Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement. RNA silencing is a revolutionary innate immunity mechanism in eukaryotes that has greatly expanded our knowledge of gene expression and regulation in plants. RNA interference (RNAi) is an important regulatory mechanism that has become an invaluable tool for plant research, especially in terms of understanding the effects of gene regulation in response to abiotic and biotic stress. RNAi has enabled researchers to gain insight into gene function, pest resistance, and physiological processes in plants. Although RNA is known to play critical roles in biology, the extensive capabilities and complexity of this nucleic acid remained elusive and not fully understood. The intrinsic nature of the relatively labile, often single-stranded RNA molecule, and limited availability of RNA-dependent enzymes had slowed characterization and progress. Traditional research focused on messenger transcript (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), but the universality of the molecule to life remained underestimated . The occSolemoviridae, one of the most devastating group of viruses worldwide for many important crops [Solemoviridae has been especially challenging as they are phloem-limited and transmitted in a persistent circulative manner by specific aphids.Plant viruses can cause major diseases in crops worldwide. Management has relied on a combination of strategies involving virus eradication and transmission prevention. Early studies reported the occurrence of cross-protection in plants where infection with a mild strain of a virus protected against infection by more pathogenic strains . A majornt crops ,5. MolecPolerovirus coat protein antisense RNA of the Potato leafroll virus produced similarly high levels of reduced virus titre and disease resistance as the corresponding sense mRNA [Myzus persicae was reduced and disease symptoms in foliage and tubers were eliminated. This showed that RNA was capable of conferring resistance as a trigger molecule and was subsequently observed in other plant virus groups [Remarkably, transformation of plants with the genus nse mRNA ,6. The rs groups . Replicas groups . The disExperiments to transiently or stably increase endogenous gene expression often unexpectedly produced a decrease in mRNA. For example, attempts to overexpress chalcone synthase (CHS) in pigmented petunia petals blocked anthocyanin biosynthesis . DevelopCaenorhabditis elegans resulted in the disappearance of the targeted message [unc-22 dsRNA per cell supporting a role as a trigger molecule. The RNAi mechanism is a naturally occurring process in most eukaryotes, conferring an ability of dsRNA to induce a sequence-specific systemic silencing process [RNA interference (RNAi) involves a sequence-specific suppression of gene expression by transcriptional or translational repression. The results of the RNAi characterization demonstrated that feeding or injecting gene-specific dsRNA into  message . Silenci process ,9,10,13.Exogenous dsRNA initiates RNAi by activating the ribonuclease Dicer enzymes that bind and cleave dsRNA into 21–24-base-pair small interfering RNA (siRNA) fragments with 3′ overhangs of 2–3 nucleotides . Dicer pThe occurrence of double-stranded RNA during viral RNA replication and hairpin RNA secondary structures regulating gene expression, indicated that ssRNA viruses have an inherent protective mechanism from RNAi ,15. SileStably or transiently expressed genes and nucleic acids in genetically engineered plants is often utilized in the study of gene function or the heterologous production of commercially valuable products. The use of full-length infectious clones (FLICs) of RNA viruses has facilitated the amplification of targeted genes, providing a convenient vector platform that can circumvent RNAi for site-directed mutations and increase or reduce gene expression to characterize PTGS and produce valuable heterologous commercial products. Application of virus-induced gene silencing (VIGS) has successfully utilized several RNA virus vectors including Tobacco rattle virus (TRV), Potato virus Y (PVY), TMV, and PLRV ,19,20,21Pseudomonas syringae, Cladosporium fulvum, and Verticillium species [Innate immunity is more complex than originally envisioned and the RNAi regulatory mechanism is independent of other recognition and signalling pathways. Identification of genes for gene receptors and avirulence proteins has advanced our understanding of cellular resistance to a wide range of pathogens, including  species ,23,24,25 species ,27. Cros species ,29. TogeExogenously introduced dsRNA to target plant pests began with the introduction of dsRNAs through microinjections ,31. MicrZea mays (corn) called SmartStax Pro that was created to target corn rootworm (Diabrotica vwirgifera virgifera) that was approved for commercial use by the U.S. Environmental Protection Agency, the U.S. Food and Drug Administration, and the U.S. Department of Agriculture [Investigators have created transgenic plants that express desired dsRNAs to cause RNAi-induced gene silencing in the target organism when it ingests plant material, referred to as Host-Induced Gene Silencing (HIGS) ,33. One iculture ,33,34. Ciculture . A similiculture .Saccharomyces cerevisiae (yeast) containing dsRNA targeting spotted wing fruit fly Drosophila suzukii [D. suzukii consumes, and therefore was seen as a viable vector to induce oral ingestion of the dsRNA. They had success and found that locomotor activity, survivorship, and reproductive fitness were all negatively impacted by the complimentary dsRNA [Microorganisms transformed to contain target dsRNA have also been evaluated as a method for exogenous application. One notable example showed that bacteria transformed to contain the target dsRNA could be fed to insects to induce RNAi . These g suzukii . This tyry dsRNA . AcceptaApplication of RNAi as a foliar spray has become of particular interest, specifically because it has promise in being turned into a viable and specific biopesticide that would be available for commercial use. This technology is referred to as Spray-Induced Gene Silencing (SIGS), and it is a method that allows non-transformative control of plant pests and pathogens ,35. ThisPublished exogenously applied RNA studies report efficacies of up to 100% with over 30 days of activity and were influenced by many parameters including the genetic target, RNA size, and environment . With alRNA silencing is an essential component of innate immunity and gene regulation in plants and a rapidly growing number of tools are available for applications. Efficacy and survivability of the dsRNA is being actively explored to meet specific environmental and industry requirements. Exogenous RNA survivability and uptake may be improved using polymeric, lipid-based, and inorganic nanoparticles . Advance


R-loops are inherent byproducts of transcription consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. These structures are of key importance in controlling numerous physiological processes and their homeostasis is tightly controlled by the activities of several enzymes deputed to process R-loops and prevent their unproper accumulation. Senataxin (SETX) is an RNA/DNA helicase which catalyzes the unwinding of RNA:DNA hybrid portion of the R-loops, promoting thus their resolution. The key importance of SETX in R-loops homeostasis and its relevance with pathophysiological events is highlighted by the evidence that gain or loss of function SETX mutations underlie the pathogenesis of two distinct neurological disorders. Here, we aim to describe the potential impact of SETX on tumor onset and progression, trying to emphasize how dysregulation of this enzyme observed in human tumors might impact tumorigenesis. To this aim, we will describe the functional relevance of SETX in regulating gene expression, genome integrity, and inflammation response and discuss how cancer-associated SETX mutations might affect these pathways, contributing thus to tumor development. Senataxin (SETX) is an RNA/DNA helicase which catalyzes the resolution of R- loops, inherent byproducts of transcription consisting of an RNA:DNA hybrid and a displaced single-stranded DNA.Gain or loss of function mutations of SETX gene underlies the pathogenesis of two distinct neurological disorders: AOA2 (ataxia with oculomotor apraxia - type 2) and ALS4 , respectively.SETX physiological functions ranging from gene expression regulation to genome integrity maintenance and inflammation response.SETX dysregulation has been observed in human tumors and several evidence pointed towards a tumor suppressive role of this helicase.Are SETX defects directly involved in tumor onset and progression?Which specific SETX functions underlie its potential tumor suppressive role?Is it possible that SETX gain of function alterations might drive tumor development, similar to what is observed in neurological context?loci, including either coding or noncoding sequences, and their accumulation/stabilization is promoted at specific DNA regions, typically enriched with purine-rich sequences, or during replication-transcription collisions [Cells have evolved an intricate network of signaling activated in response to DNA damage that ensures that DNA lesions might not be converted into heritable mutations. Paradoxically, the major sources of mutagenic events are represented by physiological processes involving DNA and RNA metabolism. During DNA replication or gene transcription DNA undergoes a series of changes which may generate structural intermediates which can facilitate DNA mutations. An example of such structural intermediates are R- loops, RNA:DNA hybrids generated during transcription when the non-coding DNA strand is displaced as the transcribed DNA strand forms a hybrid with the nascent pre-mRNA , 2. Thesllisions .R-loops formation can impact numerous biological processes, such as antibodies diversification in B cells, DNA damage response and gene expression at multiple levels, including regulation of the chromatin architecture at the promoter region, transcription elongation, and termination –10. In hAlthough physiologically relevant for many biological processes, the persistence of the R-loops might be deleterious for cell viability –21. TherSETX is one of the best-characterized R-loops resolving helicase enzyme. The human SETX gene is localized on chromosome 9 and includes 33 exons codifying a 302-kDa protein. SETX protein presents key protein domains: an N-terminal protein interaction domain, the helicase domain, and a C-terminal Nuclear Localization Signal (NLS) Fig. . SETX isThe scientific interest in this enzyme started in 2004 when two independent groups found that recessive and dominant mutations of SETX gene are responsible for the pathology of two distinct neurological disorders: AOA2 (ataxia with oculomotor apraxia - type 2)  and ALS4SETX mutations in AOA2 patients (approximately 120 mutations identified so far) are localized throughout the entire sequence and include missense, nonsense, splice site, frameshift, and insertion/deletions. ALS4 mutations are mainly localized in the N-terminal portion and in the helicase domain. The type of mutations associated with these neurological diseases supports the concept that SETX loss of function underlines the pathogenesis of AOA2, while SETX gain of function is responsible for ASL4 , 31–33.The delineation of the physiological functions of human SETX has been markedly influenced by studies done on its yeast homolog, Sen1p which shares with the human SETX a conserved