Lymph node metastasis is the main cause of poor prognosis of head and neck squamous carcinoma (HNSCC) patients. N6-methyladenosine (m6A) RNA modification is an emerging epigenetic regulatory mechanism for gene expression, and as a novel m6A reader protein, IGF2BP2 has been implicated in tumor progression and metastasis. However, not much is currently known about the functional roles of IGF2BP2 in HNSCC, and whether IGF2BP2 regulates lymphatic metastasis through m6A modification in HNSCC remains to be determined.The expression and overall survival (OS) probability of m6A-related regulators in HNSCC were analyzed with The Cancer Genome Atlas (TCGA) dataset and GEPIA website tool, respectively. The expression levels of IGF2BP2 were measured in HNSCC tissues and normal adjacent tissues. To study the effects of IGF2BP2 on HNSCC cell metastasis in vitro and in vivo, gain- and loss- of function methods were employed. RIP, MeRIP, luciferase reporter and mRNA stability assays were performed to explore the epigenetic mechanism of IGF2BP2 in HNSCC.We investigated 20 m6A-related regulators in HNSCC and discovered that only the overexpression of IGF2BP2 was associated with a poor OS probability and an independent prognostic factor for HNSCC patients. Additionally, we demonstrated that IGF2BP2 was overexpressed in HNSCC tissues, and significantly correlated to lymphatic metastasis and poor prognosis. Functional studies have shown that IGF2BP2 promotes both HNSCC cell migration as well as invasion via the epithelial-mesenchymal transition (EMT) process in vitro, and IGF2BP2 knockdown significantly inhibited lymphatic metastasis and lymphangiogenesis in vivo. Mechanistic investigations revealed that Slug, a key EMT-related transcriptional factor, is the direct target of IGF2BP2, and essential for IGF2BP2-regulated EMT and metastasis in HNSCC. Furthermore, we demonstrated that IGF2BP2 recognizes and binds the m6A site in the coding sequence (CDS) region of Slug and promotes its mRNA stability.Collectively, our study uncovers the oncogenic role and potential mechanism of IGF2BP2, which serves as a m6A reader, in controlling lymphatic metastasis and EMT in HNSCC, suggesting that IGF2BP2 may act as a therapeutic target and prognostic biomarker for HNSCC patients with metastasis.The online version contains supplementary material available at 10.1186/s13046-021-02212-1. Head and neck squamous cell carcinoma (HNSCC), which can develop in the larynx, pharynx, and oral cavity, ranks as the sixth leading malignancy worldwide, resulting in approximately 350,000 deaths each year , 2. The 6-methyladenosine (m6A) readers, which are capable of regulating and recognizing the m6A modification of target mRNAs and crucial for their oncogenic functions [An RNA binding protein (RBP) known as the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is related to the regulation of various cellular and biological processes , 9. Abnounctions .Among the hundreds of internal RNA modifications, m6A is considered to be the most prevalent type in eukaryotic mRNA –22. The The primary cause of over 90% of mortality related to cancer is tumor metastasis , 34, andIn this study, we report a direct correlation between increased expression of IGF2BP2 with lymphatic metastasis and decreased survival of HNSCC patients. Our results show that knockdown of IGF2BP2 resulted in a significant reduction in lymphatic metastasis and lymphangiogenesis in a popliteal LN metastasis model. Mechanistically, we identified that Slug is the direct target mRNA of IGF2BP2 and is critical to the EMT process and metastasis in HNSCC. More importantly, we demonstrated that IGF2BP2 recognizes and binds the m6A site in the coding sequence (CDS) of Slug and stabilizes its mRNA.https://portal.gdc.cancer.gov/), which contained 502 HNSCC cases and 44 normal cases. Analysis and visualization of the genes’ expression was conducted with R software (version 4.0.3) packages ‘ggplot2’ and ‘pheatmap’. The GEPIA website tool (http://gepia2.cancer-pku.cn/#index) was used to conduct the OS analysis. Analysis and visualization of the Kaplan-Meier survival analysis was performed with the R software packages ‘survival’ and ‘survminer’. Multivariate cox regression analysis was conducted in order to determine the fitting terms, which were used for building the nomogram. The ‘forestplot’ package, found in R, was used to identify each variable’s P value, hazard ratio (HR), and 95% confidence interval (CI). To predict the 1-, 3-, and 5-year overall recurrence, we developed a nomogram in conformity with the results of the multivariate Cox proportional hazards analysis. The nomogram is a visual representation of factors, and can be used for calculating the risk of recurrence for a distinct patient through summing up the points correlated to each risk factor in the ‘rms’ package of R. The two-gene correlation map was constructed with the ‘ggstatsplot’ package in R software based on Spearman’s correlation analysis. SRAMP (http://www.cuilab.cn/sramp/), a motif-dependent m6A site predictor, was used to predict m6A modification sites on the RNA sequences of interests.RNA-sequencing data (level 3) in raw form and related clinical data regarding HNSCC were derived online from The Cancer Genome Atlas (TCGA) dataset , and 20 fresh HNSCC tissues with or without lymphatic metastasis (consisting of 10 cases with lymphatic metastasis and 10 cases without lymphatic metastasis) were retrieved from HNSCC patients who received surgery at the Department of Otorhinolaryngology of the First Affiliated Hospital of Chongqing Medical University . Fresh tissues were washed with saline after resection, and liquid nitrogen was used to freeze them immediately after which they were kept at − 80 °C for total protein and RNA extraction. Furthermore, in total 78 HNSCC tissue specimens embedded in paraffin were collected from the Department of Pathology of the First Affiliated Hospital of Chongqing Medical University. The 78 HNSCC patients underwent surgery between January 2012 and December 2019 in the Otolaryngology department of the First Affiliated Hospital of Chongqing Medical University. The clinical features of the patients are detailed in Table 2.FaDu cells were obtained from the Chinese Academy of sciences . SCC15 cells were the kind gift of Prof. Kai Yang . FaDu and SCC15 cells were both kept in Dulbecco’s modified Eagle’s medium , to which 10% fetal bovine serum  and 1% penicillin-streptomycin was added, in an atmosphere of 37 °C and 5% COIHC was performed with a detection kit  in accordance with the manufacturer’s instructions. In short, fresh xylene was used to deparaffinize the paraffin sections, which were then hydrated in gradient alcohol. The citric acid buffer was used for antigen retrieval at 90 °C–100 °C for 30 min to which an appropriate amount of endogenous peroxidase blocker was added. The standard goat serum working solution was sealed at room temperature for 15 min, and then transferred to a refrigerator of 4 °C and incubated with IGF2BP2 and LYVE-1 primary antibodies overnight. On the second day, horseradish enzyme-labeled streptavidin was dripped into the working solution, then incubated for 15 min at room temperature, and rinsed with PBS 3 times for 3 min each time. Diaminobenzidine (DAB) was used for color development, followed by hematoxylin staining for 30 s, and rinsing with tap water for 5 min. Finally, it was dehydrated, transparent and mounted with neutral gum. The IHC stained sections were reviewed and scored independently by two superior pathologists. A final score was then calculated by multiplying the signaling intensity score and the staining distribution score. The signal intensity scores were categorized as follows: 0 , 1 (weak), 2 (moderate), and 3 (strong). The staining distribution scores were determined according to the percentage of positive cells: 0 (0–5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), 4 (75–100%). The median value was chosen as the cutoff.® Total RNA Kit I  was utilized for isolation of the total RNA from HNSCC specimens and cells according to instructions of the manufacturer. Subsequently, an Agilent 2100 Bioanalyzer  was used to determine the total RNA’s quality through absorbance readings at 260 nm. Reverse transcription of total RNA into complementary DNA (cDNA) was done with a gDNA Eraser  using a PrimerScript™ RT Reagent Kit. Next, for the amplification process an ABI 7500 Real-Time PCR System  and a SYBR Premix Ex Tag™ Kit  were utilized in accordance with instructions of the manufacturer. The conditions in which the PCR amplification was conducted was as follows: 40 cycles in total at 95 °C for the duration of 30 s, then 95 °C for 5 s, and lastly 60 °C for 1 min. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) functioned as internal control. The 2-ΔΔCt method was applied for analysis of the results, which were presented as relative expression. All experiments were performed three times to independently verify the findings. The primers for PCR analysis are listed in Additional file An E.Z.N.A.Total proteins from HNSCC specimens and cells were derived with a protein extraction kit . A bicinchoninic acid (BCA) protein assay kit  was utilized for the detection of the concentration of protein. Next, protein extracts were diluted in 5 × loading buffer and boiled for 10 min. The boiled proteins were subjected to 10% SDS-PAGE, placed onto polyvinylidene fluoride (PVDF) membranes, and blocked with 5% nonfat dry milk for the duration of 2 h. Subsequently, we incubated the membranes overnight at 4 °C with primary antibodies, of which the details are shown in Additional file 5 cells/well, in six-well plates with culture medium, and incubated till 60%–70% confluence. Then, Lipofectamine iMAX Reagent  was utilized for transfection of the cells with siRNAs at a ratio of 1:3 and incubated for 24 h in serum-free medium. 