNA-Seq was examined separately in different allelic frequency groups, we observed that 62% of high allelic frequency mutations and only 7% of low allelic frequency mutations were captured using RNA , and it remained significant after TERT mutations were excluded . The difference in the detection rate was due to more common presence of variants with low allelic frequency in the FNA samples . Indeed, among mutations present in the FNA samples at an allelic frequency of >10%, the detection rate by RNA-Seq was 80% for BRAF, 50% for RAS, and 58% for other mutations. For mutations with a 5–10% allelic frequency, the detection rate by RNA-Seq dropped to 7% for BRAF and RAS, whereas TP53 and EIF1AX mutations were not detected  were detected in expressed RNA . The detdetected .Finally, we explored the accuracy of detection of allelic frequency of gene mutations in the RNA-Seq data. Ninety-one thyroid tissue and FNA samples were used to compare the mutation allelic frequency detectable using the RNA-Seq data with those calculated using targeted DNA sequencing. Only variants that were detected by both methods were included in the comparison. Overall, correlation between the two methods was poor and showed a RMS error of 0.26 . These rThis study assessed the completeness of detecting genetic variants using RNA-Seq and revealed that only 46–49% of pathogenic variants detectable by DNA sequencing could be captured by RNA-Seq. There was no difference in the detection rate of such variants in nonthyroid and thyroid samples and when different sequencing and detection pipelines were used at the two participating study sites.et al., RNA-Seq identified 40–48% of all coding variants detected by whole-genome sequencing of DNA , confers a very high probability of cancer. Missing low-level V600EBRAF mutation in the FNA sample would decrease diagnostic accuracy of RNA-Seq for nodules with only partial cancer sampling or those with extensive infiltration by lymphocytes or other inflammatory cells, which would more often yield FNA samples containing a small proportion of cells carrying a given mutation.Yet another factor that may affect the detection of heterozygous mutations by RNA-Seq is related to expression balance and specific situations when the normal allelic of the gene is more highly expressed than the mutant allelic ,34. Deviy of >5% . Detectiet al.  of mutations were missed when using RNA-Seq (The difference in the overall detection rate of genetic variants in thyroid samples by RNA-Seq between this study and one reported by Angell et al.  (48% vs. RNA-Seq .Similar or higher rates of genetic variants missed by RNA-Seq can be expected in thyroid FNA samples compared with the tissue samples. This is because FNA samples frequently contain a significant proportion of nonneoplastic cells, including inflammatory and stromal cells, in addition to normal thyroid cells adjacent to the tumor nodule. In the tissue samples, most of these nonneoplastic cells can be excluded before molecular analysis by selecting the most cellular tumor areas using microscopic guidance. The higher fraction of normal cells in FNA samples would lead to lowering the mutant allelic frequency, which, as discussed earlier, decreases the chance for mutations to be detected by RNA-Seq.Finally, the findings of this study suggest that allelic frequency of mutations cannot be reliably calculated from the RNA-Seq data. This is not surprising in light of the known variability in expression levels of the normal and mutant allelics. Knowing the allelic frequency of mutations may help to provide a more specific cancer probability assessment in the tested nodules , althougTERT promoter mutations are missed by sequencing of RNA isolated from thyroid samples, similar to other tissue types. This should be taken into account when RNA-Seq is used in clinical practice for diagnosis, prognostication, and selection of targeted therapies for thyroid and other cancer types.In summary, the results of this study, as well as previous reports, indicate that a significant proportion of coding mutations and all noncoding


Presence of iron and eIF4F in the depleted WGE significantly enhanced translation for both IRE RNAs. This suggests that iron promotes translation by enhancing the binding rates of the eIF4F∙IRE RNA complex. eIF4F·IRE RNA binding is temperature-dependent; raising the temperature from 5 to 25°C, enhanced the binding rates of eIF4F·FRT IRE (4-fold) and eIF4F·ACO2 IRE (5-fold). Presence of Fe2+ caused reduction in the activation energy for the binding of FRT IRE and ACO2 IRE to eIF4F, suggesting a more stable platform for initiating protein synthesis. In the presence of iron, lowered energy barrier has leads to the faster association rate and slower rate of dissociation for the protein-RNA complex, thus favoring efficient protein synthesis. Our results correlate well with the observed translational efficiency of IRE RNA, thereby suggesting that the presence of iron leads to a rapid, favorable, and stable complex formation that directs regulatory system to respond efficiently to cellular iron levels.Interaction of iron responsive elements (IRE) mRNA with the translational machinery is an early step critical in the initiation of protein synthesis. To investigate the binding specificity of IRE mRNA for eIF4F, kinetic rates for the eIF4F·IRE RNA interactions were determined and correlated with the translational efficiency. The observed rate of eIF4F·FRT IRE RNA interactions was 2-fold greater as compared to eIF4F·ACO2 IRE RNA