helicase domain, suggesting that both proteins may have similar functions. Indeed, SETX and Sen1p can resolve RNA Polymerase II-driven R-loops and direct the exosome to sites of transcription-replication collisions , 35. BotSimilarly to its yeast homolog, SETX is involved in the regulation of gene expression. SETX interacts with RNA pol II and other RNA transcription and processing factors, such as poly(A)-binding proteins 1 and 2 (PABP1/2), hnRNPs, SAP155, SMN, SPT5, TAF4, KAP1/TRIM28, and TP63 –43. ThesThe function of SETX to facilitate the transcriptional termination process is more relevant in highly expressed genes such as house-keeping genes. As line of principle, actively expressed genes should be more prone to undergo mutagenic events and therefore it is not surprising that R-loops processing at the termination sites and DNA damage response are functionally linked in order to limiting DNA lesions and preserve genome integrity. A relevant example of such functional interactions is represented by the ability of SETX to interact with BRCA1 , a tumorIn addition to BRCA1, SETX might regulate transcription termination through its recruitment by other factors that might dictate gene specificity. In human keratinocytes the master epithelial transcription factor ΔNp63 is able to recruit SETX on the termination region of early epidermal differentiation genes favoring R-loops removal on those loci and allowing efficient transcriptional termination and gene expression  Fig. 2A2A. RemarSETX interacting partners can also inhibit its activity as it happens in the circadian clock oscillation where a molecular complex involving the repressor PER binds to SETX blocking subsequent processing by XRN2  Fig. 2A2A. This,In addition to positively regulate gene expression, SETX may also act as transcriptional repressor. During meiosis, SETX-BRCA1 complex is recruited to XY sex chromosomes, where it mediates transcriptional sex chromosome inactivation . FurtherAlterations of the DNA damage and DNA repair signaling are hallmarks of many, if not all, human cancers , 58–62. Defects in R-loops homeostasis might interfere with the DNA damage response and impair DNA repair mechanisms, mainly when DNA lesions occurs at transcriptionally active regions. When double strand breaks (DSB) occur at transcribed loci, the transient formation of R-loops favors DNA repair and prevents illegitimate rejoining of broken ends . R-loopsThe critical role of SETX in preserving the genome integrity is also demonstrated by its ability to promote the removal of cytotoxic DNA interstrand crosslinks (ICLs) . GeneralIn addition to impair DNA repair pathway, SETX loss might induce per se DNA damage by inducing transcription stress. Mouse and human studies have indeed demonstrated that SETX deficiency results into R-loop accumulation at promoter-proximal regions, which in turn promotes RNA Pol II pausing/stalling  Fig. 2B2B. In th+/− and Setx−/− females are characterized by a decline of the ovarian activity, known as premature ovarian ageing (POA). In detail, SETX knock-out increases the DNA damage in ovarian follicles and reduces the ovarian follicular reserve. This phenotype does not seem to be related to defects of the DNA repair pathway but rather to the acquisition of de novo DNA damage, likely due to an increase of the oxidative stress. Intriguingly, BRCA1 deficiency has also been linked to POA and SETX deletion has been observed in ovarian cancers with a similar extent than BRCA1 deletions [Another example of the functional relationship between DNA damage and SETX has been recently reported by analyzing oocyte derived from SETX knock-out mice . Subramaeletions , 73.2O2, camptothecin, and mitomycin C, but not to ionizing radiation [2O2-induced DSB and enhanced chromosomal instability in response to H2O2. Along the same line, in Hela cells SETX loss is associated with impeded mtDNA replication and increased levels of intracellular reactive oxygen species (ROS) [The functional link between SETX activity and oxidative stress is not restricted to the female germline. AOA2 cells are characterized by an increased sensitivity to oxidative DNA damage insults such as Hadiation . Importaes (ROS) . These ees (ROS) . BRCA2 ies (ROS) –79.All together, these data indicate that defects of SETX activity impact the fidelity of DNA repair and DNA damage response signaling, induce transcription stress and lead to increased levels of DNA damage, which ultimately result to genomic rearrangements and genomic instability.It is well known that transcription may constitute a barrier for DNA replication mainly when replication and transcription machinery collides in head-on conformation on the lagging strand DNA , 81. TheInflammation signaling represents an essential physiological cellular response of the innate immunity involved in many biological processes, such as tissue repairing and defense against pathogens. However, chronic inflammation response has been largely proved to be related to pathogenesis of several human diseases, including cancer –89. In tMore recently, dysregulation of R-loops homeostasis by SETX loss has been linked with the activation of innate immune response . In detaL389S+/− knock-in animals suggesting that gain or loss of function SETX mutations differently impact inflammatory response, leading likely to different biological outcomes [Notably, SETX and IRF3-dependent signaling are also interconnected during virus infection. SETX is indeed able to attenuate the transcription of IRF3-dependent antiviral genes during the early phase of viral infection . Accordioutcomes .In the tumor context IRF3 activation by R-loops dysregulation should represent a cellular defense mechanism to limit the propagation of genomically unstable cells harboring aberrant DNA or RNA metabolism. Accordingly, in cancerous and immune cells cGAS-STING signaling has been linked to induction of cell death, cell senescence, and with the activation of antitumor immunity. However, the precise role of cGAS-STING signaling on cancer occurrence and progression is controversial. Indeed, several evidence indicates that this pathway may favor tumor metastasis and facilitate cancer progression , 97. It SETX gene and a tumor suppressive screening identified SETX as a mutated gene in breast cancer [http://www.cbioportal.org/public-portal). Some SETX residues are more prone to undergo mutations. For instances, R1623X mutations have been reported in in 4 cases of Uterine Endometrioid Carcinoma, 2 cases of stomach carcinoma and 2 cases of colon adenocarcinoma. Tumors with the highest rate of SETX mutations are uterine endometrial carcinoma , skin melanoma  and colon adenocarcinoma . Remarkably, cutaneous SCC patients harboring SETX mutation showed a decrease of the overall survival compared to wild-type counterpart. Three AOA2-related SETX mutations, Q868X, R1363X, and P413L have been reported in Uterine Carcinoma, colon carcinoma and skin melanoma, respectively, suggesting that loss of SETX function might be beneficial for tumor development and progression  SETX gene is frequently mutated or its expression is downmodulated in different malignancies; (ii) R-loops are generally considered oncogenic events since their unscheduled formation affects genome integrity; (iii) SETX controls DNA damage response and DNA repair mechanisms whose dysregulation is frequently observed in many, if not all, tumors; (iv) SETX physically interacts with the tumor suppressor gene BRCA1 to promote DNA repair at the transcribed loci; (v) SETX together with the exonuclease SAN1 is able to induce the repair of ICLs whose defects are associated with the cancer susceptibly syndrome Fanconi anemia (FA); (vi) SETX attenuates the replication stress, which in the tumor context is an early event triggered by the activity of many oncogenes; (vii) SETX activity is involved in inflammatory response which is often deregulated in many human tumors.Over the past years, multiple evidence have clearly established the critical role of the R-loops resolving enzyme SETX in different physiological and pathological processes. The most compelling evidence that SETX dysregulation affects human health is the finding that gain or loss of function mutations of Although these observations suggest a potential tumor suppressive role of SETX, the genetic and molecular evidence of the direct involvement of SETX defects in tumor onset and progression has not been provided so far. The lack of the cancer susceptibility phenotype in AOA2 patients as well as the absence of spontaneous tumorigenesis in SETX knock-out mice further complicates our comprehension of the potential impact SETX on tumor development. Furthermore, it is not clear which specific SETX functions underlie its potential tumor suppressive role. Analysis of AOA2 and ALS4 cells has clearly indicated that the unscheduled turnover of R-loops is a central hallmark of those neurological disorders. However, as discussed above, alterations of R-loops homeostasis by SETX dysregulation impinge on many processes, each of them potentially contributing to the development of neurological disorders as well as tumor development. Furthermore, it is possible that not only loss of function but also gain of function alterations might drive tumor development, similarly to what is observed in the neurological context. While AOA2 patients do not show an increase of cancer susceptibility, a cohort of ALS4 patients harboring SETX L389S mutation show the presence of benign and malignant intestinal lesions linking colon carcinoma development with SETX gain of function mutations. Although this phenotype has been reported in a single study and the molecular pathways underlying this phenotype are missing, we can argue that the biological outcome exerted by an efficient SETX enzyme, as much as one that removes R-loops inefficiently, could be instrumental for tumor development. For instances, in contrast to AOA2, ALS4 mutations affect the TGF-beta pathway which is commonly dysregulated in human colon adenocarcinoma. ALS4 mutations do not interfere with SETX sumoylation, which is required of its interaction with the RNA exosome complex at sites of replication-transcription conflicts , 44. It Another consideration emerging from these observations is that SETX levels need to be tightly regulated since slight alterations of its expression or activity may induce dramatic effect on cell homeostasis. Our understanding of the mechanisms regulating SETX expression is still limited. A recent report demonstrated that similarly to yeast Senp1, SETX protein levels are regulated by the ubiquitin proteasome system , a regulSETX is ubiquitously expressed. Nevertheless, SETX mutations affect only limited types of human tissues leading mainly to neurological disorders and defects in germline homeostasis. It is possible that these tissues are more sensitive to R-loop unscheduled turnover due to the type of transcribed genes or to the lack of an efficient compensatory mechanism. The same scenario might take place during tumor onset and only specific tissues might be sensitive to SETX dysregulation.In conclusion, these observations will certainly fuel future research aimed to formally clarify the impact of SETX on tumor evolution and enlighten the ambiguous boundary between the physiological and pathological outcome of SETX-driven R-loops resolution. The analysis of the functional consequences of SETX loss in mice models of cancer research will help our understanding of how and whether this important DNA/RNA helicase may impact tumor development and progression.