24 h later, we discarded the transfection medium, and fresh complete culture medium without siRNA and Lipofectamine iMAX was added. At the timeframe of 48 h or 72 h following transfection, we collected the cells for protein or RNA extraction to conduct RT-qPCR and western blotting to validate the transfection efficiency. The IGF2BP2 target and negative control sequences are listed in Additional file To reduce the off-target effect of the siRNAs, three independent siRNAs targeting IGF2BP2 and a negative control siRNA were constructed and generated by GenePharma . In brief, HNSCC cells were seeded, at a concentration of 2 × 104 cells/well and incubated until roughly 30% confluence was reached. Then, the cells were infected with shRNA or overexpression lentiviral vectors with HiTransG A infection-enhancing solution according to the multiplicity of infection . After incubating for 16 h at 37 °C, the viral medium was discarded and substituted by fresh culture. Then, the cells were screened in culture medium containing puromycin (2 μg/ml) for 1 week and stably silenced- and overexpressed-IGF2BP2 HNSCC cells were generated.For knockdown, short hairpin RNA (shRNA) of human IGF2BP2 was cloned into a hU6-MCS-Ubiquitin-firefly_Luciferase-IRES-puromycin lentiviral vector . To achieve overexpression, human full-length cDNA of IGF2BP2 was cloned into a Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin lentiviral vector . The target sequences of IGF2BP2 have been detailed in Additional file Wound-healing and transwell assays were carried out in vitro for the detection of cell migration and invasion. The detailed procedures of these assays have been described in our previous study .6 FaDu cells were transfected with sh-IGF2BP2-luc and sh-NC-luc, suspended in 60 μl PBS, and then injected into the footpads of the mice. Six weeks after injection, mice were subjected to bioluminescence imaging to evaluate lymphatic metastasis. For bioluminescence imaging, mice were anesthetized by inhaling 2% isoflurane for approximately 5 min, injected intraperitoneally with D-Luciferin potassium salt , and imaged with a bioluminescence system . All the primary tumors and popliteal LNs were harvested and embedded in paraffin for IHC analysis. The LN volumes were calculated based on this formula: LN volume (mm3) = length × width2 × 0.5. All the experiments were authorized by the Laboratory Animal Use Management Committee of the Experimental Animal Center of Chongqing Medical University.Male BALB/cA nude mice aged 4–6 weeks and with a weight between 18 and 22 g were obtained from Huafukang Biotechnology Co., , and accommodated in specific pathogen free (SPF) barrier facilities. To construct the metastasis model, 5 × 10FaDu and SCC15 cells seeded on sterile coverslips were fixated with 4% paraformaldehyde for the duration of 20 min, then permeabilized with 0.1% Triton X-100 for 15 min, and lastly blocked with goat serum at room temperature for a total of 30 min. Subsequently, primary antibodies were utilized for incubation of the cells overnight at 4 °C, of which the details are shown in Additional file 7 cells each plate), were accumulated and then lysed with IP lysis buffer  supplemented with protease inhibitor (100×) and RNase inhibitor (40 U/μl) on ice for the duration of 30 min. The cell lysates, pipetted up and down several times, were stored in − 80 °C for 5 min, then allowed to thaw on ice, and centrifuging at 12,000 g for 10 min. Then the cell lysates were divided into two parts, one was saved as input group for the whole cell extraction, and the other was used for the following immunoprecipitation (IP) treatment (IP group). For the IP group, cell lysates were incubated with 5 μg anti-IGF2BP2  or IgG antibody , respectively, which was alternated continually throughout the night at 4 °C. Protein A/G magnetic beads  were rinsed five consecutive times with 0.1% Tween-20 in PBS, and then mixed with cell lysate-antibody complexes and rotated continuously at 4 °C for 6 h. Next, the RNA-protein complexes were rinsed five consecutive times with elution buffer  and treated with proteinase K at 55 °C for 1 h. Bound RNAs were extracted and subjected to RT-qPCR for quantitative analysis. Relative enrichment was normalized to the input. The primers for RT-qPCR were showed in Additional file HNSCC cells at 90% confluence, cultured in 10-cm plates  or mouse IgG were immobilized on magnetic beads using the Dynabeads™ Antibody Coupling Kit  according to the manufacturer’s instruction. Subsequently, total RNA was incubated with antibody-conjugated beads in 500 μl binding buffer  and rotated continually for 4 h at 4 °C. M6A-modified mRNAs were eluted from the beads with elution buffer and then purification was carried out for further analysis by RT-qPCR. Relative enrichment was normalized to the input. The primers for RT-qPCR were showed in Additional file 5 HNSCC cells were first seeded in plates with 24-wells, followed by culturing for 24 h. Then plasmids carrying wild-type or mutated-type Slug CDS were transfected into the cells. After transfection for 12 h, cells were re-seeded into a plate with 96-wells and then for 24 h incubated. The Dual-Luciferase® Reporter Assay System  was used to analyze the luciferase activities. Renilla Luciferase (R-luc) was used to achieve normalization of the firefly luciferase (F-luc) activity.2 × 10HNSCC cells were seeded in plates with six-wells and grown to approximately 50% confluence following incubation for 24 h. Then, actinomycin D  was used to treat the cells, which were harvested at 0 h, 3 h, and 6 h. The total RNA was derived and then analyzed with qRT-PCR. The mRNA value of each group was calculated and normalized to GAPDH at the designated time. The degradation rate of mRNA was approximated based on previously published protocols .t test to compare results between two different groups, and the one-way ANOVA was conducted for multiple comparisons. All the data are presented in the form of mean ± SD and are the result of no less than 3 independently performed experiments. Statistical significance was determined as values of P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not statistically significant.All the statistical analysis were conducted with GraphPad Prism  and SPSS 21.0 software . The correlation between IGF2BP2 expression and clinicopathologic parameters were analyzed with the Chi-Square test. Univariate and multivariate regression analyses were employed with the Cox proportional hazards model to identify independent factors affecting the survival of HNSCC patients. The Kaplan-Meier method was carried out to produce survival curves, and any significant differences in survival probability between groups were compared by log-rank statistics. We applied the two-tailed Student’s We analyzed the expression profiles of 20 m6A-related regulators in 502 HNSCC and 44 normal tissues from the TCGA database in order to study the role of m6A modification in HNSCC. As shown in Fig. We initially examined IGF2BP2’s expression in HNSCC and NATs with RT-qPCR and western blot to evaluate its role in HNSCC. The RT-qPCR analysis demonstrated that IGF2BP2 was significantly overexpressed in HNSCC tumor samples compared with NATs Fig. . UnivariLN metastasis is a complex process involving multiple alterations, including an enhanced ability in cell migration and invasion , 49. To To further determine the role of IGF2BP2 in lymphatic metastasis of HNSCC, we used nude mice to establish a popliteal LN metastasis model Fig. A, as menEMT is a critical step in the initiation of tumor metastasis . To furtSubsequently, we determined the role of IGF2BP2 in HNSCC cells undergoing EMT. RT-qPCR and western blot analyses showed that IGF2BP2 depletion downregulated N-Cadherin and vimentin’s expression levels, whereas that of E-Cadherin was upregulated at the mRNA as well as protein levels in FaDu and SCC15 cells treated with TGF-β Fig. . Taken thigh-expressed HNSCC tissues to IGF2BP2low-expressed HNSCC tissues to normal adjacent tissues were observed based on the TCGA database  , such as Snail, Slug, ZEB1, and Twist , 38, we ase Fig. B. In ordase Fig. C-F. Meanase Fig. C. Althouase Fig. G. Converase Fig. G. Furthease Fig. H. To evaase Fig. I. Based 01) Fig. J. CollecSince IGF2BP2 is considered to be an RNA binding protein, also known as “reader” in m6A RNA modification , 9, 19, The primary cause of a poor prognosis in HNSCC patients is LN metastasis , 48. AltIGF2BP2, which acts as a novel m6A reader, is related to different biological processes, for instance, cell self-renewal, stemness maintenance, proliferation, and metastasis –12. PrevDuring the LN metastasis process, one important step is that tumor cells gain the capability of migration and invasion, and then move toward the lymphatic capillaries , 49. It The biological importance of m6A modification is determined by m6A readers that can recognize and directly bind to the m6A modification sites upon its target mRNAs, thus affecting their fate , 21. To There are also some limitations in this study. First, the overall number of specimens is limited because access to tissue is invariably difficult. Second, although our findings reveal the oncogenic role of IGF2BP2 in promoting the lymphatic metastasis and EMT process, the regulatory mechanism between LN metastasis and EMT is not yet fully explained. This is probably because cancer cells undergoing EMT acquire the capacity to hijack a variety of chemotactic signals and apply them to disseminate through the lymphatic system . HoweverIn summary, our study reveals for the first time the clinical and biological function of IGF2BP2 in facilitating lymphatic metastasis in HNSCC, and demonstrated that IGF2BP2 promotes EMT and cell metastasis via stabilizing Slug mRNA in an m6A dependent-manner. These outcomes indicate that IGF2BP2 could function as a predictive biomarker of LN metastasis and potential target for anti-metastasis therapies for HNSCC patients.Additional file 1.Additional file 2.