binding. Addition of iron enhanced the association rates and lowered the dissociation rates for the eIF4F binding to both IRE RNAs, with having higher preferential binding to the FRT IRE RNA. The binding rates of both eIF4F·IRE RNA complexes correlated with the enhancement of protein synthesis  Deletion of the 30-nt IRE leads to the removal of the IRP modulation, and represses IRP independent rate of protein synthesis  is the universal gas constant, T (Kelvin) is the absolute temperature, and A is the Arrhenius pre-exponential factor. The value of Ea was calculated from the slope of the fitted linear plot of lnk versus T-1.To assess the activation energy of the protein-RNA complexes with and without addition of iron, the temperature-dependent observed rate constants  buffer in the cell; dissociation was monitored as the fluorescence signal increase. The increasing signal in the dissociation portion of fluorescence data was fit to the single-exponential function using Kaleida Graph software (version 2.1.3) as described previously  and eIF4F∙ACO2 IRE RNA [(23.0 ± 0.8) x 10−3 s-1] complexes. These results indicate that iron can selectively decrease the dissociation rates of both IRE RNA∙eIF4F complexes and increase binding rates. This suggests that eIF4F·IRE RNA complex forms more rapidly and dissociates slower in the presence of iron.To further understand the kinetic mechanism of this reaction, the effect of iron on the dissociation rates . Addition of iron, lowered the activation energies for eIF4F·FRT IRE and eIF4F·ACO2 IRE complexes. Diminished temperature suggests that not only the IRE RNAs compete in binding to the eIF4F protein in the presence of iron, but also provides a path with a substantially lower energy barrier. Addition of iron to the eIF4F·FRT IRE or eIF4F·ACO2 IRE complexes decreased activation energies, suggesting that iron promotes conformational changes within the eIF4F∙IRE RNA complex. Previously [in vitro translation of FRT and ACO2 IRE mRNA in WG extracts and depleted WG extracts supplemented with eIF4F. These results indicate that addition of exogenous eIF4F and iron restores translation in depleted WG extracts, confirming the importance of iron for eIF4F binding to IRE RNA; this interaction favors positively the eIF4F-dependent protein synthesis. Iron-stabilized binding and more rapid formation of the eIF4F∙IRE RNA complex leads to an enhanced in vitro protein synthesis of ferritin and mitochondrial aconitase IRE RNA, demonstrating biological importance of this binding during cellular environment changes. These results point to the significance of iron on the eIF4F·IRE RNA complex formation and reflect a kinetic advantage over IRP∙IRE RNA complex.The Arrhenius activation energies for the binding of both types of IRE RNAs with eIF4F protein showed a significant difference (eviously , 48, we eviously . These ceviously  that eIFeviously . Here wein vitro iron-induced protein synthesis for IRE RNAs and the differences in their binding [Structural differences between ferritin and mitochondrial IRE RNAs are reflected in the stability and kinetic differences of the protein/IRE RNA complexes observed here, since eIF4F protein is present in both complexes and only the IRE RNA is differs. IRE RNA structural differences then explain, at least in part, for the significant difference that occur during an  binding . We foun


Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA–RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention.Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1–S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the  Genome packaging is an essential process in the virus life cycle, as at least one genome (or one set of RNAs of a multi-segmented genome) must be incorporated into the limited space available in the capsid. Two strategies are used by viruses for genome packaging; one is the assembly/recruitment of the genome prior to capsid formation, and the other is the formation of empty protein capsids into which the genome is packaged. The latter strategy is particularly notable in the double-stranded DNA (dsDNA) viruses, such as tailed bacteriophages and herpesviruses, and the double-stranded RNA (dsRNA) viruses  ,2,3. ForOrthomyxoviridae (influenza), Phenuiviridae , Arenaviridae , Birnaviridae , Hantavirus, Nairovirus, Herbevirus, and Reoviridae , the virus not only needs to selectively package viral RNAs but must also ensure that at least one complete set of genomic segments is recruited within each capsid. With viruses where the genomic segment number is low, for example the two-segmented (Birnaviridae) or three-segmented (Phenuiviridae) genome viruses, the packaging of segments appears to be stochastic with a significant portion of virus particles containing an incomplete genome complement . Th. Th26]. To substantiate this model further, a second in vitro ssRNA interaction assay based on a biotinylated primer-coated streptavidin bead was developed, which did not require the presence of any viral protein a. Using These data suggest that the genome segments may be pre-assembled prior to being packaged into the capsid and that this assembly is likely based on a network of segments, which is initiated by the smaller segments. trans-acting RNA–RNA interactions have also been found to play an important role in genome packaging . Mo. Mo32]. (S6–S10) . The com(S6–S10) . Both in(S6–S10) . This inThese proof-of-principle data demonstrate a new concept for how multipartite genomic RNA segments might be combined prior to, or during, genome