RNA modifications, particularly N6-methyladenosine (m6A), are pivotal regulators of RNA functionality and cellular processes. We analyzed m6A modifications by employing Oxford Nanopore technology and the m6Anet algorithm, focusing on the HepG2 cell line. We identified 3968 potential m6A modification sites in 2851 transcripts, corresponding to 1396 genes. A gene functional analysis revealed the active involvement of m6A-modified genes in ubiquitination, transcription regulation, and protein folding processes, aligning with the known role of m6A modifications in histone ubiquitination in cancer. To ensure data robustness, we assessed reproducibility across technical replicates. This study underscores the importance of evaluating algorithmic reproducibility, especially in supervised learning. Furthermore, we examined correlations between transcriptomic, translatomic, and proteomic levels. A strong transcriptomic–translatomic correlation was observed. In conclusion, our study deepens our understanding of m6A modifications’ multifaceted impacts on cellular processes and underscores the importance of addressing reproducibility concerns in analytical approaches. RNA modifications, also known as epitranscriptomic modifications, play a crucial role in regulating RNA function and cellular processes. Among the over 300 identified RNA modifications, N6-methyladenosine (m6A) stands out as the most prevalent and well-researched modification in eukaryotic mRNA ,2. NotabThe identification of RNA modifications via conventional approaches, such as immunoprecipitation techniques , enzyme-dependent methods , or chemical detection methods (Pseudo-Seq and AlkAniline-Seq), is associated with several limitations. While these techniques facilitate the generation of detailed transcriptome-wide maps of RNA modification sites, their applicability is constrained by the accessibility of antibodies or chemical agents specific to particular modifications. Furthermore, all these methodologies necessitate the reverse transcription and sequencing of cDNA followed by short-read sequencing to identify modifications. Yet, during each of these phases, a fraction of modifications may be lost. Additionally, these approaches lack single-nucleotide resolution and cannot identify modifications for single RNA molecules ,10.Oxford Nanopore technology (ONT) provides a novel approach to directly sequence RNA molecules without converting them to cDNA or utilizing PCR amplification. Direct RNA-Seq concludes the RNA sequence from the current intensity as RNA molecules traverse through the nanopores. Modified nucleotides generate a distinct signal intensity compared to their unmodified counterparts, enabling the computational determination of modification sites on each RNA molecule using either supervised  and “DNA-binding transcription factor binding” (FDR < 0.001). It has also been established that these genes participate in processes associated with “modification-dependent protein binding” (FDR < 0.001), indicating their involvement in protein binding during post-translational modification of target proteins .Since the data we previously obtained are technical replicates of direct RNA-seq performed using Oxford Nanopore technology on the same HepG2 sample, as with gene expression, we expected a high level of reproducibility in m6A modifications . HoweverIt is noteworthy that genes for which possible m6A sites were common for all technical replicates maintained associations with ubiquitination processes, such as “ubiquitin protein ligase binding” (FDR = 0.002) and “ubiquitin-like protein ligase binding” (FDR = 0.003) B, as welThe majority (64.7%) of m6A sites are unique to each gene, with a maximum number of five modified sites per gene, an average of 1.5, and a median of 1 B. It is These findings underscore the need to validate and improve algorithms for detecting modifications from a single sample, especially considering potential quality variations associated with Oxford Nanopore technology limitations. Nevertheless, we observe the preservation of functional trends, allowing us to integrate all detected potential m6A modification sites into further analysis.https://github.com/GoekeLab/sg-nex-data, accessed on 29 October 2023) )2Cl, 0.3% CHAPS, 0.01% heparin, and 2.5 mM DTT). The fractionation was performed on an Agilent 1100 Series chromatograph at 4 °C with a flow rate of 0.8 mL/min. The representative chromatogram is shown in Total RNA was isolated with the RNA Clean & ConcentratorTM-25 kit , according to the manufacturer’s instructions. Polysomes were isolated using the Ribo Mega-SEC method  based onThe preparation of sequencing libraries was carried out with the PCR-cDNA Sequencing kit , strictly following the manufacturer’s protocol. The 14 cycles of PCR were conducted for both total RNA and polysome RNA. The long-read sequencing was carried out on a MinION nanopore sequencer (ONT) in 48 h single runs, using FLO-MIN106 flow cells.https://www.ncbi.nlm.nih.gov, accessed on 1 September 2023) under accession number PRJNA972889.The raw sequencing data for the HepG2 translatome were deposited in the NCBI SRA  under accession number PRJNA765908. The raw proteome data were uploaded to Mendeley Data . Data for the K562 cell lines were obtained from the Singapore Nanopore Expression Project  [The transcriptome and proteome data were described in Pytnitskiy et al.  and Poveer 2023) . Gene exer 2023) . ExpressWe used Nanopolish (version 0.14.0) for the resquiggling step . To idenThe R software environment was used for computations and visualization (ver. 4.1) . We usedIn conclusion, our analysis has highlighted the active involvement of genes with detected m6A modification sites in critical cellular processes, particularly in ubiquitination, aligning with prior research linking m6A modification to histone ubiquitination in cancer. Additionally, these genes play roles in regulating transcription factors and protein folding processes.Furthermore, our examination of various expression levels, including the transcriptomic, translatomic, and proteomic levels, revealed a robust correlation between the transcriptomic and translatomic levels. However, the correlation with the proteomic level was slightly lower, underscoring the intricacies involved in translating mRNA levels into functional proteins and other regulatory processes.Our study sheds light on the intricate interplay between technical nuances and biological functions in epitranscriptomic research. It underscores the need for methodological rigor and highlights the importance of considering both technical and biological factors in the identification of m6A modification sites.


Caenorhabditis elegans PUF protein fem-3 binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5′ end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities.  gld-1 mRNA expression to maintain C. elegans germline stem cells. The authors show that an intrinsically-disordered region of FBF-2 autoinhibits its RNA binding. LST-1 antagonizes this interaction to promote RNA binding.FBF-2 and LST-1 repress  Despite the lack of well-defined structures, they engage in molecular recognition and molecular assembly and play important roles in cellular signaling and regulation3. RNA-binding proteins are enriched with IDRs4. IDRs can influence RNA-binding activities and promote formation of higher-order ribonucleoprotein complexes, such as membrane-less granules10Intrinsically disordered regions (IDRs) are prevalent in eukaryotic proteomes13. RNA recognition can be modulated by physical interaction of RNA-binding proteins with partner proteins via globular domains or IDRs15. These collaborative RNA recognition complexes produce distinct regulatory capabilities not seen with the individual proteins18. Understanding the mechanistic details and the impact that partner proteins have on RNA-binding proteins is critical for deciphering the cellular functions of the complexes. Here we use the C. elegans PUF protein FBF-2 (fem-3 Binding Factor-2) and its partner protein LST-1  as model RNA regulatory proteins to explore the molecular mechanism underlying their collaboration, focusing on the roles of their IDRs 24. A mechanistic understanding of the LST-1–FBF interaction is therefore of particular importance.Proper control of mRNA localization, translation, and stability requires recognition of sequence or structural motifs by RNA-binding proteins to select specific target mRNAs31. The signature PUF RNA-binding domain (RBD), also called the Pumilio-Homology Domain (PUM-HD), is flanked by extended N-terminal and short C-terminal IDRs  with each repeat binding to one nucleotide33. C. elegans FBF-1 and FBF-2 (referred to collectively as FBF) are more flexible in their RNA sequence recognition37. A well-studied FBF-binding element (FBE) in the gld-1 3′ untranslated region, called FBEa, has a 9-nt core sequence beginning with UGU and is preceded by an upstream cytosine that increases RNA-binding affinity, 5′-CAUGUGCCAUA-3′40. More recently, an 8-nt compact FBE (cFBE) was identified, 5′-CUGUGAA(A/U)n-3′36. Although FBF-2 is sufficient to recognize gld-1, its interaction with LST-1 is required for GSC self-renewal22.FBF-2 has an architecture characteristic of all PUF proteins, including Drosophila Pumilio and human PUM1 and PUM2DRs Fig. . The PUF20. The LST-1 protein is largely unstructured , LST-1 A and LST-1 B, that interact with FBF-2, each with a KxxL sequence22. Crystal structures of FBF-2 bound to LST-1 A or B identify a seven-residue core region, including the KxxL sequence, that binds to FBF-2 with similar peptide backbone structure18. The two LST-1 PIMs are biologically redundant for GSC maintenance in nematodes22, but they have distinct FBF-2-binding affinities and distinct effects on FBF-2 RNA-binding affinity in vitro. An LST-1 B peptide (LST-167–98) binds tightly to FBF-2 and decreases RNA-binding affinity of its RBD, while an LST-1 A peptide (LST-119–50) binds with lower affinity to FBF-2 and has no effect on RNA-binding affinity18.LST-1 is expressed in GSCs in response to signaling from the stem cell nichered Fig. , includigld-1 RNA. We report a 2.1-Å crystal structure of FBF-2 in complex with RNA that identifies a PIM within the intrinsically disordered FBF-2 C-terminal tail (CT) bound to the same site on the RBD as LST-1. When comparing RNA binding by the FBF-2 RBD to RNA binding by the RBD plus the CT (RBD + CT), we discover that the CT is autoinhibitory. Addition of the CT to the RBD decreases RNA-binding affinity by increasing the off-rate. We demonstrate that interaction between the FBF-2 CT PIM and RBD is required for autoinhibition. This interaction between the CT PIM and RBD appears to restrain an electronegative cluster near the 5′ end of the bound RNA. Consistent with this notion, alanine substitutions in the electronegative cluster diminish autoinhibition. LST-1 A and B increase the RNA-binding affinity of FBF-2 RBD + CT, and we find that LST-1 A and LST-1 B bind more tightly to the RBD than the CT. Together, these findings suggest that LST-1 enhances FBF-2 RNA-binding affinity by displacing its CT, thereby alleviating autoinhibition. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in GSC self-renewal.Here we investigate the mechanism by which LST-1 and FBF-2 interact to control 164–575, a fragment with the RBD that lacks its intrinsically disordered C-terminal tail but retains its in vivo RNA-binding specificity  was generated in an fbf-1 loss-of-function background. Approximately 11% of the mutant progeny were sterile due to failure to initiate oogenesis, indicating a germline defect. In contrast, worms expressing full-length FBF-2 in an fbf-1 loss-of-function background were 0% sterile. To explore possible roles of the FBF-2 CT, we expressed and purified an FBF-2 fragment containing both RBD and CT (FBF-2164–632). This FBF-2 RBD + CT had increased thermal stability compared to the RBD alone  were disordered.Our previous structural and biochemical studies relied on FBF-2ity Fig. 17,18,36,607–613) align structurally with the core regions of LST-1 A (residues 32-38) and LST-1 B (residues 80-86) and made remarkably similar contacts to the RBD  or LST-1 B 18. The binding of LST-1 B to RBD + CT was ~50-fold weaker than to the RBD alone  . This substantial decrease is greater than the effect of LST-1 B on RBD  Fig. . Indeed,17. Like L610A, the Y479A substitution decreased the thermal stability of FBF-2 RBD + CT  or in the C-terminal tail L610A). Y479 is a critical part of the hydrophobic pocket that binds to key leucine residues in partner proteins Fig. , and its0A. Y479 79A or in18. The FBF-2 CT IDR is flexible and therefore residues other than the PIM were not visualized in our crystal structure of FBF-2 RBD + CT bound to RNA  to adopt a conformation similar to that seen in our crystal structure. The disordered region between the RBD and PIM forms a loop, which is constrained at the PIM, but the loop conformations are heterogeneous with low confidence scores of pLDDT<50. This loop places an electronegative cluster N-terminal to the FBF-2 CT PIM near the 5′ end of the bound RNA  assays to measure the kinetics of FBF-2 binding to 18, both LST-1 A and LST-1 B increased the RNA-binding affinity of FBF-2 RBD + CT. LST-1 A increased the RNA-binding affinity of FBF-2 RBD + CT to FBEa RNA 3-fold  FBF-2 in vitro RNA-binding affinity is autoinhibited by its intrinsically disordered CT, and (2) this autoinhibition is relieved by interaction with either PIM in the partner protein LST-1. We further show that autoinhibition by the CT is imposed by interaction of the RBD with an intramolecular PIM in the CT. Yet how does this intramolecular interaction result in reduced RNA-binding affinity? We propose that the FBF-2 PIM acts as Velcro®, attaching to the RBD and constraining the disordered loop to be located near the 5′ end of the bound RNA where an electronegative cluster of acidic residues in the CT weakens RNA-binding affinity by electrostatic repulsion.Here we identify a molecular mechanism for the FBF-2 and LST-1 interdependence in FBF-2 requires LST-1 to maintain GSCs, and this partnership depends on LST-1’s two PIMs, LST-1 A and LST-1 B. We suggest that LST-1 interaction relieves FBF-2 autoinhibition resulting in increased RNA-binding affinity. LST-1 A and LST-1 B bind with higher affinity to FBF-2 than its CT PIM and will outcompete the FBF-2 CT PIM. When the FBF-2 CT PIM is released by LST-1 competition, we propose that the FBF-2 CT loop is no longer formed and therefore the CT with its electronegative cluster does not interfere with RNA binding.45, and others, such as the C-terminal tail of Drosophila stem-loop-binding protein (SLBP) and an N-terminal extension to the YTH domain of fission yeast Mmi1, promote or stabilize RNA-bound protein conformations to increase RNA-binding affinity47. IDRs in RNA-binding proteins can also inhibit RNA binding, typically via electrostatic interactions mediated by acidic residues. For example, the C-terminal acidic tail of the E. coli RNA chaperone protein Hfq inhibits non-specific RNA binding by interacting with basic patches on the protein48, and a poly D/E sequence in yeast Nop15 stabilizes the neighboring RRM and suppresses non-specific RNA binding49. FBF-2 also utilizes an IDR with an electronegative cluster to interfere with RNA binding. In this case, however, inhibition controls its specific RNA-binding activity, and LST-1 interaction reverses autoinhibition and increases RNA-binding affinity.Although many RNA-binding proteins contain IDRs that influence RNA binding, FBF-2 autoinhibition by its CT and relief by LST-1 is distinct from other reported control mechanisms. IDRs, such as serine/arginine-rich regions in SR proteins and arginine-glycine repeats in FMRP, have been shown to directly interact with RNA51. Both CPB-1 and GLD-3 harbor consensus KxxL motifs suggesting they interact with FBF-2 via the same surface as LST-1 does54. Therefore, CPB-1 and GLD-3 likely collaborate with FBF-2 similar to LST-1: they could also displace the FBF-2 CT and modulate the RNA-binding activity of FBF-2. Different interaction strengths of the FBF-2 CT vs partner proteins with the FBF-2 RBD appear to be key to regulating the RNA-binding activity of FBF-2. The equilibrium of competitive binding can be shifted by altering the local abundance of partner proteins, although the effective concentration of the intramolecular CT is constant. Thus, the RNA-binding affinity of FBF-2 can be finely tuned in different regions of the C. elegans germline.Moreover, FBF-2 and its paralog FBF-1 function throughout the germline along with additional PUF partner proteins, such as CPB-1 and GLD-3C. elegans express nine PUF proteins, including FBF-2. Are the other eight PUF proteins regulated by partner proteins with PIMs and are any of them regulated via an intramolecular PIM? A C. elegans ‘PUF hub’ identifies additional protein partnerships mediated by PIMs that are essential for germline stem cell self-renewal55. This hub includes four PUF proteins  and two partner proteins (LST-1 and SYGL-1). LST-1 and SYGL-1 interact with the four PUF proteins55. Two PIMs have been identified in both LST-1 and SYGL-1, and they are essential for germline stem cell maintenance55, yet the molecular interactions and impact on RNA-binding affinity have been studied only for the FBF-2 and LST-1 partnership18. Below we summarize what is known about C. elegans PUF proteins and their potential to be regulated by inter- or intramolecular PIMs , including its PIM, is longer than the FBF-1 CT (residues 568–614). The FBF-1 CT includes an electronegative cluster whose sequence is almost identical to FBF-2. This highly conserved sequence is followed by a sequence that might be a PIM, 605NLRLMRT611, but it is quite different from the FBF-2 PIM  were identified by testing in vivo function, but SYGL-1 B has a valine rather than a lysine in the first position. It appears that PIM leucine residues must be in a sequence or structural context, but we do not yet understand it. For example, SYGL-1 PIMs were not functional when the critical leucines were mutated to alanine, despite the presence of neighboring leucine residues23. We also observed here that FBF-2 L608  than to FBF-1 and FBF-2 . Nevertheless, their RNA sequence specificities are similar to FBF-1 and FBF-2. An in vitro selection experiment identified three classes of RNA sequences bound by PUF-1160, and we now recognize that two of these classes are comparable to the 9-nt gld-1 FBEa and the 8-nt cFBE36. This overlapping sequence specificity suggests that the four hub PUF proteins can regulate similar target mRNAs. However, the mechanistic details of the partnership between PUF-3/-11 and LST-1/SYGL-1 appear to be different than for FBF-2. Although the residues that are important for the recognition of LST-1 by FBF-2 are also found in PUF-3 and PUF-11  was cloned into the pSMT3 vector (kindly provided by Dr. Christopher Lima), which encodes an N-terminal His6-SUMO tag followed by a Ulp1 protease cleavage site63. The FBF-2 RBD + CT protein was overexpressed in E. coli BL21-CodonPlus (DE3)-RIL competent cells (Agilent). A 1-L LB culture with 50 µg/mL kanamycin was inoculated with a 5-mL overnight culture and grown at 37 °C to OD600 of ~0.6. Protein expression was induced with 0.1 mM IPTG, and the culture was grown at 16 °C overnight. The cell pellet was resuspended in 40 ml lysis buffer containing 20 mM Tris, pH 8.0; 0.5 M NaCl; 20 mM imidazole; 5% (v/v) glycerol; and 0.1% (v/v) β-mercaptoethanol and disrupted by sonication. After centrifugation, the His6-SUMO-tagged FBF-2 RBD + CT protein was purified from the soluble fraction of the E. coli cell lysate in lysis buffer using Ni-NTA resin (Qiagen), and was eluted with a buffer containing 20 mM Tris pH 8.0, 50 mM NaCl, 0.2 M imidazole and 1 mM DTT. The eluted fusion protein was incubated with Ulp1 protease overnight to remove the His6-SUMO tag. Subsequently, the FBF-2 protein was purified with a Hi-Trap Heparin column (Cytiva), eluting with a 5–100% gradient of buffer B. Heparin column buffer A contained 20 mM Tris pH 8 and 1 mM DTT, and buffer B contained an additional 1 M NaCl. The peak fractions from the heparin column were pooled and concentrated to 5 ml and loaded onto a HiLoad 16/60 Superdex 75 column (Cytiva) in a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, and 0.5 mM TCEP. Purified FBF-2 RBD + CT protein was concentrated and snap-frozen for later binding experiments.A cDNA fragment encoding TM software  was used to analyze protein melting curves and calculate melting temperatures (Tm).Mutants of FBF-2 RBD + CT: Y479A (5′-GACGAGATTTTCGACGGAGCCATTCCACATCCGGACAC), L610A (5′-GGAAGGAAGCCTGATGGCAGAGCCACGGAGCAAT), and Loop6A  were generated by site-directed mutagenesis with the primers indicated. Plasmid sequences were verified by DNA sequencing (Azenta Life Sciences). Protein variants were expressed and purified like wild type proteins. Melting temperatures of wild type and mutant proteins were assessed by differential scanning calorimetry. Reaction mixtures  contained ~0.3 mg/ml FBF-2 proteins and Sypro Orange dye (1:1000 dilution) (Sigma). Fluorescent intensity was collected from 25 °C to 95 °C (3 °C increment/min) with a real time PCR instrument (Applied Biosystems™ QuantStudio 7 Flex System) using excitation and emission wavelengths of 470 nm and 586 nm, respectively. Protein Thermal Shift61, using a plasmid with a cDNA fragment encoding the FBF-2 RBD cloned into the pSMT3 vector (kindly provided by Dr. Christopher Lima). This plasmid encodes an N-terminal His6-SUMO fusion tag followed by a TEV protease cleavage site63. TEV protease was used to cleave the His-SUMO tag, instead of Ulp1 protease, because it was generated from a previous pGEX6p FBF-2 RBD construct. Otherwise, the purification procedure was the same as the FBF-2 RBD + CT protein described above.The FBF-2 RBD protein (residue 164-575) was purified as described previously18, using plasmids with cDNA fragments encoding LST-1 A (residues 19–50) and LST-1 B (residues 67–98) cloned into the pSMT3 vector. A 1 L culture of TB media with 50 µg/mL kanamycin was inoculated with a 5-mL culture grown overnight at 37 °C. The 1-L culture was grown at 37 °C. At OD600 of ~1.0, protein expression was induced with 0.4 mM IPTG, and the culture was grown at 22 °C for ~20 h. After lysis by sonication and centrifugation, the soluble fraction of E. coli cell lysate in a buffer containing 20 mM Tris, pH 8.0; 0.5 M NaCl; 20 mM imidazole; and 5% (v/v) glycerol was mixed with 5 mL Ni-NTA resin (Qiagen) for 1 h at 4 °C. After extensive washing the LST-1 proteins were eluted with a buffer of 20 mM Tris, 50 mM NaCl, and 200 mM imidazole, pH 8. Ulp1 protease was added to the eluant and incubated at 4 °C for 2 h or overnight to remove the His6-SUMO tag from LST-1 A or B. LST-1 A protein was separated from His6-SUMO with a HiTrap Q column (Cytiva), and the column flow-through, which contained LST-1 A, was collected and concentrated using Amicon Ultra-15 filters (3 K MWCO). LST-1 B protein was purified with a HiTrap Heparin column (Cytiva) and eluted with a 5–100% NaCl gradient . The peak fractions containing LST-1 B were pooled and concentrated. Both LST-1 A and B were further purified with a HiLoad 16/60 Superdex 75 column (Cytiva).LST-1 A and B peptides were purified as described previouslyLST-1 protein concentrations were determined by NanoDrop based on UV absorption at 280 nm.E. coli BL21-CodonPlus (DE3)-RIL competent cells (Agilent). A 1-L TB culture was inoculated with a 5-mL overnight culture and grown at 37 °C to OD600 of ~1.0 and then grown at 22 °C overnight after induction with 0.4 mM IPTG. The soluble fraction of E. coli cell lysate in a buffer containing 20 mM Tris, pH 8.0; 0.5 M NaCl; 20 mM imidazole; and 5% (v/v) glycerol was mixed with 5 mL Ni-NTA resin for 1 h at 4 °C. After extensive washing the FBF-2 CT proteins were eluted with a buffer of 20 mM Tris, 50 mM NaCl and 200 mM imidazole, pH 8. The Ulp1 protease was added to the eluant and incubated at 4 °C for 1 h to remove the His6-SUMO tag. FBF-2 CT protein was purified with a HiTrap Q column, and the column flow-through containing FBF-2 CT was collected