EML4-ALK, CCDC6-RET, and TPM3-NTRK1) were successfully identified. We confirmed the fusion breakpoint sequences of the three fusions from HD796 by Sanger sequencing. Regarding the limit of detection, this panel could detect the target fusions from a tumor sample containing a 1% fusion-positive tumor cellular fraction. Taken together, our ThyChase panel would be useful to identify gene fusions in the clinical field.In addition to mutations and copy number alterations, gene fusions are commonly identified in cancers. In thyroid cancer, fusions of important cancer-related genes have been commonly reported; however, extant panels do not cover all clinically important gene fusions. In this study, we aimed to develop a custom RNA-based sequencing panel to identify the key fusions in thyroid cancer. Our ThyChase panel was designed to detect 87 types of gene fusion. As quality control of RNA sequencing, five housekeeping genes were included in this panel. When we applied this panel for the analysis of fusions containing reference RNA (HD796), three expected fusions ( Thyroid cancer is one of the most rapidly increasing cancers throughout the world, including South Korea . AlthougRET oncogene can be overactivated through gene fusion [RET/PTC rearrangement in papillary thyroid cancer (10%–30%) and PPARG-PAX8 rearrangement in follicular thyroid cancer (30%–60%) [RET, THADA, NTRK1, NTRK3, ALK, BRAF, MET, and FGFR2 [NTRK1/3 fusions have been reported to be associated with advanced tumor stage and aggressive lymphovascular invasion [ALK fusions have been suggested to have a higher likelihood of dedifferentiation [Next-generation sequencing (NGS) technology and efforts to identify genetic alterations in cancers, such as the Cancer Genome Atlas, have revealed genetic alteration profiles in diverse cancers . In addie fusion . The mosnd FGFR2 ,10. For invasion -13. Tumontiation . TherefoVIT-ALK in lung adenocarcinoma [From a technical standpoint, the detection of fusion genes by DNA-based NGS is almost impossible due to the presence of diverse-sized intronic sequences between the fusion target exons. Therefore, RNA-based NGS panel analyses are commonly conducted to detect the target fusions in thyroid cancer in addition to the use of DNA-based NGS to detect somatic mutations. RNA-based NGS panels should include housekeeping genes. Since housekeeping genes are expressed in all tissue compartments and cell types, they can be used for quality control and normalization of NGS data . In addiarcinoma ,17. Multarcinoma . Among tarcinoma . This paIn this study, we aimed to develop a custom RNA-based NGS panel to identify the important fusion events in thyroid cancer. In addition to the key fusions in thyroid cancer, uncommonly reported fusions and fusion subtypes were also included in this panel.EML4-ALK, CCDC6-RET, SLC34A2-ROS1, TPM3-NTRK1, and ETV6-NTRK3 fusions. HD783 is an FFPE sample that does not contain those fusions. RNA was extracted from the FFPE sample using an FFPE Total RNA Miniprep System kit . The quality and quantity of RNA samples were determined using a NanoDrop 2000c spectrophotometer .In this study, we used two standard materials: HD796  as a fusion-positive control and HD783 (Horizon Discovery) as a fusion-negative control. HD796 is a formalin-fixed paraffin-embedded (FFPE) tissue that contains the cDNAs were synthesized using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) and used for NGS library preparation. The libraries were manually constructed using our custom thyroid fusion panel, ThyChase. The amplicon library was prepared with the Ion Plus Fragment Library Kit  and the Ion Xpress Barcode Adapters Kit (Life Technologies) according to the manufacturer's instructions. In detail, 10 μL of cDNA was amplified in reaction mixtures of 59 μL containing 45 μL of Platinum PCR SuperMix High Fidelity and 4 μL of ThyChase panel. Polymerase chain reaction (PCR) was performed with a GeneAmp 9700 thermal cycler (Thermo Fisher Scientific) under the conditions of 95°C for 2 min followed by 35 cycles of 95°C for 15 s, 58°C for 15 s, 68°C for 10 s, and a final hold at 4°C. Libraries were purified using 106 μL of AMPure XP Reagent  on a magnetic stand (Thermo Fisher Scientific) and eluted with 25 μL of low tris-EDTA buffer. Then, adapter ligation and nick repairing were performed to make barcode sequencing adapters . Finally, the libraries were quantified using quantitative PCR  on a QuantStudio 12K Flex Real-Time PCR System qPCR machine (Thermo Fisher Scientific).Emulsion PCR, bead enrichment, and chip loading procedures were automatically performed on an Ion Chef instrument (Thermo Fisher Scientific) using Ion 510, 520, and 530 Kits (Thermo Fisher Scientific). A planned run was created for each chip within Ion Torrent Suite Software v5.12.1 (Thermo Fisher Scientific) with the template size set at 200 bp. The NGS libraries were then sequenced on an Ion S5 XL sequencer (Thermo Fisher Scientific) .Raw sequence data were analyzed with the Torrent Suite . A custom reference genome was assembled to contain sequences of the 87 designed target fusions and five housekeeping genes based on hg19. To call the mapped sequence data, we used Torrent Coverage Analysis (version 5.12.0.0). More than five support reads were considered as fusion-positive. The identified fusions were then manually inspected in the Integrative Genomics Viewer . The mean sequencing depth was 5,189×  across the entire target region . The datEML4-ALK fusion-positive) by mixing it with the RNA extracted from the FTC-133 cell line (EML4-ALK fusion-negative) from 100% to 0.5%. The RNAs were subjected to RNA sequencing using the ThyChase panel. To verify the fusions identified by the ThyChase panel, we performed Sanger sequencing of the fusion amplicons.To determine the limit of detection (LOD) of fusions, we diluted the RNA extracted from the NCI-H2228 cell line  are known to have multiple fusion partners, while the other seven are known to have a single fusion partner. Details of the fusion genes and their fusion partners are listed in EML4-ALK fusion   . In tota of ALK) . In addiEML4-ALK, CCDC6-RET, TPM3-NTRK1, SLC34A2-ROS1, and ETV6-NTRK3 gene fusions [EML4-ALK, CCDC6-RET, and TPM3-NTRK1) were included in our ThyChase panel. The ETV6-NTRK3 gene fusion was also included in ThyChase; however, the ETV6-NTRK3 fusion breakpoint of the HD796 RNA was different from the fusion breakpoint covered by ThyChase. Therefore, we targeted the three gene fusions for optimization and validation of ThyChase. HD783 RNA was used as a fusion-negative control that did not harbor any of the above-mentioned gene fusions [We used HD796 RNA as a fusion-positive standard material. HD796 contains  fusions . Of the  fusions .2, suggesting that our custom thyroid gene fusion panel is suitable for RNA sequencing  fusions in the HD796 reference RNA. RNA panel sequencing was performed based on the optimized reaction conditions described above. As expected, all three fusion targets were successfully detected in the HD796 RNA, whereas they were not identified in the HD783 RNA , which is known to harbor the K fusion . As expeell line . ThroughEML4-ALK fusion-positive) samples that were diluted with FTC-133 (EML4-ALK fusion-negative) by different dilution factors (100% to 0.5%). As a result, the read count of EML4-ALK fusion decreased in a dose-dependent manner from 100% to 0.5%, and we could identify fusion-supporting reads from the 0.5% fusion-positive tumor cellular fraction , CCDC6-RET (exon 1 of CCDC6 and exon 12 of RET), and TPM3-NTRK1 (exon 7 of TPM3 and exon 10 of NTRK1) fusions  were successfully identified. We also confirmed that this fusion-focused panel could identify the target fusions from a cancer cell line in addition to the fusion-positive reference RNA. In terms of the LOD, this panel could detect the target fusions from a tumor sample containing a 1% fusion-positive tumor cellular fraction. We finally verified the fusion breakpoint sequences of the three fusions from HD796. Although we could not verify all of the designed fusions in this study due to limitations of the fusion reference materials, all the data in this study indicate that the ThyChase panel can reliably identify the key fusions in thyroid cancer. Taken together, the ThyChase panel would be useful to identify gene fusions in the clinical field.In conclusion, we developed an RNA-based sequencing panel focused on identifying fusions in thyroid cancer. The ThyChase panel was designed to detect 87 gene fusion types. As quality control for RNA sequencing, five housekeeping genes were included in this panel. When we applied this panel for the analysis of fusions contained in the reference RNA (HD796), the three expected fusions (


Coral reefs are the poster child for ocean biodiversity—a marine equivalent of the terrestrial tropical rainforest in their scientific importance and public perception. Compared with most of the ocean, coral reefs harbour an incredible variety of biodiversity, and this diversity is also fairly well studied so that many of the basic interactions making up the ecosystem are understood at some level. The foundation of the system is obviously coral, particularly reef-building, scleractinian corals. The calcium carbonate skeletons secreted by these animals are literally the bedrock of the reef and create habitats that other reef creatures depend upon . At thisToxoplasma, Cryptosporidium, or the malaria agent Plasmodium) and are now called Corallicolida . Th. Th15]. Chlorophyll is a member of the porphyrin family of molecules, and several other products of this pathway serve various roles in the cell  , so one


GAPDH, GPI, and HPRT1) across all samples. Of these, 48 samples were used for RNA sequencing, which had moderate quality based on MultiQC evaluation . Analysis of clot histology showed that more acellular samples yielded RNA of lower quantity and quality. We obtained useful mRNA from AIS clot samples stored in RNALater. qPCR analysis could be performed in almost all cases, while sequencing data could only be performed in approximately two-thirds of the samples. Acellular clots tended to have lower RNA quantity and quality.Mechanical thrombectomy (MT) for large vessel acute ischemic stroke (AIS) has enabled biologic analyses of resected clots. While clot histology has been well-studied, little is known about gene expression within the tissue, which could shed light on stroke pathophysiology. In this methodological study, we develop a pipeline for obtaining useful RNA from AIS clots. A total of 73 clot samples retrieved by MT were collected and stored in RNALater and in 10% phosphate-buffered formalin. RNA was extracted from all samples using a modified Chemagen magnetic bead extraction protocol on the PerkinElmer Chemagic 360. RNA was interrogated by UV–Vis absorption and electrophoretic quality control analysis. All samples with sufficient volume underwent traditional qPCR analysis and samples with sufficient RNA quality were subjected to next-generation RNA sequencing on the Illumina NovaSeq platform. Whole blood RNA samples from three patients were used as controls, and H&E-stained histological sections of the clots were used to assess clot cellular makeup. Isolated mRNA was eluted into a volume of 140 µL and had a concentration ranging from 0.01 ng/µL to 46 ng/µL. Most mRNA samples were partially degraded, with RNA integrity numbers ranging from 0 to 9.5. The majority of samples (71/73) underwent qPCR analysis, which showed linear relationships between the expression of three housekeeping genes ( Acute ischemic stroke (AIS) is the fifth leading cause of death worldwide. In the U.S., about 7 million people over the age of 20 have had a stroke, and every year approximately 800,000 Americans will experience a stroke, causing 130,000 deaths and persistent neurological deficits in many survivors. The use of mechanical thrombectomy (MT) in the treatment of AIS is now mainstream therapy for large vessel occlusions. The advent of this technology has enabled the biologic investigation of resected clot material. Histological analyses have demonstrated clot composition could be used to inform clinical management of AIS patients. For example, the fibrin/platelet (FP), red blood cell (RBC), and white blood cell (WBC) content of the clot have been associated with AIS etiology , and thuTranscriptome profiling is a powerful tool that