packaging. Thus, there is a unique opportunity to explore exactly how such RNA networks are formed and what role they play in genome recruitment into nascent assembling capsids in other Reoviruses such as Rotavirus.Although the structural conformation of each RNA segment and RNA–RNA interactions between segments play pivotal roles in regulating BTV genome sorting and packaging, viral proteins may also be involved in this process. Our current understanding is that the 10 positive-sense ssRNA segments of BTV first associate with the TC prior to encapsidation by the VP3 layer of 10 decamers, resulting in the subcore particle. Of these four proteins, VP6 and VP3 appear to play key roles in genome recruitment and localization in the assembling capsid.Orbivirus genus in the Reoviridae. This internal core protein has a strong affinity for both ssRNA and dsRNA molecules, suggesting that it is closely associated with the viral genome . WhCryo-EM and asymmetric reconstructions of the BTV vertex have allowed obtaining an atomic model of the large polymerase protein VP1, but not VP6, suggesting that it may be either too flexible to obtain a location or masked by genomic RNA . HoweverVarious in vitro assays have demonstrated that BTV non-structural proteins are not directly involved in RNA sorting, recruiting, or packaging. However, the phosphorylated non-structural protein 2 (NS2) of Orbiviruses has a high affinity for viral ssRNA over host RNAs, and phosphorylation is not necessary for the RNA-binding properties ,40. NS2 We have summarized how multipartite genomic ssRNA segments are combined prior to, or during, packaging using an experimental model virus, BTV, with direct implications for related medically important viruses  and many other complex dsRNA viruses that infect human, animals, and plants. An extensive background knowledge coupled with multidisciplinary approaches, including reverse genetics and a cell-free RNA packaging assay, have facilitated our understanding of the process. It includes the dynamic nature of the segmented RNA genome, the importance of the UTRs for driving the conformations of each RNA necessary for the next level of interaction, how exactly such RNA networks are formed, how they are recruited into nascent assembling capsids, and what capsid proteins might be involved. However, certain key questions are still not clear. For example, although it is evident that RNA–RNA interactions are based on complementary sequences, the precise sorting signals required for BTV RNA packaging have not been identified. Nevertheless, the current studies have uncovered a completely new concept of packaging in which a reduced number of RNA segment complexes is packaged rather than each segment individually. Such fundamental mechanisms of RNA network formation by multiple segmented genomes offer new possibilities for future antiviral design which block this essential and conserved process for BTV and related pathogenic viruses of humans and animals or for designing defective interfering particles containing an incomplete set of genomic segments that may act as effective vaccines.


To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17gag, integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF “family” clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 110 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina ( One of the most distinctive features of HIV-1 evolution is its high recombinogenic potential, possibly the greatest among human pathogens, which is reflected in the high frequency of unique recombinant forms (URFs), each generated in a dually- or multiply-infected individual, found wherever different genetic forms circulate in the same population . Some ofPlasma samples from HIV-1-infected individuals were collected in 14 Spanish regions for antiretroviral drug resistance tests and for a molecular epidemiological study. The study was approved by the Committee of Research Ethics of Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. The study did not require written informed consent by the study participants, as it used samples and data collected as part of routine clinical practice and patients’ data were anonymized without retaining data allowing individual identification.pol fragment in protease-reverse transcriptase (Pr-RT) was amplified from plasma RNA by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) followed by nested PCR as described previously .Sequences were aligned with MAFFT v7 . InitialMosaic structures were analyzed by bootscanning  with Simnd, and 3rd) (–4 – 2 x 10–2 subs/site/year) was used for the substitution rate. We also specified an uncorrelated lognormal relaxed clock and a Bayesian SkyGrid coalescent tree prior (The time and the location of the most recent common ancestor (MRCA) of the identified CRF was estimated using Pr-RT sequences with the Bayesian Markov chain Monte Carlo (MCMC) coalescent method implemented in BEAST v1.10.4 , using aand 3rd) . A unifoee prior . In the n = 4), Spain (n = 3), Paraguay (n = 1), Brazil (n = 1), and Italy (n = 1) (n = 4), Argentina, and Equatorial Guinea] and all 3 other database sequences from samples collected in Spain were from Latin Americans, one each from Paraguay, Argentina, and an unspecified country. Transmission was predominantly heterosexual, but 7 were men who have sex with men (MSM), the sequences of 5 of whom branched in a subcluster  . Pr-RT s (n = 1) . Epidemibcluster .gag, integrase, gp120, gp41-rev overlap, and nef  of total infections, 48.3% (14 of 