and concentrated using Amicon Ultra-15 filters (3 kDa MW cutoff). It was further purified with a HiLoad 16/60 Superdex 75 column. The FBF-2 CT protein concentration was determined using the Qubit protein assay kit (Invitrogen) because it lacks aromatic residues.A cDNA fragment encoding FBF-2 CT (residues 601–632) was cloned into the pSMT3 vector. The protein was overexpressed in 280 = 3.62) was incubated with cFBE RNA (5′-CUGUGAAUG-3′) at a molar ratio of 1:1.2 on ice for 1 h prior to crystallization. Crystals of the protein–RNA complex were grown in 30% PEG 400, 0.1 M Tris pH 8.5 by hanging drop vapor diffusion at 20 °C with a 1:1 ratio of sample:reservoir solution. Crystals were directly flash frozen in liquid nitrogen.Purified FBF-2 RBD + CT protein  was used as a search model for molecular replacement with Phaser65 as implemented in Phenix version 1.20–4459. For the FBF-2 RBD + CT/cFBE complex, the C-terminal peptide of FBF-2 (residues 607–613) was built manually into the electron density. Residues 570–606 were disordered. The model was improved through iterative refinement and manual building with Phenix version 1.20–4459 and Coot (version 0.9.6 EL)67. Data collection and refinement statistics are shown in Supplementary Table X-ray diffraction data were collected at a wavelength of 1.0 Å using SERGUI at beamline 22-ID (SER-CAT) of the Advanced Photon Source. Data sets were scaled with HKL2000Experiments were performed at 20 °C using a MicroCal PEAQ-ITC Automated  with a 200-μL standard cell and a 40-μL titration syringe. FBF-2 and LST-1 variants were prepared in a buffer of 20 mM HEPES pH 7.4, 150 mM NaCl, and 0.5 mM TCEP by gel filtration. LST-1 (100–400 µM) was titrated from the syringe into the cell containing FBF-2 (10–30 µM) in 2 µl aliquots. Experiments were performed in duplicate due to the limitation of the amount of protein needed. Data were analyzed using the MicroCal PEAQ-ITC Analysis Software (version 1.40) provided by the manufacturer with the one-site model Table .gld-1 FBEa, 5′-AUCAUGUGCCAUAC-3′). The FBF-2 proteins were serially diluted 2-fold from the highest concentration of 10 µM in the buffer used for gel filtration . 10 µl of the serially diluted protein samples were mixed with 10 µl RNA (10 nM) in a 2× buffer of 10 mM HEPES pH 7.4, 0.01% (v/v) Tween 20, 0.1 mg/ml BSA, 0.1 mg/ml yeast tRNA, and 2 mM DTT. For experiments in the presence of LST-1, LST-1 proteins at constant concentration were preincubated with FBF-2 at 4 °C for 2 h. The LST-1 concentrations were chosen based on their Kd’s for interaction with FBF-2. The final FBF-2 concentrations ranged from 5000 nM to 0.6 nM, and the LST-1 A or B concentrations were constant at 150 μM or 50 μM, respectively. The protein-RNA mixtures were incubated at 4 °C overnight. The samples were resolved on 10% TBE polyacrylamide gels run at constant voltage (100 V) with 1× TBE buffer at room temperature for 35 min. The gels were scanned and visualized with a Typhoon FLA 9500 imager using the Cy5 channel (excitation wavelength 635 nm). Band intensities were quantified with ImageQuant 5.2 (Cytiva). The data were fit with GraphPad Prism (version 9.2.0) using nonlinear regression with a one-site specific binding model. Mean Kd’s and standard error of the mean (SEM) from three or more technical replicates are reported /cFBE RNA complex. Four additional ribonucleotides, AUAU, were added to 5′ end, and this protein-RNA complex was used as the starting model for the molecular dynamics (MD) simulation. An MD simulation for the Apo form of FBF-2 RBD + CT was also run for comparison.To build a model containing the FBF-2 RBD + CT bound to RNA, including residues 570–606 that were disordered in our crystal structure, we obtained the AlphaFold structure of FBF-2 protein from UniProt (UniProt Entry: Q09312). We selected residues 168–613 of the AlphaFold structure68. The charge of the complex or apoprotein was neutralized with corresponding amount of Na+ or Cl− and then solvated with 150 mM NaCl (Supplementary Table 69: (1) steepest descent energy minimization of the solvent water with restraints on the protein and ions; (2) 20 ps constant number-pressure-temperature (NPT) MD simulation at 50 K and 1 atm to equilibrate solvent water with restrains on the protein and ions; (3) heating up the system to 300 K via a series of 10-ps constant number-volume-temperature (NVT) MD simulations at 50, 100, 150, 200, 250, and 300 K; (4) 120-ns production NPT MD simulation at 300 K and 1 atm. In the production MD simulations, a 2-fs time step was used with SHAKE constraints on all bonds involving hydrogen. Long-range electrostatic interactions were treated with the particle-mesh Ewald method. The cutoff for the Lennard-Jones potential was set at 1.0 nm. The simulations were run three times with different starting conformations, and the RMSF analysis is shown in Supplementary Fig. The Amber20 package with AMBER force fields ff19SB, RNA.OL3 and water.opc was used for MD simulationsgld-1 FBEa RNA were measured using a 2-channel OpenSPR instrument (Nicoya). A 3′-biotin-labeled synthetic RNA was ordered from Horizon Discovery . The 3′-biotinylated RNA was immobilized on streptavidin-treated biotin sensor chips (Nicoya). All experiments were performed at 20 °C with a buffer of 20 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM DTT. SPR data were collected using OpenSPR . FBF-2 proteins were injected with a flow rate of 20 µl/min. Data were obtained at five different protein concentrations . Sensor chips were regenerated using 0.05% or 0.1% (v/v) SDS. For each experiment, five binding curves were simultaneously fit using a 1:1 binding model with the TraceDrawer software . Mean values and standard deviation were calculated from two technical replicates. Despite our efforts, the fit of the binding curves for the association phase of FBF-2 RBD and FBF-2 RBD + CT mutants is poorer than the fit for FBF-2 RBD + CT WT. The appearance of the curves is consistent with the derived kon and koff values, and importantly, the curve fit for the dissociation phase is better.Kinetics of FBF-2 binding to the Further information on research design is available in the Peer review fileSupplementary Information fileDescription of additional supplementary filesSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Movie 1Reporting SummarySource Data File


Analysis of the AS landscape also revealed cell type specific AS signatures for some genes  involved in key functions in PCa tumorigenesis. Finally, we also validated the clinical relevance of MKNK2 AS events in PCa patients and pointed to the possible regulatory mechanism related to m6A in the exon14a/b region and SRSF1. Overall, we characterize the role of METTL3 in regulating PCa-associated AS programs, expand the role of METTL3 in tumorigenesis, and suggest that MKNK2 AS events may serve as a new potential prognostic biomarker.Although the role of METTL3 has been extensively studied in many cancers, its role in isoform switching in prostate cancer (PCa) has been poorly explored. To investigate its role, we applied standard RNA-sequencing and long-read direct RNA-sequencing from Oxford Nanopore to examine how METTL3 affects alternative splicing (AS) in two PCa cell lines. By dissecting genome-wide METTL3-regulated AS events, we noted that two PCa cell lines  behave differently in exon skipping and intron retention events following METTL3 depletion, suggesting AS heterogeneity in PCa. Moreover, we revealed that METTL3-regulated AS is dependent on N The mol6A modification is ubiquitous in most eukaryotes (6A is regulated by the methyltransferase complex (writers), demethylases (erasers), and reader proteins   provides the possibility to study splicing variants and alternative polyadenylation events , which d6A) .+ and AR- PCa cells. The different effects of METTL3 on exon skipping and intron retention might be related to distinct splicing partners.In this study, we combined standard RNA-Seq and long-read DRS of Oxford Nanopore to comprehensively dissect the impact of METTL3 on RNA splicing in the setting of two PCa cell lines with distinct AR sensitivities. We revealed that METTL3 potentially had shared and unique mechanisms affecting alternative splicing in AR22.1Normal epithelial prostate cell line (RWPE-1) and PCa cell lines (LNCaP and DU145) were obtained from Prof. Andrew Chan’s laboratory in the School of Biomedical Sciences, The Chinese University of Hong Kong. RWPE-1 cells were cultured using the KSFM-1 medium kit  with supplementary bovine pituitary extract (BPE) and human recombinant epidermal growth factor (rEGF). LNCaP cells were maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS and 1% PS, and DU145 cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS and 1% PS. All cells were incubated at 37°C and 5% CO2.2.2The siRNA against METTL3  or control was transfected into LNCaP and DU145 cells using RNAiMAX reagent (Invitrogen). Total RNA was extracted with a Pure-link mini kit (Invitrogen) 48 h after transfection. RNA concentration was measured via a Nanodrop 2000, and RNA integrity (RIN) was detected using an RNA 6000 nanochip (Agilent Technologies) (Requirement: RIN>8.8). PolyA RNA was isolated from 75 μg of total RNA using a Dynabeads™ kit.2.3cDNA transcription was performed using a SuperScript III synthesis kit (Invitrogen) and then used for qPCR based on SYBR (Applied Biosystems). Two siRNAs (siM3-1 and siM3-2) targeting METTL3 were designed, and we observed that the knockdown efficiency of siM3-1 was only 50%, which we considered insufficient for further analyses. Therefore, the more effective target-2 (siM3-2) was selected for the following analysis  of siControl- and siMETTL3-transfected cells was performed on a HiSeq 4000 sequencing platform (Illumina). Clean reads were aligned to the human hg38 genome (UCSC) using HISAT2 (v2.2.1) software . PCA of 2.5https://portal.gdc.cancer.gov/projects/TCGA-PRAD), wherein we also retrieved the clinical data of 499 primary PCa patients. This FPKM table was used for expression profile analysis of m6A regulators. The association between METTL3 expression and overall survival and disease-free survival was determined using the GEPIA2 web server .As previously described, each 1 μg purified polyA RNA was subjected to library preparation following the SQK_RNA002 kit procedure and consequently sequenced on a Nanopore GridION platform for a 48-h runtime . Base-ca2.7Splicing analysis of DRS data was performed with the FLAIR (v1.5) pipeline . SpecifiDifferential AS events of RNA-Seq data were identified using rMATS (v4.1.0) with a strict parameter of cstat=0.05 , 29 and th percentile, dividing the PCa patients into high PSI and low PSI groups. The survival difference between high and low PSI groups was calculated by using “survival” (3.5) and “survminer” (0.4.9) R software.The PSI index of MKNK2a/b isoforms in PCa was downloaded from the TCGA SpliceSeq database. The MKNK2_AA_46570 event represents the PSI index of sequencing reads covering between exon 13 (e13) and exon 14a (e14a), indicating the expression of the MKNK2a isoform. While the MKNK2_AT_46567 represents the PSI index of the sequencing reads covering between exon 13 and exon 14b (e14b), producing the MKNK2b isoform. According to the previous reference , we set 2.86A sites between the siControl and siMETTL3 groups were detected using ELIGOS2 and DRUMMER   . The genomic features of m6A sites were annotated with the ChIPseeker R package  . The cri package . The det2.9http://homer.ucsd.edu/homer/motif/) software. The resulting motifs were visualized using the ggseqlogo R package.The regions of differential intron retention and exon skipping events were extracted for motif analysis using HOMER  and prostate adenocarcinoma (n=499). Most m6A “writer” and “reader” proteins were overexpressed in PCa tissues compared to normal tissues  and DU145  compared to normal prostate cells (RWPE-1)  . To expl3.2-) and LNCaP (AR+) cells with METTL3 knockdown. First, the knockdown efficiency of siRNA against METTL3 was assessed in the two PCa cell lines by qPCR and western blotting and shown in To gain further insight into the molecular mechanism by METTL3 in PCa, we performed RNA-Seq in DU145 .We next explored the consistency between LNCaP and DU145 cells in response to METTL3 knockdown. There were 458 genes commonly upregulated, and 224 genes downregulated, respectively , 38 and LIP data , 40 (Fi+) and DU145 (AR-) cells  cells . Taken t3.3+) and DU145 (AR-) cells. Overall, 0.6-1.5 million reads were generated for each sample, with an average read length of 1.0 kb and average read quality of 11  performed using the Oxford Nanopore long-read sequencing platform to generate METTL3-dependent AS profiles in LNCaP , metabolic pathways, and proliferation hallmarks   and DU145 (AR-) cells have different functional enrichments upon METTL3 depletion  and downregulated IR events (11 up/62 down) .