reveals RNA expression patterns that define cellular responses associated with biologic processes and disease states. These data are valuable in both understanding biologic and pathobiologic cellular mechanisms and developing useful biomarkers. Hence, clot mRNA could be extremely useful for such purposes. However, since retrieved clot samples are often small and may be acellular (in the case of fibrin-rich clots), isolating high volumes of high-quality RNA may be difficult. To our knowledge, only two reports in the published literature have demonstrated analysis of RNAs extracted from retrieved AIS clot samples ,6. HowevHerein, we sought to demonstrate an RNA isolation pipeline that we tailored to the PerkinElmer Chemagic 360 platform. Combining RNALater-based collection/storage, manual sample disruption, and Chemagen magnetic bead extraction, RNA was isolated for use in downstream applications. We performed both traditional qPCR and next-generation RNA sequencing on clot RNA and RNA isolated from peripheral whole blood, as a control. Furthermore, we investigated the histology of the clots to determine if RNA quality was affected by clot composition. We hope this report provides a useful pipeline that can enable further investigations of RNA expression in clot samples retrieved from large vessel AIS cases.This study was approved by the University at Buffalo Institutional Review Board (STUDY00002092). All methods were carried out in accordance with the approved protocol and written informed consent was obtained from all subjects. We included patients in this study if they were receiving MT procedures for the treatment of AIS and had sufficient clot material collected. Patients without usable clot tissue samples were excluded.From 9/2018 and 11/2020, clot samples were collected from patients receiving MT by either stent retriever, aspiration, or combined therapy at the Gates Vascular Institute in Buffalo, NY. Immediately after the procedure, participating clinicians placed portions of the retrieved clot material into production-sterile FluidX tubes . One FluidX tube was filled with RNALater RNALater , a storage reagent that quickly penetrates tissue samples to stabilize and protect RNA and inactivate RNases. Another FluidX tube was filled with 10% phosphate-buffered formalin to facilitate downstream histological analyses. After 24 h at room temperature, samples in RNALater were stored at 4 °C for 24 h, then transferred to −80 °C until further processing. Samples in formalin were allowed to fix at room temperature for 24–48 h then transferred into 70% ethanol until further analysis. For a subset of patients, arterial whole blood was collected from the access sheath during the procedure and placed into one 2.5 mL PAXgene Blood RNA tube . After 24 h at room temperature, these samples were stored at −20 °C for 24 h, then transferred to −80 °C until further processing.3 to 10 mm3) was removed from the RNALater and placed directly into lysis buffer, followed by manual disruption via shearing for ~5 min. Disrupted samples were then transferred to a 24-well plate, incubated in lysis buffer with proteinase K, then processed on the high-throughput PerkinElmer Chemagic 360 instrument that uses Chemagen Technology, as described by the manufacturer . In brief, RNA was captured through highly specific binding to DNAseI-treated M-PVA magnetic beads that are attracted to transiently magnetized metal rods. To avoid contamination in the procedure, the rods brought the RNA-bound beads through a series of process solutions (including DNAseI treatment), serial wash buffers, and elution buffers that ultimately yield purified RNA. Whole blood RNA was processed using a complementary protocol and extracted directly from 500 µL aliquots of PAXgene blood RNA tubes. For both clot and whole blood, RNA was eluted in a final volume of 140 µL.For RNA isolation, the clot material  or a NanoDrop 2000 spectrophotometer . A ~50 µL aliquot was reserved for sequencing. For library preparation, RNA concentration was first more precisely measured via the Qbit Assay  with a TBS-380 Fluorometer . The quality of the RNA samples was measured on an Agilent 2400 Tape Station or an Agilent 2100 BioAnalyzer . RNA samples to be sequenced needed to have acceptable quality, as measured by the RNA integrity number (RIN > 3.0). In the case of unreadable and lower RIN (<3.0), samples were still sequenced if inspection of the electropherogram showed rRNA peaks with minimal gDNA contamination (the average RIN of sequenced RNA was 3.1).GAPDH, GPI, and HPRT1. For each gene, oligonucleotide primers with an ~60 °C melting temperature and a length of 15–25 nucleotides were designed to produce PCR products with lengths of 50–250 base pairs via Primer3 software and Primer-BLAST . The replication efficiency of each primer had been tested by qPCR on serial dilutions of pooled cDNA  as previously described ,8,9. PriFor reverse transcription, cDNA was generated from total RNA using SuperScript III Reverse Transcriptase Kit  according to the manufacturer’s instructions. Quantitative PCR was run with 10 ng of cDNA in 25 μL reactions in duplicate in the Bio-Rad CFX Connect system  using the qScript One-Step SYBR Green Master Mix Kit , and gene-specific primers at a concentration of 0.02 μM each. The temperature profile consisted of an initial step of 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and then a final melting curve analysis from 60 °C to 95 °C for 20 min. Gene-specific amplification for these primers had been demonstrated by a single peak using the Bio-Rad dissociation melt curve. The Ct values for each housekeeping gene were recorded for all samples.For RNA sequencing, we used the Illumina TruSeq RNA Library Preparation Kit  that employed a poly-A pull-down for mRNA isolation. Library preps were assessed by the Agilent 2100 BioAnalyzer RNA 6000 Pico Chip . RNA libraries were then subjected to 100-cycle, paired-end sequencing in a NovaSeq6000 system  and demultiplexed using Bcl2Fastq. Per-cycle base-call (BCL) files generated by the Illumina NovaSeq6000 were converted to per-read FASTQ files using bcl2fastq version 2.20.0.422 using default parameters. The quality of the sequencing was reviewed using FastQC version 0.11.5. Detection of potential contamination was done using FastQ Screen version 0.11.1 [To analyze the quality of the RNA sequencing data, we performed several analyses in R. We used DESeq2 version 1.32.0   under the default settings, as described elsewhere [For histology, formalin-fixed clots (stored in 70% ethanol) were embedded in paraffin blocks, and sectioned at 4 µm on a microtome. Sections were stained with Hematoxylin and Eosin (H&E) stain, as described previously . The stalsewhere . Prior tA total of 73 AIS patients  treated by mechanical thrombectomy met the inclusion criteria for this study . The clip = 0.020, Student’s t-test). Bioanalyzer analysis showed that clot RNA had an average RIN of 3.1 (range = 0–9.5) and whole blood RNA had an average RIN of 6.8 (range = 4.8–8.1). Based on the concentration, the RIN, and assessment of the Bioanalyzer traces to rule out genomic DNA contamination, we selected 48 (66%) clot RNA samples and all three whole blood samples for RNA sequencing analysis.2 = 0.84 for GPI vs. GAPDH, R2 = 0.70 for HPRT1 vs. GPI, and R2 = 0.64 for GAPDH vs. HPRT1).To determine clot RNA viability in downstream analyses, we performed qPCR on all samples except 2 that did not have sufficient sample volume for the reactions. Only seven of the samples failed qPCR testing, meaning that for two or more housekeeping genes, their Ct values were >35  prior to sequencing. From the FastQC and MultiQC reports , the seqn = 52 of the cases. p = 0.042, Student’s t-test)  C,D. Ther t-test) .The advent of thrombectomy therapy for large vessel AIS has enabled analyses of the retrieved clot material . The bioIL-1β, IL-6, IL-8, IL-18, TNFα, MCP-1, MMP-2, and MMP-9. However, they did not provide any RNA quantity and quality metrics and did not follow standard guidelines for qPCR .Transcriptome profiling is a powerful tool for revealing expression patterns that are associated with complex diseases ,15,16, sGAPDH expression in all isolated RNA samples using qPCR.Other groups have reported successful isolation of quality RNA from other compromised vascular tissue samples. For example, Zakaria et al. used TRIzol and a column-based method (Qiagen QIAamp) to isolate RNA from frozen coagulated blood samples (average concentration = 26.3 ng/μL and average 260/280 = 1.71) and demonstrated qPCR analyses . AdditioBased on these previous reports, and our own unsuccessful attempts using column-based RNA extraction kits and phenol–chloroform-based methods, we chose to implement a magnetic bead-based method for isolating RNA that was combined with manual tissue disruption and proteinase K treatment. This protocol reduced human errors and low extraction efficiencies that can accompany phenol–chloroform and spin column-based techniques, because of the high binding affinity of the beads. This also aids in the isolation of nucleic acids from smaller tissues with low cellular composition. Based on our QC analyses, this method was able to extract RNA with comparable concentration, 260/280, and RIN values to that of Zakaria et al.  and AhmeGAPHD expression was detectable in all samples, but GPI and HPRT1 could not be detected in several samples, indicating a higher concentration of cDNA may be needed. Overall, linearity between Ct values among different housekeeping genes demonstrates stability in expression in the majority of RNA samples, indicating they can be used for downstream qPCR. On the other hand, fewer samples were amenable to RNA sequencing. Due to the fairly degraded nature of the samples, we opted to implement a library preparation assay to enrich mRNA using a poly-A pulldown. This may have been less ideal than rRNA depletion techniques , but preliminary tests (not shown) demonstrated better quality libraries using a poly-A selection. Despite this compromise, all libraries had greater than 20M reads that mapped to the human genome (or multiple genomes), and all but four had greater than 10M unique reads. Additionally, due to the amount of degradation in the samples, qPCR, which is more reliable (depends less on poly-A selection and only requires amplification of the target sequence), should be performed on significant genes identified by RNA sequencing as validation of expression.The primary goal of this work was to determine if the isolated RNA could be used in downstream applications. We found that the vast majority of RNA samples were viable for qPCR analysis using 10 ng of cDNA in 25 µL reactions. This work has several limitations. First, we could not control the biologic composition or the size of the collected clot. These factors may influence the amount of starting material and the quality of the RNA we can extract. Second, due to procedural limitations, we stored retrieved clots in RNALater. Immediate storage in liquid nitrogen may better limit RNA degradation and yield higher quality extractions. Third, the RNA quality of many clot samples was low. While this does not pose a prohibitively significant problem for qPCR analysis, it may skew RNA sequencing results, particularly those sequencing of libraries created by poly-A selection. Therefore, in future studies, confirmatory qPCR of target genes identified via RNA sequencing will be necessary to validate differential expression. Lastly, we demonstrated success using a bead-based extraction method on the PerkinElmer Chemagic 360. This instrument and protocol are costly, which may limit widespread adoption of the procedure and could limit larger, future studies.This is the first demonstration of the isolation and downstream application of RNA from AIS blood clots. Overall, nearly all of the 73 cases had enough RNA for qPCR analysis, while 48/73 were amenable for RNA sequencing. Analysis of both housekeeping gene amplification curves and standard RNA sequencing QC demonstrated the feasibility of downstream quantification applications for AIS RNA. Analysis of histology showed that acellular (FP dominant) clots tended to have lower RNA quantity and quality. These results lay the groundwork for future investigations into the molecular biology of AIS clots and their relationship to stroke pathophysiology, which are currently underway.