29) of non-subtype B infections, and 60.1% (14 of 23) of F1/BF1 infections.At the Los Alamos HIV Sequence Database, there are HIV-1 sequences from samples collected in Paraguay from only 23 individuals, of which 12 are NFLG sequences from samples collected in 2002 or 2003 and 11 are env V3 region sequences from samples with unknown collection years. In phylogenetic analyses, 2 of 12 NFLG and 1 of 11 V3 sequences  branched in the clade formed by CRF66_BF viruses . Howeverr2 = 0.5871). In the BEAST analysis, for the sequences corresponding to South American individuals residing in Spain, the assigned location trait was their country of origin, rather than their place of residence. This was done because most individuals harboring CRF66_BF identified in Spain were of South American origin (mostly from Paraguay) and because we found no definitive evidence of the local circulation of CRF66_BF in Spain, as reflected in clusters mainly comprising Spanish individuals. Therefore, we assumed that South Americans harboring CRF66_BF viruses had acquired HIV-1 in their countries of origin. In this analysis, the substitution rate was estimated at 1.987 x 10–3 subs/site/year  and the time of the MRCA of CRF66_BF was estimated around 1984 , with its most probable location in Paraguay (PP = 0.76), with Argentina in second place (PP = 0.22)  . Conside = 0.22) .th reported CRF derived from subtypes B and F. Samples harboring CRF66_BF were collected in 5 countries, in South America  and Western Europe , with a majority collected in Spain. However, of samples collected in Spain, a great majority were from Paraguayan individuals. Bayesian coalescent analyses , pointed to a most probable origin of CRF66_BF in Paraguay (PP = 0.76), with Argentina being the second most probable location (PP = 0.22). When the analysis was performed assigning the most recently diagnosed samples of clusters found in a single Spanish city to Spain as the location trait, irrespective of the country of origin of the individual, the PPs for a MRCA in Paraguay or Argentina were not very different . Therefore, the results point to a South American origin of CRF66_BF, either in Paraguay or Argentina, without a definitive support for either country. However, given the great predominance of Paraguayans among CRF66_BF-infected individuals living in Spain, we cannot rule out that the same could happen in Argentina, where Paraguayans represent the largest immigrant national group   and is sAmong HIV-1-infected Paraguayans residing in Spain studied by us, there was relatively high prevalence (21%) of CRF66_BF infections, which suggests that CRF66_BF could be one of the major HIV-1 genetic forms circulating in Paraguay. A better knowledge of the current prevalence of CRF66_BF in Paraguay would require sequencing a representative sample of recent HIV-1 diagnoses in the country. However, HIV-1 sequences from only 23 patients sampled in Paraguay are available at the Los Alamos HIV Sequence database, and the most recent molecular epidemiological study published to date involves the analysis of sequences from 55 samples collected 18 to 19 years ago , which aNotably, CRF66_BF, unlike all other non-Brazilian CRF_BFs identified to date in South America , is unrelated to CRF12_BF, as deduced from the lack of breakpoint coincidence and of phylogenetic clustering with CRF12_BF. This implies that CRF66_BF originated independently from viruses of the CRF12_BF family, with a presumable ancestry in Brazil, where B and F1 viruses are circulating .2), as well as on the diffusion of South American HIV-1 variants in Europe.CRF66_BF is the 5th CRF of South American ancestry originally identified in Western Europe [after CRF42_BF , CRF47_BIt is interesting to note that although transmission of CRF66_BF is predominantly via heterosexual contact, most individuals in a cluster are MSM , which sOne of the essential tasks of Biology is naming and classifying organisms. In this work, we have accomplished this task by identifying a new HIV-1 circulating recombinant form, derived from subtypes B and F1, named CRF66_BF. CRF66_BF most likely originated in South America, either in Paraguay or Argentina, and, unlike all non-Brazilian South American CRFs identified to date, is unrelated to CRF12_BF. The identification and genetic characterization of HIV-1 variants is the first and necessary step for molecular epidemiological studies examining their geographic dissemination, growth dynamics, and epidemiological associations, as well as for analyzing their biological properties, such as pathogenic and transmissibility potentials, response to antiretroviral therapies, and susceptibility to immune responses inducible by vaccines. Such studies on CRF66_BF and other South American CRFs may be the subject of future work.The names of the repository and accession numbers can be found in the article (Materials and Methods and The studies involving human participants were reviewed and approved by Committe of Research Ethics, Instituto de Salud Carlos III, Madrid, Spain. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MT and ED conceived the study and supervised the experimental work. JB, MT, and ED processed sequences and performed phylogenetic analyses. MT performed phylodynamic analyses. HG performed data curation. JB, SB, MM-L, VM, MS, EG-B, and JEC performed experimental work. MN-T, JM, MZ-S, EU, JR, CR, IR-A, LE-O, JP, JG-C, AO, and JJC obtained samples and epidemiological data from patients. MT, ED, and HG wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.