+ and AR- cells is associated with SFs. By analyzing the proteomic data of METTL3 reported by Yue et al.  . Next, we et al. , we disc3.66A, we first analyzed differential m6A sites from DRS data using ELIGOS2 software with strict criteria, focusing primarily on decreased m6A sites  and intronic regions (10.3% or 11%), indicating the potential involvement of m6A in IR and ES events  and 280 (13.8%) overlapping genes between DEGs and m6A-modified genes in DU145 and LNCaP cells, respectively    –53, but in vitro and in vivo  resulted in more introns being preserved and more exons being skipped, whereas the opposite phenomenon was observed in AR+ cells (LNCaP). It has been shown that m6A promotes exon inclusion or exon exclusion. A breast cancer study showed that depletion of METTL3 causes exons of specific genes to be retained  at serine 209, which is a key rate-limiting molecule in the mRNA translation phase . Phospho6A modification near the cleavage site can affect the binding of cleavage factors to transcripts, thereby changing the way the transcripts are cleaved. Our results also showed that m6A sites overlap significantly with splice junctions, suggesting that m6A is involved in the regulation of splice site recognition. Further, the data revealed that the methylation level of the exon14a/b region of the MKNK2 gene in PCa cells was significantly higher than that in normal cells. And after METTL3 knockdown, the predicted methylation level of exon exon14a/b region was significantly reduced. Therefore, the declining m6A level in MKNK2 caused by METTL3 knockdown potentially reduces the binding of splicing factors. In the future, further experiments are needed to verify the mechanism of METTL3 regulating MKNK2 AS events.Finally, we explored the possible mechanism of METTL3 in regulating MKNK2 splicing. The variable splicing process of genes is controlled by a variety of regulatory elements, mainly including splicing factors and splicing sites . It is k- and AR+ PCa cells. However, which factors are functionally related to these responses remains unanswered, which may enhance the understanding of the carcinogenesis process of different PCa stages.This study has some limitations. First, while offering advantages such as long read lengths and RNA modification analysis, the DRS approach has limitations of higher error rates and more complex bioinformatics analysis. Our results are mainly based on bioinformatic analysis, and further research in experimental cohorts is warranted. It is worth mentioning that our conclusions are supported by other independent datasets and qPCR experiments, demonstrating the validity. Second, we uncovered an intriguing mechanism of METTL3 in AR+ and AR- PCa cell lines via a combination of standard RNA-Seq and long-reads direct RNA-Seq of Nanopore. We revealed both common and phenotypic heterogeneity of different PCa cells regulated by METTL3, which were potentially associated with m6A modification and SFs. The study identified the clinical relevance of MKNK2 AS events in PCa and explored the potential mechanism of METTL3 in modulating MKNK2 AS events. These findings provide new insights into RNA modifications in PCa and could serve as a favorable molecular basis for novel treatment strategies in androgen-sensitive and androgen-resistant prostate cancer.In summary, our study used genome-wide analyses to investigate METTL3-regulated AS events in ARThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.LW: Study design, Data processing, and Writing-Original draft preparation. LYW, YW, and CF: discussed the data and assisted in the sequencing library preparation. LS, YL, JN and CY: validated the differentially expressed or spliced genes. JH, PC, and CN: Writing-Reviewing and Editing. ST: Supervision, Writing-Reviewing and Editing. All authors contributed to the article and approved the submitted version.


C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei. Tethering the nuclear Argonaute WAGO-9/HRDE-1 induces heterochromatin formation and independently induces small RNA amplification. Consistent with this finding, HRDE-1, while predominantly nuclear, also localizes to peri-nuclear nuage domains, where amplification is thought to occur. Tethering a heterochromatin-silencing factor, NRDE-2, induces heterochromatin formation, which subsequently causes de novo synthesis of HRDE-1 guide RNAs. HRDE-1 then acts to further amplify small RNAs that load on downstream Argonautes. These findings suggest that HRDE-1 plays a dual role, acting upstream to initiate heterochromatin silencing and downstream to stimulate a new cycle of small RNA amplification, thus establishing a self-enforcing mechanism that propagates gene silencing to future generations.Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that induction of heterochromatin silencing in  C. elegans. They demonstrate that the nuclear Argonaute HRDE-1 induces subnuclear-co-localization of target genes in heterochromatin. Heterochromatin formation subsequently triggers de novo HRDE-1 guide RNA loading. Finally, HRDE-1 enters nuage and activates small RNA amplification. Thus, HRDE-1 effects multiple steps of a self-enforcing transgenerational silencing process.Ding and colleagues investigate inherited silencing in   However, how the amplification system in nuage communicates with and drives the nuclear events during the initiation and maintenance of transgenerational silencing is not well understood.In many animal germlines, small RNA/Argonaute pathways function transgenerationally to install and re-inforce chromatin silencing essential for fertility. For example, in flies, worms, and mammals, members of the PIWI Argonaute family engage genomically encoded small RNAs termed PIWI-interacting RNAs (piRNAs) that silence transposons to maintain genome integrity.C. elegans, transgenerational silencing can be initiated by the PIWI pathway, by the canonical double-stranded RNA (dsRNA)-induced RNAi pathway, or by intronless mRNA.11 Inherited silencing is maintained by a family of related downstream worm-specific Argonautes (WAGO Argonautes) guided by small RNAs (22G-RNAs) produced by cellular RNA-dependent RNA polymerase. Once established, inherited silencing can be propagated independently of the initiating cues via continuous cycles of WAGO 22G-RNA amplification and transmission of the WAGO Argonautes and their small RNA co-factors to progeny.14In C. elegans.16 HRDE-1 is thought to engage nascent transcripts at target loci to induce heterochromatin and transcriptional silencing through the nuclear RNAi pathway.17 HRDE-1 promotes the transgenerational silencing of many genes18 and is thought to do so by recruiting chromatin remodeling factors, including the nucleosome remodeling and deacetylase complex (NuRD) and histone methyltransferases .19 The nuclear RNAi pathway is also required for the spreading of secondary small RNAs from piRNA target sites.20The nuclear WAGO Argonaute, HRDE-1/WAGO-9, plays a central role in transgenerational silencing in 25 to recruit—i.e., tether—HRDE-1 or the nuclear silencing factor NRDE-2 to a reporter mRNA. In principle, tethering enables initiation of silencing in the absence of upstream initiators such as piRNAs or dsRNA and, with appropriate genetic tests, can be used to order events in the pathway. We show that tethering either HRDE-1 or NRDE-2 can induce a complete silencing response, including small RNA amplification and transgenerational silencing that persists even after the λN-fusion protein is crossed from the strain. Tethering NRDE-2 initiates chromatin silencing through nrde-4 and independently of hrde-1 but requires hrde-1 for small RNA amplification. By contrast, tethering HRDE-1 stimulates chromatin silencing through NRDE-2 and NRDE-4 but can elicit small RNA amplification independently of both these chromatin-silencing factors. Mutations that block HRDE-1 from binding small RNA disarm silencing and cause HRDE-1 to become cytoplasmic, but tethering HRDE-1 in these mutants nevertheless initiates a strong silencing response that requires small RNA amplification proximal to the tether site. The small RNA amplification machinery is recruited to the tether site by sequences in the N-terminal half of HRDE-1 . Like full-length HRDE-1 protein, HRDE-1 NTD co-localizes with MUT-16 in Mutator foci, subdomains of cytoplasmic nuage where the small RNA amplification machinery resides.26 Our findings suggest that HRDE-1 lies at a nexus in the silencing pathway, shuttling from the nucleus to the nuage and back, to coordinate the nuclear and cytoplasmic events of transgenerational silencing.Transgenerational silencing requires a series of events that are thought to occur in the nuage, nucleus, and cytoplasm. Because all of these events are essential for the cycle of inherited silencing, their order has been difficult to determine. For example, it is not known whether the nuclear Argonaute HRDE-1 directly triggers RdRP recruitment and amplification of small RNAs or whether it must first induce heterochromatin at its targets to elicit small RNA amplification. Here, we use the phage lambda N (λN)-boxB tethering systemTo order events in inherited silencing, we sought to uncouple initiation and maintenance of silencing. To do this, we used the phage λN-boxB tethering system to recruit nuclear silencing factors HRDE-1 or NRDE-2 to a target reporter that is robustly expressed in the germline . We hypohrde-1 or nrde-2 loci  and nuage-localized factors  and components of the chromatin nuclear silencing machinery (nrde-2 or nrde-4)  –S2K.nrde-2 mutants exhibited a 4-fold increase in reporter pre-mRNA both with and without HRDE-1 tethering  compared with wild type, but removing nrde-2 activity in the context of tethering caused spliced RNA levels to increase , reaching levels of approximately 40% of wild-type mRNA levels. It is important to note that the qPCR assay cannot distinguish mRNA from template RNA being silenced, as template RNAs derive from spliced RNAs. Moreover, the high levels of spliced RNA in λN::HRDE-1;nrde-2 worms correlate with a marked accumulation of reporter RNA localized in nuage . Thus, the accumulated spliced RNA likely reflects template RNA engaged in amplifying the small RNA silencing signal, perhaps to compensate for the loss of heterochromatin silencing. Further study is needed to understand the effects of nrde-2 mutants on pre-mRNA levels, such as whether increased pre-mRNA levels in nrde-2 mutants reflect processing defects.30 Nevertheless, in the nrde-2 background, HRDE-1 tethering reduces mRNA and pre-mRNA levels by 2-to 3-fold, suggesting that tethered HRDE-1 can exert effects on both mRNA and pre-mRNA levels independently of NRDE-2. Taken together, our findings suggest that HRDE-1 functions twice during inherited silencing—upstream of nuclear silencing to recruit NRDE-2 and NRDE-4 and again downstream of these factors to induce small RNA amplification and post-transcriptional clearance of mRNA. While these events likely occur sequentially and thus depend on each other during the normal course of inherited silencing,31 tethering HRDE-1 initiates both modes of silencing independently, either of which is sufficient to prevent reporter GFP expression.HRDE-1 tethering in wild-type worms reduced the unspliced pre-mRNA reporter level by 2-fold and the spliced RNA level by 100-fold, as measured by quantitative PCR (qPCR) . For unkethering  but exhinrde-2 and nrde-4, while NRDE-2 requires both nrde-4 and hrde-1. A likely explanation for these findings is that heterochromatin silencing directed by NRDE-2 and NRDE-4 induces the de novo synthesis of small RNAs that engage HRDE-1 and that HRDE-1 can further amplify these small RNAs to propagate silencing to offspring. Indeed, whereas we detected very few small RNAs targeting the reporter in the absence of tethering  background to segregate away λN::HRDE-1, the reporter remained silent for at least 13 generations, with no change in penetrance. Moreover, we observed only a slight reduction in small RNA levels primarily in regions juxtaposed to the boxB hairpins  and mut-16(+)   and 3M. cing see .rde-3 and mut-16 mutants, which are defective in small RNA amplification, suggests that the unloaded Argonaute can direct chromatin silencing when tethered. To further test this idea, we monitored silencing (1) by λN::HRDE-1 in an hrde-2 mutant, which is defective in HRDE-1 small RNA loading13 and (2) by a λN::HRDE-1(Y669E) mutant, predicted by structural work to be defective in guide RNA binding  caused elevated pre-mRNA levels as measured by qRT-PCR  mutant was defective in silencing a piRNA reporter (hrde-1(null)  animals   and S4C.reporter  and 4C,  animals  and 4F.  compare E–3G—i.e.