The synthetic pathways of life’s building blocks are envisaged to be through a series of complex prebiotic reactions and processes. However, the strategy to compartmentalize and concentrate biopolymers under prebiotic conditions remains elusive. Liquid-liquid phase separation is a mechanism by which membraneless organelles form inside cells, and has been hypothesized as a potential mechanism for prebiotic compartmentalization. Associative phase separation of oppositely charged species has been shown to partition RNA, but the strongly negative charge exhibited by RNA suggests that RNA-polycation interactions could inhibit RNA folding and its functioning inside the coacervates. Here, we present a prebiotically plausible pathway for non-associative phase separation within an evaporating all-aqueous sessile droplet. We quantitatively investigate the kinetic pathway of phase separation triggered by the non-uniform evaporation rate, together with the Marangoni flow-driven hydrodynamics inside the sessile droplet. With the ability to undergo liquid-liquid phase separation, the drying droplets provide a robust mechanism for formation of prebiotic membraneless compartments, as demonstrated by localization and storage of nucleic acids, in vitro transcription, as well as a three-fold enhancement of ribozyme activity. The compartmentalization mechanism illustrated in this model system is feasible on wet organophilic silica-rich surfaces during early molecular evolution. Prebiotic compartmentalization could prove essential for the evolution of life. Guo et al. show that liquid-liquid separation in an aqueous two-phase system driven by evaporation may already suffice to facilitate chemical processes required for the RNA world hypothesis. Microdroplet reactors facilitate thermodynamically unfavorable synthetic reactions in bulk solution, where one product molecule is formed from two or more reactant molecules with a high loss of entropy4. In particular, evaporation could have played a critical role in these prebiotically relevant processes, such as concentrating reagents to enhance the reaction rates inside microdroplets7, and regulating conditions for biopolymer synthesis through wet-dry cycling10. Despite overcoming the inherent thermodynamic unfavorability in chemical synthesis, how prebiotically synthesized polymers assemble into cellular and subcellular-like compartments capable of autocatalysis11 and self-replication13 remains unresolved.Microdroplets have been demonstrated as prebiotically plausible reactors15. Besides their biological functions in modern cells, the existence of membraneless organelles in cells provides a new perspective on prebiotic compartments on the early Earth16. Such non-membrane-bound compartments may represent “remnants” of ancient structures for spatiotemporally regulated biochemical reactions17. This concept dates back to Alexander Oparin’s arguments in the 1930s18. LLPS is a thermodynamically transient, nonequilibrium process leading to phase segregation (nonassociative or segregative phase separation) or the formation of coacervates (associative phase separation) in an initially well-mixed macromolecular-containing system. LLPS can be tuned by varying polymer concentration, charge density, pH, and temperature. With the ability to concentrate biopolymers and form primordial compartments in the dilute “primordial soup”19, LLPS has been reported to guide the transition from prebiotically synthesized polymers to highly organized structures. This may have driven the evolutionary engine of the first living cells under prebiotic conditions20.Liquid-liquid phase separation (LLPS) has recently been shown as the route for the formation of intracellular membraneless organelles21) of oppositely charged polymers. A unique advantage of coacervate droplet formation as a prebiotic pathway of compartmentalization is its spontaneous assembly in dilute solution. Coacervate droplets could help transition from prebiotically synthesized biopolymers to fatty acid membranes encapsulated protocells24. However, limitations still exist for complex coacervates as prebiotic reactors. Limitations include the structural stability and inhibited function of RNA due to the complex ion-pairing interactions between RNA and polycations in coacervate droplets. For example, RNA polymerization has shown to be inhibited in polyamine-containing coacervates25, and the rate constant of RNA cleavage is 60-fold slower inside complex coacervates than that in buffers26. In contrast, a similar RNA catalytic reaction exhibits a 70-fold increased rate of reaction through segregative phase separation of polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS)27. It has also been reported that nucleic acid duplexes can be destabilized inside complex coacervate droplets28. Hence, a prebiotically plausible pathway for nonassociative phase separation of two neutral polymers would provide an important missing piece of the puzzle for prebiotic compartmentalization. However, the prebiotic pathway for emergence of nonassociative phase separation faces a major challenge to reach such high polymer concentrations in the dilute and homogeneous “primordial soup”29.Currently, most proposed prebiotically plausible membraneless compartments are poly-electrolyte-rich coacervate droplets formed by associative LLPS triggered by specific interactions  must be introduced to enhance nonspecific molecular interactions or reduce mixing entropy. NES has been widely applied in prebiotic chemistry to maintain thermodynamically unfavored processes such as accumulation of biomolecules34. A recent example is that, to mimic molecular evolution within porous volcanic rocks on the early Earth, heated microbubbles were used as reactors for assembly and localization of prebiotic molecules35. Nevertheless, despite segregative LLPS of ATPS being hypothesized to play important roles in origins of life chemistry36, how nonassociative phase separation can be achieved under prebiotically plausible NES remains largely unexplored.In the free energy landscape, nonassociative phase separation occurs when mixing entropy is overcome by nonspecific molecular interactions and thus polymer incompatibility appears in solutionInspired by the widely introduced evaporation process in wet-dry cycling and prebiotically plausible microdroplet reactors, here we report droplet evaporation-assisted nonassociative phase separation for prebiotic compartmentalization inside a sessile droplet of ATPS. By formulating the LLPS mechanism under the NES, we show that the nonuniform evaporation flux and initial composition of the sessile droplets determine the formation and evolution of self-organized membraneless compartments. The evolution of phase-separated membraneless compartments inside the aqueous sessile droplet looks and behaves similar to intracellular phase separation. Most importantly, the membraneless compartments formed in our model system can serve as spatially functional reactors for enrichment of informational molecules and flow of genetic information, validated by in vitro transcription (IVT) and enhanced ribozyme activities. Our model can validate nonassociative phase separation in moderate conditions with no heating, cooling, or osmotic shock needed. Thus, we elucidate a plausible pathway for prebiotic compartmentalization via nonassociative phase separation. Furthermore, the simple yet robust system can capture both the macromolecularly crowded interior environment and the nonequilibrium features of extant cells, providing cell-mimicking conditions essential for a variety of intracellular functions.40, is prepared and used as the stock solution for nonassociative phase separation. To achieve the NES required for LLPS, a droplet extracted from the stock solution is pipetted onto a glass slide then it starts to evaporate. Similar scenarios with such NES, that aqueous sessile droplets dispersed on the organophilic silica-rich surfaces, could be found ubiquitously on the early Earth  inside the evaporating droplet, which is defined as the front of the phase-separated regions inside the sessile droplets  the velocity of PSF is almost constant and calculated to be about 6 µm s−1, in the same scale as that of Marangoni-driven fronts of thin spreading films;53 (iii) as a water-soluble polymer, PEG is a surface-active agent capable of decreasing surface tension of the solution  due to evaporation. In the PEG/dextran system, an increase of TLL can lead to the increase of the density difference, the interfacial tension, the viscosity of the dextran-rich phase, as well as partitioning strengths of a third species like nucleic acids and proteins into one of the two phases63. Hence, through understanding the kinetic pathway and increasing TTL, our model system can be further harnessed to concentrate biopolymers inside phase-separated compartments, and to serve as the reactor that can enhance a series of biochemical reactions.Summarizing, our results present a robust NES for nonassociative phase separation inside a sessile droplet. Due to the highly nonuniform evaporation flux, phase separation is triggered at the rim of the sessile droplet, exhibiting a self-organized pattern decided by both original composition and the concentration change due to the water loss, as demonstrated by the kinetic pathway of phase separation. Meanwhile, the emergence of phase separation can enlarge the compositional gradient inside the droplet, triggering Marangoni flow that convects the phase-separated compartments to the center of the droplet. Hence, the microstructures of phase separation and the hydrodynamic flow are strongly coupled inside the evaporating droplet. With the appearance of rich dynamics, a full understanding on the phase behavior and pattern evolution during droplet evaporation would require the coupling of the Navier–Stokes equation and Cahn–Hilliard equation15 (i) They all undergo a transition from a homogeneous state to a phase-separated state, and that only happens above a threshold concentration of the components; (ii) they are both surrounded by the aqueous macromolecular-crowding environment and governed by nonequilibrium thermodynamics, and (iii) the immiscible compartments formed by LLPS both exhibit properties, such as round morphology, fusion by contact, and wetting properties, that characterize typical liquid structures. These similarities make it possible to bridge prebiotic LLPS to protocells with subcellular membraneless compartments by using our model system.Here we point out some common features of polymer self-organization and compartmentalization in our model system with that of the intracellular LLPS:64. As RNA is a polymer capable of serving as both information storage and a functional catalyst, it is hypothesized to be intrinsic to the origins of life. RNA can undergo associative LLPS via complex coacervation in the presence of polycations, as it has a polyanionic backbone. Likewise, RNA building blocks nucleoside triphosphates (NTPs) can undergo LLPS along with polyphosphate, which is hypothesized to be a primordial energy molecule65. Associative LLPS has previously been shown capable of concentrating such biomolecules, however the structural stability and functionality of highly-charged biopolymers like DNA and RNA are inhibited due to their complex coacervation with polycations28. Thus a nonassociative phase separation model with plausible prebiotic conditions is required, where biopolymers can be concentrated without the loss of stability and functionality.The RNA world is a leading hypothesis for the origins of life36. For both the Cy-5 labeled DNA and the Broccoli RNA aptamer/DFHBI-1T, the ratio of the fluorescence intensity of dextran-rich compartments  ssDNA sequence was used in combination with FITC-dextran (green fluorescence) to label the dextran-rich phase. The DNA localized to the dextran-rich phase by LLPS Fig. . This inion Fig. . LocalizThese DNA/RNA-containing dextran-rich liquid droplets can range from hundreds of nanometers to several microns. Similar to intracellular membraneless compartments composed of RNA and proteins, the DNA/RNA-containing dextran-rich liquid droplets have interfacial tension-directed movement and coarsening behaviors. This implies that polymer self-organization and compartmentalization could have played a significant role in driving the evolutionary engine of the first live cells under prebiotic conditions.To demonstrate the advantages of maintaining functionality of nucleic acids through nonassociative phase separation, we perform IVT inside our model system Fig. . The seq66. Various functions of catalytic RNA under different prebiotic conditions have been demonstrated. In particular, it has been suggested that water evaporation could have led to a viscous environment under model prebiotic conditions, thereby enhancing RNA replication and catalytic activities68. We suggest that our model system could provide a prebiotic strategy for the concentration of catalytic RNAs, thus promoting their functionality. To demonstrate this argument, we performed an RNA substrate cleavage reaction using hammerhead ribozyme (HHR)69 in the evaporating ATPS droplet. The substrate (HHS) is labeled terminally with a FAM fluorophore and a TAMRA FRET pair quencher. Under catalysis by HHR, the substrate is cleaved into two fragments and emits fluorescence /poly A11 RNA (rA11) coacervates25. Hence, more work on the generalization of the impact of complex coacervates on RNA folding and reaction rates is needed to fully understand how coacervates formed through associative phase separation could serve as primitive compartments. There may also exist a synergistical mechanism, where both associative and nonassociative phase separation could play roles in prebiotic compartmentalization. For example, by the addition of PEG, the RNA oligomer partitioning into the polyU-spermine coacervates was increased by more than two orders of magnitude75. Likewise, a more than fivefold enhancement of transcription rate was achieved inside PEG-containing coacervates of cell lysate protein76. It is worthwhile to explore new model systems where multiple mechanisms could work synergistically to enhance prebiotically relevant reactions.An alternative pathway for prebiotic compartmentalization could be achieved inside the complex coacervates that formed through associative phase separation of oppositely charged species. RNA partitioning into the coacervates would be mainly achieved by ion-pairing interactions. However, the highly negatively charged nature of RNA indicates that there would always be complex RNA-polycation interactions that may be unproductive for RNA folding inside the coacervates. Previous studies have identified and tested a variety of complex coacervates formed by polyelectrolytes with different size and sequences. The impact of these coacervates on the prebiotically relevant processes, including RNA partitioning, RNA structural stabilities, and enzymatic reactions, varies and largely relies on the physicochemical properties of charged molecules that trigger phase separation. For example, the partitioning coefficient of poly U15 inside the poly U-spermine coacervate is about 60-fold less than that of poly A15 RNA14. These structures reside in the nonequilibrium environment of living cells and regulate chemical reactions far from equilibrium. Hence, our model system may also lend a deeper understanding to mechanisms of compartmentalization in modern-day cell biology.There are also implications of our model system for modern-day cells. LLPS has been recognized as an essential cellular compartmentalization strategy in both prokaryotes and eukaryotic cells. Non-membrane-bound liquid droplets rich in protein and/or RNA, also called biomolecular condensates or membraneless organelles, are formed. For example, RNA can be compartmentalized in a variety of membraneless organelles including P-bodies, P granules, the nucleolus, and Cajal bodies−1 fluorescein isothiocyanate-dextran , The sessile droplet consisted of poly(ethylene glycol) . The contact angle of the droplet varied between 45° and 25° during the whole evaporation process, measured by bright-field imaging (Nikon) from the side view. The microscope glass slides (ISOLAB GmbH) were used as solid substrates for the sessile droplets. The glass slides were firstly wiped by ethanol wetted tissue to mechanically get rid of contaminants on the surfaces. Then the slides were sonicated in ethanol for 15 min, followed by successively washing with isopropyl alcohol and Milli-Q water, to remove organic contaminants on the surfaces. Then we dried the slides by nitrogen flow and put them into oven (65 °C) for 1 h.The bright-field images were captured by a high-speed camera  coupled with an inverted microscope (Motic AE2000). The fluorescence images were captured by a Nikon Ti2-E Widefield microscopy. For imaging channels of FITC-dextran and Rhodamine B, the excitation wavelength were 475 and 575 nm, respectively. The captured images are processed using ImageJ (NIH) software. Droplet deposits after fully drying on the silica wafer were sputtered with gold before imaging under scanning electron microscope .A PCR mixture was assembled containing 1x PCR buffer, 300 μM dNTPs, 400 nM forward and reverse primers, 1 nM template DNA, and 3 units of PFU polymerase  in a 100 μL reaction. Thermal cycling consisted of one cycle of 96 °C for 1 followed by 10 cycles of 96 °C for 15 s, 50 °C for 30 s, and 72 °C for 45 s, followed by one cycle of 72 °C for 1 min and hold at 4 °C. The PCR product was purified using a QIAquick PCR Purification Kit . To generate Cy-5 BrocT dsDNA template, Cy5 labeled forward primer was used in the PCR reaction.IVT mixture  was assembled containing 1× transcription buffer, NTPs, DTT, T7 RNA polymerase mix, 50 nM Broccoli dsDNA template, and 200 μM DFHBI-1T. The transcription mix was incubated at 37 °C for 1 h to allow transcription of Broccoli RNA. The ATPS solution is made up of 9 wt% PEG and 4 wt% dextran. The transcription product containing Broccoli RNA aptamers was then added to the ATPS solution with the volume ratio of 1:10. A 0.5 μL droplet of the mixture was pipetted on the glass substrate for evaporation and imaging.IVT mixture  was assembled containing 1× transcription buffer, NTPs, DTT, T7 RNA polymerase mix, and 200 μM DFHBI-1T. The ATPS solution is made up of 11 wt% PEG and 2 wt% dextran. Then the transcription mixture and 50 nM dsBroccoli DNA template solution were added into the ATPS solution with the volume ratio of 1:1:10. A 5 μL droplet of the mixture was pipetted on the glass substrate for evaporation and imaging.Both HHR and X-motif ribozyme are used for the cleavage reaction inside the evaporating droplet. The ribozyme cleavage experiments were carried out with RNAse free reagents in an RNAse free environment. The ATPS solution is made up of 11 wt% PEG and 2 wt% dextran. IVT mixture  was assembled containing 1× transcription buffer, NTPs, DTT, T7 RNA polymerase, and 50 nM ribozyme DNA template. The transcription mix was incubated at 37 °C for 1 h to allow transcription of ribozyme RNA. The final mixture is prepared by adding 1 μL of ribozyme substrate (10 μM) and 1 μL of ribozyme RNA transcription product into 38 μL ATPS solution. The ribozyme reaction is started by pipetting a 5 μL droplet of the final mixture on the glass substrate.2. The non-ATPS solution was 5 mM Tris pH 7.4, 1 mM MgCl2. The reaction mixture was made up of 38 uL ATPS/non-ATPS solution, 1 μL of ribozyme substrate (10 μM) and 1 μL of ribozyme RNA transcription product. For ATPS and non-ATPS condition, twelve 2-µL aliquots of reaction mixture were made, six aliquots for with wet-dry cycling time points and six aliquots for without wet-dry cycling time-points. The wet-dry cycling samples were incubated in open cap centrifuge tubes in a desiccant filled box to aid evaporation. For the first time-point of 0 h 0.4 μL of RNA PAGE loading dye with 2 mM EDTA was added to the sample and then transferred to −20 °C for storage. Every subsequent time-point  0.4 μL of RNA PAGE loading dye was added to the corresponding time point samples before storage at −20 °C. Additionally 1 μL of water was added to each of the remaining wet-dry cycle samples. After the time course experiments, all samples were loaded onto a 10% TBE PAGE gel for separation before fluorescent imaging analysis at both ex490/em525 and ex556/em573 using a ChemiDoc MP Imaging System (Bio-Rad).The HHR was used to further demonstrate ATPS induced reaction increase under wet-dry cycling conditions. The experiments were carried out with RNAse free reagents in an RNAse free environment. The ribozyme reactions were done with both ATPS and no ATPS buffer systems. The ATPS solution was made up of 11 wt% PEG and 2 wt% dextran in 5 mM Tris pH 7.4, 1 mM MgClSupplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution. Genome packaging is the process whereby viruses assemble their genomes into capsids . The priInterestingly, many RNA viruses have evolved genome organizations that greatly complicate viral assembly and packaging. Segmented viruses, such as influenza and rotavirus, need to incorporate multiple genome segments for their virions to be infectious . On the cis acting packaging signals