λN::hrde-1 truncation mutants  and hrde-1(CTD) alleles failed to silence a piRNA sensor —i.e., dramatically increased levels of small RNAs proximal to the boxB sites and reduced levels of small RNAs targeting GFP sequences. Interestingly, the magnitude of small RNA accumulation induced by λN::NTD at the boxB sites was ~4-fold greater than that induced by either λN::HRDE-1 in nrde-2 mutants or by λN::HRDE-1(Y669E)    and 5H. 1(Y669E)  and 5H. elements  and 5H. To further analyze changes in target RNA caused by tethering, we used qRT-PCR. Surprisingly, whereas tethering wild-type λN::HRDE-1 reduced the reporter pre-mRNA by 50% and mRNA by 99% , λN::NTDhrde-1 null allele to generate animals heterozygous for the tethering construct. Finally, we crossed these λN::NTD/null heterozygotes  to a hrde-1(+) reporter strain, resulting in two types of cross progeny—λN::NTD/+ or null/+ heterozygotes. Remarkably, although the λN::NTD/null parents lacked a functional HRDE-1 RISC, they nevertheless robustly transmitted silencing to the next generation  in worms that also express either mCherry::GLH-1, which localizes broadly within nuage, or MUT-16::mCherry, which localizes prominently in Mutator foci. GFP::HRDE-1 co-localized to a subset of peri-nuclear mCherry::GLH-1 foci, especially in association with late pachytene germ nuclei , thus HRDE-1 and MUT-16 localize within a nuage subdomain (or domains) independently of each other.MUT-16 is required for the co-localization of small RNA amplification factors within Mutator foci.f HRDE-1  and 7B. f HRDE-1  and 7B. activity  and 7B. C. elegans germline. In addition to its known role in directing heterochromatin silencing downstream of RNAi15 and Piwi Argonaute silencing,14 our tethering studies have shown that HRDE-1 is also de novo loaded with small RNA, downstream of heterochromatin silencing, enabling it to prime a new round of small RNA amplification within nuage  and that small RNA levels also increase dramatically to compensate and silence mRNA expression. Taken together, our findings suggest that when the nuclear heterochromatin pathways are inactive, the target mRNA is silenced by a combination of cytoplasmic clearance or trapping in the P granule.The nuclear silencing events that depend on HRDE-1 cause the target alleles to co-localize into a single focus of DNA FISH signal  and S6E.S. pombe, the RNAi-induced transcriptional silencing complex (RITS), which includes an RdRP and a nuclear Argonaute AGO1p, resides in heterochromatin. A previous study showed that tethering of AGO1p to RNA via a boxB reporter system, similar to the one used here, was sufficient to recruit the RITS complex, induce small RNA amplification, and drive reporter silencing25.In the yeast 38 How heterochromatin leads to de novo programming of HRDE-1 is nevertheless unknown. In C. elegans, the RdRP EGO-1 has been shown to associate with germline chromatin,40 and several of our findings would be consistent with a cycle of nuclear small RNA transcription and de novo HRDE-1 loading within heterochromatin. Such a mechanism could explain why tethering NRDE-2 in the absence of HRDE-1 initiates heterochromatin silencing but not small RNA amplification  to induce silencing that transmits to offspring via either the sperm or the egg  was partially dependent on wago-1  during each germline life cycle. For example, small RNAs guide heterochromatin formation in the zygote, and heterochromatin then propagates silencing before feeding back into the ations15 –S7F. Giv11) could trigger default recruitment of the same RNA silencing factors that mediate physiological silencing in response to bona fide Argonaute-guided silencing. To control for such possibilities, we used genetics to dissect the nature of the silencing pathways induced by tethering and found that tethering different factors elicited different genetic dependencies for silencing. For example, λN::NRDE-2 required nrde-4(+) activity for silencing but λN::HRDE-1 tethering did not. We have controlled for possible artifacts by initiating parallel studies on untethered factors and by using a combination of genetics, microscopy, and RNA-expression profiling. Together, these studies give us high confidence that tethering, in these instances, has faithfully replicated actual physiological steps in silencing.In this study, we use an artificial mechanism to recruit RNA silencing factors to their targets. Recruiting, factors via the λN/boxB system may elicit non-physiological mechanisms that block gene expression. For example, tethering factors to the reporter UTR could prevent proper recruitment of translation-initiation machinery or 3′ end processing factors. Transcripts that are not processed properly .Further information and requests for resources and materials should be directed to and will be fulfilled by the All materials generated in this study are available from the Original small-RNA sequencing datasets are publicly available in NCBI under the accession number BioProject: PRJNA874806.This study did not generate any new code, but the scripts used in the study are available from the Any additional information required to reanalyze the data reported in this paper is available from the C. elegans Bristol N2 (CGC) and cultured on nematode growth media (NGM) plates with E. coli OP5043 or E. coli HT115 for RNAi experiments. Strains used in this study were generated by CRISPR-cas9 method or Cross (see All the strains used in this study were derived from 44 were used to edit the genome. Plasmid pRF4 containing rol-6 (su-1006) was used as co-injection marker. For short insertions like λN and deletion mutations, synthesized single-strand DNAs were used as the donor; for long insertions like GFP, mCherry, and 5xBoxB, the annealed PCR products were used instead. The gRNA and donor sequences were listed in Ppie-1:GFP::his-58:unc-54UTR (WM701). The 5xBoxB sequence amplified from a previously published strain JMC00222 was inserted before the unc-54 UTR.The Cas9 ribonucleoprotein (RNP) CRISPR strategyYoung adult worms were transferred to glass slide in M9 buffer with 0.4mM Tetramisole. Epifluorescence and differential interference contrast (DIC) microscopy were performed on a Zeiss Axio Imager M2 Microscope and images were processed with ZEN Microscopy Software (Zeiss). Confocal images were taken by a Andor Dragonfly Spinning Disk confocal microscope. Confocal images were processed with Imaris Microscopy Image Analysis Software.2O once. Total RNA was extracted with TRIZOL and treated with DNase I to remove DNA contamination. First strand cDNA was synthesized by Superscript IV with random hexamers. Quantitative PCR was performed on a Quant studio 5 Real-time PCR machine together with Fast SYBR Green Master Mix. Actin was used as internal reference (primer set S5265 and S527). Primer set of oYD826 and oYD827 were used for reporter. All primers used were listed in Young adult worms were collected and washed with M9 for three times and ddH45 was applied to the young adult worm samples. Anti H3K9me3 antibody (Upstate 07523) and CHIP grade IgA/G magnetic beads were used for the immunoprecipitation. During elution, RNase A and Protease K were used to remove RNA and proteins. For qPCR, actin was used as internal reference. All primers used were listed in A traditional worm CHIP method6 Synchronized young adult worms were collected and total RNA were purified with Trizol. Two biological repeats were included for each strain. Small RNAs were enriched using a mirVana miRNA isolation kit. Homemade PIR-1 was used to remove the di or triphosphate at the 5′ to generate 5′ monophosphorylated small RNA. Adaptors of 3’ (DA35) and 5’ (DA4) were ligated to the small RNA by T4 RNA ligase 2 (NEB) and T4 ligase 1 (NEB) sequentially. Reverse transcription was performed with SuperScript III and RT primer (DA5). After PCR amplification, productions around 150 bp were separated by 12% SDS-PAGE and equally mixed. Libraries were sequenced on a NextSeq 550 sequencer with the illumina NextSeq 500/550 high output kit in 75bp single-end sequencing mod. Reads were trimmed by cutadapt and mapped using Bowtie2.42 For small RNAs mapped to the reporter, total reads with length longer than 16 nt were used to normalized between samples. Plots were generated by R and R studio.Small RNA cloning was conducted as previously reported.hrde-1(null) or hrde-1(Y669E) compared to WT.To determine the genes with increased or decreased antisense small RNAs  and S4C,33 with default setting. HRDE-1 structure was aligned with hAgo2 by PyMOL46 and its domains were annotated based on the alignment.The 3D structure of HRDE-1 was predicted by I-TASSER online server27 total RNAs were extracted with Trizol. SuperScript IV was used to generate the first strand DNA with reverse transcription primer oYD1001. A pair of outer primers (oYD998 and oYD1002) were used for the first round PCR amplification with Taq DNA polymerase. After 100-fold dilution, another round of PCR was performed with a pair of inner primers (oYD256 and oYD1003). PCR products were analyzed by 1.5% agarose gels. DNA bands were purified, cloned with TOPO TA Cloning Kit and sent for sanger sequencing. gsa-1 served as a control for pUG PCR analysis.As previously reported,Worms at young adult stage were dissected in Happy Buffer  . Dissected gonads were transferred to poly-lysine treated dish with 80 μl of Happy Buffer and fixed by adding equal volume of 5% formaldehyde in PBST (PBS+0.1% Tween 20) for 30 min. After one wash with PBST, gonads were treated with PBST-Triton (PBST+0.1% Triton) for 10 min, washed with PBST again and emerged in 70% ethanol for 30 min to overnight. Before hybridization, samples were washed with fresh wash buffer (2xSSC +10% formamide) for 5 min hybridization was performed at 37°C for 18 h to overnight in hybridization buffer (900 μl Stellaris RNA FISH Hybridization Buffer+ 100ul formamide) with 10 pmol RNA FISH probes. Samples were washed with wash buffer, once quick wash, one wash for 30 min at 37°C and two quick washes. Mounting medium with DAPI was added to preserve the signal. Confocal images were taken with an Andor Dragonfly Spinning Disk confocal microscope and processed with Fusion and Imaris.Same to RNA FISH, gonads were dissected, fixed and washed with PBST and treated with 70% ethanol. Then, samples were washed with wash buffer three times, one at room temperature for 5 min, one at 95°C for 3 min, and one at 60°C for 20 min. Hybridization was performed in hybridization buffer  + detection probe ) at 95°C for 5 min and then transferred to 37°C for 3 h to overnight. After hybridization, samples were wash with 2xSSC for 20 min at 60°C, and then 2xSSCT (2xSSC +0.3% Triton X-100) for 5 min at 60°C and another 20 min at 60°C.47After another wash with 2xSSCT for 5 min at room temperature, samples were preserved in the mounting medium with DAPI. Confocal images were taken with an Andor Dragonfly Spinning Disk confocal microscope and processed with Fusion and Imaris. Primary probes of DNA FISH were picked from the oligo lists generated by OligoMiner.gfp RNAi food. After 24 h, worms were dissected for the FISH experiment. RNA FISH and DNA FISH were performed as described above.Synchronous L1 worms of the reporter strain were plated on NGM plates for 48 h. Then the worms were collected and washed with M9. About 100 worms were plated on every IPTG plate with the 1