that serve as high affinity binding sites for the viral capsid (or nucleocapsid) proteins ,2416,24]Complex viruses that express sub-genomic or spliced viral RNAs have an additional challenge: they must not only distinguish their genomic RNA from cellular RNA, but also from non-genomic viral RNAs . One simCoronaviruses (CoV) have extraordinarily large genomes (~30 kb) that presumably pose additional difficulties for packaging, yet genomic RNA is efficiently and selectively incorporated into virions ,59,60,61Packaging sites may also include motifs necessary for the correct presentation of the RNA molecule in time and space. For example, influenza viruses have a segmented genome of negative sense viral RNAs (vRNAs) that are replicated in the nucleus via complementary RNA (cRNA) intermediates. Long-range interactions between the 5′ and 3′ termini, in some cases over distances of thousands of nucleotides, construct the promoter structure that is involved in transcription, replication and packaging . InteresGenome packaging occurs during the late stages of replication when sufficient genomes and structural proteins have been replicated and produced to ensure effective viral assembly. Sometimes even, the same viral RNA molecule must carry out several competing functions. It is not surprising therefore that viruses heavily regulate the translation, replication, and packaging of their genomes. RNA viruses achieve this, in part, by exploiting the dynamic and flexible properties of RNA. Namely, RNA molecules can spontaneously fold into multiple, mutually exclusive structures, acting as riboswitches with each structure having a different function . RNA cancis-acting replicating element (CRE) in the coding region of the NS5B protein forms a long distance base pairing with the highly conserved X-region in 3′ UTR [Hepatitis C virus (HCV) is a model of such complex RNA based regulation ,86,87. Tn 3′ UTR ,90,91,92n 3′ UTR   that are packaged into viral particles as viral ribonucleoprotein (vRNP) complexes [A well-studied example is influenza A virus of the omplexes . Becauseomplexes ,133. Furomplexes ,135,136.omplexes ,139,140.omplexes ,149,150.omplexes ,152, andomplexes ,154. Theomplexes . This woomplexes ,149,155.omplexes ,156,157.omplexes . Collectomplexes  or ligatomplexes . Furtheromplexes ,162,163.Reoviridae family, which includes rotavirus and bluetongue virus. Rotavirus has a genome composed of eleven dsRNA segments of different sequences and lengths (0.7 to 3.1 kb) [Similar principles seem to apply to dsRNA segmented viruses of the  3.1 kb) . Paralle 3.1 kb) . Disrupt 3.1 kb) . Studies 3.1 kb) ,167,168. 3.1 kb) ,169,170. 3.1 kb) . How thiRNA based packaging signals play a much broader role in viral life cycles than assembly, and it is now appreciated that RNA virus genome structures enhance virus ‘evolvability’ by facilitating gene exchange during co-infection . One widRetroviruses package two near identical copies of the genome as a non-covalently associated dimer . One evoPackaging signal incompatibilities are also thought to be a major restriction to reassortment in segmented viruses ,185,186.Generally, viral RNA packaging is assumed to be a process dependent on a few clearly defined RNA structural motifs specifically recognized by a viral protein. However, when evaluating binding affinities and specificities of those RNA-protein complexes in vitro, they often do not show the specificity that is observed for the packaging process in vivo . Thus, iExcitingly, methods to characterize RNA virus packaging signals are being developed and refined to better resolve the details of these integrative processes. Approaches to study viral RNA packaging spans disciplines and can now shed light on this process across multiple scales. For example, advanced cryo-EM techniques promise to determine RNA structures at high resolution in three-dimensions ,189,190.


Alphaviruses are positive-sense RNA arboviruses that are capable of causing severe disease in otherwise healthy individuals. There are many aspects of viral infection that determine pathogenesis and major efforts regarding the identification and characterization of virulence determinants have largely focused on the roles of the nonstructural and structural proteins. Nonetheless, the viral RNAs of the alphaviruses themselves play important roles in regard to virulence and pathogenesis. In particular, many sequences and secondary structures within the viral RNAs play an important part in the development of disease and may be considered important determinants of virulence. In this review article, we summarize the known RNA-based virulence traits and host:RNA interactions that influence alphaviral pathogenesis for each of the viral RNA species produced during infection. Overall, the viral RNAs produced during infection are important contributors to alphaviral pathogenesis and more research is needed to fully understand how each RNA species impacts the host response to infection as well as the development of disease. Alphaviruses are single-stranded, positive-sense RNA viruses that are naturally transmitted between a mosquito vector and a vertebrate host. Epizootic spillover events result in the infection of humans and horses, potentially resulting in severe disease. Alphaviruses are largely classified as either arthritogenic or encephalitic based on the symptoms of infection. Arthritogenic alphaviral infection causes disease varying from mild to severe multi-joint arthritis and can persist for several months to years past the acute phase of infection . This inThe primary purpose of this review is to summarize what is known about how the viral RNAs (vRNAs) produced during alphaviral infection influence pathogenesis . While mAlphaviruses produce primarily three RNA species during viral replication: the genome, which encodes the nonstructural proteins; the minus-strand RNA, which serves as the template for viral replication and transcription; and the subgenome, which encodes the structural proteins . The speFrom a compositional perspective, there are two features of the alphaviral RNAs that arise in a non-templated fashion during replication/transcription. These two features are the 5′ cap structure and the 3′ poly(A) tail. Despite being non-templated, these RNA features arise from the specific activities of the alphaviral replication complex; with the addition of the 5′ cap being due to the concerted activities of nsP1 and nsP2, and the addition of the poly(A) tail is due to nsP4. While, to date, there have been no reported virulence determinants associated with the 3′ poly(A) tail, the 5′ cap structure, in contrast, has been associated with virulence at several levels.The alphaviral cap structure is different from host cap moieties, creating a major means by which the host is able to distinguish between self and viral RNAs. More specifically, eukaryotes incorporate a Type-1 cap that is 2′O methylated . CytosolNonetheless, the alphaviral capping machinery is not absolutely efficient. Recent work from our lab has demonstrated that a significant proportion of the genomic RNAs produced during alphaviral infection lack a 5′ cap structure . In lieuWhile the direct mechanistic impact of the noncapped genomic RNAs remains a topic of ongoing assessment, altering the ratio of capped/noncapped genomic vRNA produced during infection is known to have significant impacts on alphaviral pathogenesis. More specifically, increasing the proportion of capped genomic RNA produced during SINV infection of 4-week old C57BL/6 mice resulted in almost complete ablation of morbidity and mortality as well as significantly reduced levels of inflammatory cytokine transcripts compared to wild type SINV infection . ImportaDue to the genetic arrangement of the alphaviruses, the genomic and subgenomic viral RNAs possess shared and nonshared untranslated regions. Specifically, the 5′ untranslated regions (UTRs) of the genomic and subgenomic RNAs are different from one another due to the utilization of an internal initiation site on the minus strand template RNA for subgenomic RNA synthesis, whereas synthesis of the genomic RNA begins at the 3′ terminus of the template minus-strand RNA . In contThe alphaviral 5′ UTR varies in both length and sequence among the different alphaviruses and is typically between 27 and 85 nucleotides long . AlthougNonetheless, the 5′UTR of the alphavirus genomic RNA does have structural elements which are directly involved in virulence, and the importance of these features has been demonstrated in multiple alphaviruses through point mutations of specific residues. Specifically, mutation of nucleotide 3 in VEEV has been shown to result in avirulence in immunocompetent mice but does not affect pathogenesis in IFNAR-/- mice ,24,25. SSimilar to the alphaviral genomic RNA, the 5′ UTR of the subgenomic RNA and its adjacent coding region also has a functional role in alphaviral pathogenesis through the presence of cis-acting features. However, unlike the genomic RNA, the 5′ features of the subgenomic RNA focus largely on the regulation or enhancement of translational activity. The most notable feature is the Downstream LooP (DLP), which is a stable stem-loop structure present in the subgenome located approximately 27–31nt downstream of the initiation codon of the capsid protein  32]. Th. Th32]. Like the 5′ UTRs, the canonical 3′ UTRs of the alphaviruses vary greatly in both sequence and length, being anywhere from ~80 to over 700 nucleotides long depending on the species ,39. In aA notable element of the alphaviral 3′ UTR is the presence of Repeated Sequence Elements (RSEs) which form stem-loop structures. Although the composition, number, and length of the RSEs vary among the alphavirus genus as a whole, the RSEs of alphaviruses in the same clade retain a large degree of similarity . In CHIKA second readily identifiable element of the alphaviral 3′UTR is the U-Rich Element (URE), which is typically found immediately 5′ of the 3′CSE sequence. While not strictly conserved across the genus, this element was found to interact with the host HuR protein. thereby promoting viral RNA stability via preventing transcript-specific deadenylation ,47. AlphIn addition to the viral cis-features of the alphavirus 3′UTRs, there are instances whereby aspects of the host RNA regulatory pathways influence alphaviral pathogenesis. These include host derived MicroRNAs (miRNA) which are short host RNAs that regulate protein translation in a transcript specific manner. These regulatory RNAs can have both pro-viral and antiviral functions and can influence viral replication directly by targeting viral RNA or