RNA viruses have developed notable strategies to evolve and escape host immunity. For non-segmented viruses, which represent most viral species, cumulative mutations (antigenic drift) are the main way of evolution. However, when two different RNA viruses infect the same cell, they can undergo two forms of recombination, both leading to antigenic shifts: RNA recombination and reassortment, with the latter restricted to viruses with segmented genomes . For exaDespite being theoretically possible in any RNA virus, the rates of recombination vary markedly among RNA viruses. Some viruses, particularly those with negative-sense single-stranded genomes, exhibit such low rates of recombination that they are effectively clonal. Although recombination is often represented as a form of sexual reproduction, there is little evidence that recombination in RNA viruses evolved as a way of creating advantageous genotypes or removing deleterious mutations. In particular, there is no association between recombination frequency and the burden of a deleterious mutation. Similarly, there is little evidence that recombination could have been selected as a form of genetic repair.Some retroviruses such as HIV recombine rapidly because their virions contain two genome copies and template switching between these copies is unavoidable during the viral replication cycle. Recombination in RNA viruses is hence likely a mechanistic by-product of the processivity of the viral polymerase that is used in replication, and it varies with genome structure. Negative-sense single-stranded RNA viruses may recombine at low rates because of the restrictive association of genomic RNA in a ribonucleoprotein complex, as well as a lack of substrates for template switching. Some positive-sense single-stranded RNA viruses exhibit high rates of recombination that can exceed the rates of single nucleotide variations. Comparison across multiple viruses suggests an inverse correlation between genome length and recombination rate. Patino-Galindo et al. detected high levels of recombination in Sarbecoviruses, HBV, HEV, Rhinovirus A, and HIV. Noteworthy recombination hotspots are found in MERS-CoV and Sarbecoviruses  . Of inteDifferent sublineages should exist, including a growing share of first-generation recombinants;Different sublineages should circulate at high levels (syndemic) to facilitate co-infection in different lineages;High levels of viral surveillance (genome sequencing), making detection and tree reconstruction feasible.Second-generation recombinants are defined as recombinants of (first-generation) recombinant lineages. To have second-generation recombinants, several conditions are needed:○HIV-1: at present, 106 circulating recombinant forms (CRFs) have been identified worldwide, with many more unique recombinant forms (URF). URFs are responsible for at least 20% of HIV-1 infections worldwide—a proportion that reaches 40% in some African countries . Inter-s○HTLV .Retroviruses:HBV X/C  and comp○Enterovirus .○Alphacoronavirus: Clade B (Serotype II) Feline coronavirus (Alphacoronavirus 1) .○▪SARS-CoV-2 ,34: whenBetacoronavirus:ssRNA viruses.XBL is an XBB.1* where part of ORF1a has been replaced with BA.2.75.* .XBY is XBF x BR.2.1.XBW is XBB.1.5 x BQ.1.14.XBV is XBB.1 x CR.1.Second-generation recombinants have been so far documented for several viruses:In conclusion, while reconstructing, further-generation recombination might sound like a mere style exercise, but it has huge implications for vaccine preparedness and seroepidemiology, recombination being a key driver of immune escape. With widespread endemic circulation, more and more second- and further-generation SARS-CoV-2 recombinant lineages are expected, with some of them posing a theoretical concern for increased disease severity and vaccine efficacy.


Metatranscriptomic analysis of RNA viromes on built-environment surfaces is hampered by low RNA yields and high abundance of rRNA. Therefore, we evaluated the quality of libraries, efficiency of rRNA depletion, and viral detection sensitivity using a mock community and a melamine-coated table surface RNA with levels below those required (< 5 ng) with a library preparation kit .Good-quality RNA libraries were obtained from 0.1 ng of mock community and table surface RNA by changing the adapter concentration and number of PCR cycles. Differences in the target species of the rRNA depletion method affected the community composition and sensitivity of virus detection. The percentage of viral occupancy in two replicates was 0.259 and 0.290% in both human and bacterial rRNA-depleted samples, a 3.4 and 3.8-fold increase compared with that for only bacterial rRNA-depleted samples. Comparison of SARS-CoV-2 spiked-in human rRNA and bacterial rRNA-depleted samples suggested that more SARS-CoV-2 reads were detected in bacterial rRNA-depleted samples. We demonstrated that metatranscriptome analysis of RNA viromes is possible from RNA isolated from an indoor surface (representing a built-environment surface) using a standard library preparation kit.The online version contains supplementary material available at 10.1186/s13104-023-06417-9. A growing interest in the presence of microbes and viruses on built-environment surfaces  has encoHowever, RNA virome analysis of built-environment surfaces is associated with several challenges. First, the amount of RNA obtained is markedly lower (in the range of picograms or less) than the input recommendations of standard library preparation kits (in the range of nanograms) owing to the extremely low biomass detected –7. StandTherefore, in this pilot study, we evaluated the impact of rRNA depletion methods on the quality of sequencing libraries, community composition, and sensitivity of virus detection using mock community RNA (0.1–10 ng) and a melamine-coated table surface RNA (< 1 ng), which had RNA amounts below the recommended input RNA amount (< 5 ng) for the library preparation kit used here. We also investigated the detection limit of SARS-CoV-2 using serial dilutions of synthetic viral RNA spiked into RNA samples from the table surface.Please refer to Additional file We used a combination of NEBNext rRNA Depletion Kit for rRNA depletion and NEBNext Ultra II Directional RNA Library Prep Kit for metatranscriptomic library preparation. The minimum recommended combined amount of total RNA for these kits is 5 ng. To test the feasibility of library preparation from RNA amounts below the protocol requirements, total RNA extracted from ZymoBIOMICS Microbial Community Standard cells was serially diluted to 10, 1.0, and 0.1 ng, and rRNA depletion and library preparation were performed. Since this mock community primarily consisted of bacteria, rRNA depletion kits for human RNA were used to preserve the abundant bacterial rRNA.Bacillus subtilis. This indicates that the combined use of the rRNA depletion kit and library preparation kit can produce high-quality libraries, even when the RNA input is reduced to 0.1 ng (100 pg). Our results are consistent with the 250–500 pg of RNA input reported previously [All three samples  were successfully prepared for library construction and sequenced. Adapter concentration and number of PCR cycles were optimally adjusted, resulting in high-quality reads  into RNA samples from the table, followed by rRNA depletion of either human or bacterial rRNA, and then performed library preparation. Abundant bacteria were assumed to be present on the table surface [5 copies of SARS-CoV-2 was also prepared.To evaluate the feasibility of library preparation from built-environment surface RNA samples with less than the recommended RNA amount for the library preparation kit, we used RNA samples from a melamine-coated table used daily by students. Simultaneously, we evaluated the impact of human or bacterial rRNA depletion on community composition and virus detection sensitivity. We spiked serial dilutions of synthetic SARS-CoV-2 RNA 101–10 copies i surface ; therefoAlthough the RNA concentrations of the table surface samples were below the limit of detection of Qubit RNA HS Assay Kit 0.25 ng/µL), all samples (except the negative control) allowed generation of sufficient libraries for sequencing Table . Thus, t5 ng/µL, All samples, including negative controls, were successfully sequenced Table . The aveWe evaluated the effect of different rRNA depletion methods on community composition Table . The estCutibacterium acnes was the most abundant. This is likely because table surfaces frequently come in contact with the human skin. In contrast, in bacterial rRNA-depleted samples, mites and nematodes  and seaweed (Ulva expansa/prolifera) were the most abundant. Since seaweed is commonly used in Japanese foods, it may have originated from meal contaminants present on table surfaces.We then focused on the community composition of the table surface , the bacterial rRNA-depleted sample (TB-s100k) showed a ~ 5 fold increase in the number of SARS-CoV-2 reads compared with the human rRNA-depleted sample .To assess the detection limit of SARS-CoV-2 RNA, we evaluated the number of SARS-CoV-2 reads obtained for the depleted samples of spiked human and bacterial rRNA using a k-mer (Kraken2–Bracken) and an alignment-based (BWA-MEM) approach. Kraken2–Bracken results indicated that no reads were detected in the negative control and non-spiked samples; however, SARS-CoV-2 reads were detected at > 103 copies were attributed to false positive results. Compared with those in the samples with 105 copies of SARS-CoV-2 (TH-s100k vs. TB-s100k), SARS-CoV-2 reads were slightly higher in the bacterial rRNA-depleted sample, although the difference was smaller than that obtained using Kraken2-Bracken. When comparing bacterial rRNA-depleted samples, SARS-CoV-2 genome coverage was 43.3% and 98.0% for the 103 (TB-s1k) and 104 (TB-s10k) copies of SARS-CoV-2 spiked-in samples, respectively (Table 3 (TB-s1k) and 104 (TB-s10k) copies of SARS-CoV-2 spiked-in samples, respectively.While Kraken2-Bracken did not detect any reads classified as SARS-CoV-2 in negative controls and non-spiked samples, BWA-MEM results revealed 81–1,437 reads mapped to the SARS-CoV-2 reference genome in those samples, signifying false positive results. Majority of these reads mapped to a polyA-containing region in the 3′ UTR of low complexity  and genome coverage (> 98%) needed to detect mutations in the SARS-CoV-2 genome in this experimental system, and the detection limit for SARS-CoV-2 RNA is 103 copies. This value corresponds to that reported in previous studies on SARS-CoV-2 surface swabbing using qRT-PCR [2 surface is required to ensure successful virus recovery and detection.In summary, at least 10 qRT-PCR . AlthougDepletion of both human- and bacteria-derived rRNA may further increase the number of virus-derived reads; therefore, we employed simultaneous depletion of both human and bacterial rRNA . The percentage of non-rRNA reads was 61.6% (THB1) and 62.6% (THB2) in the simultaneously depleted samples . Table S5 Library preparation and sequencing summary metrics of both human and bacterial rRNA-depleted samples.Additional file 3: Figure S1 Graphical view of reads mapped to the reference genome of SARS-CoV-2. The blue tracks represent the SARS-CoV-2 reference strain, and the subsequent four tracks represent Bam files of bacterial rRNA-depleted samples spiked with synthetic RNA of SARS-CoV-2 . Figure S2 Sankey diagrams of the Kraken 2 report based on human and bacterial rRNA-depleted samples (THB1 and THB2).