indirectly by targeting host mRNAs to alter the cellular environment. In situations where the miRNA directly interacts with viral RNA, viral replication is either restricted due to miRNA inhibition of viral genomic translation or enhanced due to the miRNA increasing the stability of the viral RNA ,50,51.The most well-described miRNA known to impact alphaviral pathogenesis is miR-142-3p. This hematopoietic-specific miRNA has been shown to bind to the 3′UTR of both EEEV and WEEV and restrict viral replication in myeloid-lineage cells  49,52].,52.49,52While most miRNA:vRNA interactions occur in the 5′ and 3′ UTRs, an interaction site in the structural open reading frame (ORF) of SINV and CHIKV has been recently described. Specifically, the interaction of the neuronal-specific miR-124 with the E1 region of SINV and CHIKV has been shown to positively regulate viral replication  54]. Al. Al54]. The nonstructural and structural open reading frames comprise the vast majority of the genomic and subgenomic RNAs and the sequences of these regions are much less varied between the different alphaviruses, especially in comparison with the 5′ and 3′ UTRs. Relatively little is known about how the RNA structures, modifications, and RNA:Protein interactions that occur in these portions of the viral RNA contribute to pathogenesis outside of influencing the nonstructural and structural proteins they encode. Furthermore, whether there are differences between the structural open reading frames of the genome and subgenome that influence pathogenesis in different ways is also largely unknown. In spite of all that is unknown, there are a number of studies that illustrate several aspects of the viral genome and subgenome that are known to be important determinants for alphaviral pathogenesis.In studies assessing the usage of dinucleotide motifs and codon pairs in viruses relative to their hosts, it was found that many arboviruses utilize codon pairs which are overrepresented in their vertebrate hosts as opposed to the invertebrate vector . AlphaviZAP suppression of alphaviral replication is important for host type-I IFN mediated antiviral defense, as suckling mice deficient in ZAP are unable to adequately control SINV replication and have increased mortality . In miceA key feature of many alphaviruses is the presence of an Opal stop codon in between the nsP3 and nsP4 coding regions. Several studies have identified the Opal stop codon as a virulence determinant, but the overall impact of the Opal stop codon is, interestingly, highly dependent on the particular alphavirus species. Mutation of the Opal stop codon in CHIKV results in reduced pathogenicity, as mice infected with a virus where the Opal codon has been replaced with arginine experienced decreased footpad swelling and reduced inflammation in the joints . In contIn addition to the Opal stop codon, there is a proximal stem-loop structure that promotes read-through by the ribosome. Several studies have shown that the presence and stability of this stem-loop structure influences the expresion of nsP4, but the overall impact of this secondary structure motif on alphaviral pathogenesis is unclear as direct studies involving animal models have not been pursued ,70. RegaDue to their nature as single-stranded RNA viruses, the alphaviral RNAs are capable of folding into a diverse array of secondary structures during the viral lifecycle. Indeed, recent studies have shown the presence of extensive RNA secondary structures throughout the viral genome . HoweverIn addition to the generalized structured nature of the alphaviral genome and the likely implications of such, there are several key secondary structures with specific identified roles during infection. These include the Packaging Signal (PS) located in the nonstructural ORF, and the aforementioned DLP element, and the 6K/TF Frameshift structures located in the structural ORF.The primary role of the PS is to select the genomic RNA for encapsidation during viral infection. The precise location of the packaging signal varies across the members of the genus, with the majority of the alphaviruses having a packaging signal in nsP1, and SFV and RRV having their packaging signal in the coding region of the nsP2 protein ,75. The The second known structural element in the structural ORF of the subgenomic RNA that contributes to pathogenicity is the stem-loop which causes a frameshift during the translation of the 6K protein resulting in the production of the transframe (TF) protein  76]. Th. Th76]. While numerous viral and host protein interactions with the alphaviral RNA have been described, the best-characterized interactions are those that impact viral translation or vRNA synthesis. Interfering with the interactions would certainly affect alphaviral pathogenesis, as efficient translation and synthesis of the alphaviral RNA are vital for viral replication as well as for impairing the host immune response. However, because these interactions only seem to indirectly affect alphaviral pathogenesis because they deal with important aspects of the viral life cycle, they are beyond the scope of this review and will not be discussed in detail.In addition to the two major positive-sense RNAs produced during alphaviral infection, there are several other RNA species with known and/or unknown functions that are produced at non-trivial amounts during infection. These include the negative-sense minus-strand RNA, the cryptic RNA II positive-sense RNA species, and defective viral RNAs. In this section, the known functions and contributions of each of these “other” viral RNA species are described.Functionally, the minus-strand RNA serves as the template for the synthesis of all other positive-sense viral RNAs. It is a primary product of the viral replication cycle and is synthesized by the earliest forms of the alphaviral replication complex. Indeed, the maturation of the alphaviral nonstructural polyprotein rapidly diminishes the synthesis of the minus-strand RNA in favor of the two positive-sense RNA species.Even though the function of the minus-strand RNA is believed to be well understood, the role of the minus-strand RNA in pathogenesis is less clearly defined. What few RNA structures and protein:RNA interactions have been described for the minus-strand RNA are in the context of viral replication and transcription. While disruption of features that promote efficient production of the viral genome and subgenome would have obvious impacts on the development of disease. As the mechanistic effects of these mutations involve fundamentally altering viral replication, they are beyond the scope of this review. Thus, the full extent of the RNA secondary structures present in the minus-strand RNA as well as any connected protein:RNA interactions and how they impact alphaviral pathogenesis has not yet been characterized. One aspect of the minus-strand RNA important to viral pathogenesis is that it can activate the various innate immune sensors which target non-self RNAs, such as RIG-I and MDA-5. To avoid being detected by these sensors, alphaviruses sequester the minus-strand RNA into replication spherules formed from host membranes . HoweverThe fourth viral RNA species that is generated during alphaviral infection is the positive-sense RNA species RNA II. RNA II consists of the first ~7.6kb of the genome and ends at the subgenomic promoter  81]. RN. RN81]. Defective viral genomes (DVGs) consist of viral RNAs which have large deletions that render them unable to initiate infection. During viral replication, truncation, recombination, and/or rearrangement events of the viral genome can result in the production of DVGs. Alone, DVGs are unable to replicate as they lack a full complement of replication machinery and structural proteins. However, in the presence of the wild-type virus, the DVGs can be replicated and packaged into viral particles. During alphaviral infection, DVGs are typically produced during high MOI serial passaging studies and were found to repress viral replication ,86. In pGiven the capacity for DVGs to restrict viral replication in tissue culture, there is the potential that DVG production could affect alphaviral pathogenesis in animal models as well. In SFV, mice inoculated intranasally with tissue culture-derived DVGs were protected from lethal infection, presumably because the DVGs interfered with viral dissemination and replication in the brain . HoweverAlthough the addition or excess production of DVGs during alphaviral infection is largely antiviral, the DVGs naturally produced over the course of infection may have different effects on viral pathogenesis. While the role DVGs play in the development of disease has not yet been shown for alphaviruses, it has been characterized for other RNA viruses. In the Sendai virus, DVGs have been shown to be immunostimulatory, as viral strains that produce higher levels of DVGs induce a greater IFN response than strains producing relatively few DVGs. In mice infected with the Sendai virus, DVGs produced by the virus have been found to be responsible for inducing the production of type-I IFN and triggering the host immune response . In tissAs summarized above, the alphaviral RNAs directly contribute to virulence and pathogenesis to a significant extent. These contributions are in addition to the obvious linkage between viral replication/RNA synthetic fitness and pathogenesis, and typically involve multiple aspects of the host/pathogen interface. The mechanisms by which the viral RNAs contribute to virulence are both direct, as in acting to directly evade or resist aspects of the host innate immune response, and indirect via the modulation of the production of viral proteins during infection. The alphaviral RNA virulence determinants are often, but not always, associated with secondary structures and may be found throughout the entire length of the viral RNA. Furthermore, virulence determinants that lack defined secondary structures often act as interaction sites for host and viral RNA-binding proteins. In these instances, the virulence determinant is directly linked to the primary sequences of the viral RNAs themselves.Overall, the critical contributions of the alphaviral RNAs to pathogenesis have been established, but much work remains to identify the full extent to which the alphaviral RNAs are intertwined with pathogenesis and the precise mechanisms by which the viral RNA influence disease. While the knowledge compiled above represents the current state of understanding in this regard, it also highlights the presence of significant critical gaps in the understanding of the role of viral RNAs in pathogenesis.