Escherichia coli, E. coli-derived outer membrane vesicles (OMVs) and five other bacterial strains . vsRNAs of 8–15 nt in length [RNAs (8-15 nt)] were found to be more abundant than RNAs of 16–30 nt in length [RNAs (16–30 nt)]. vsRNA biotypes were distinct and varied within and across bacterial species and accounted for one third of reads identified in the 8–30 nt window. The tRNA-derived fragments (tRFs) have appeared as a major biotype among the vsRNAs, notably Ile-tRF and Ala-tRF, and were selectively loaded in OMVs. tRF-derived vsRNAs appear to be thermodynamically stable with at least 2 G-C basepairs and stem-loop structure. The analyzed tRF-derived vsRNAs are predicted to target several human host mRNAs with diverse functions. Bacterial vsRNAs and OMV-derived vsRNAs could be novel players likely modulating the intricate relationship between pathogens and their hosts.RNA sequencing (RNA-seq) is the gold standard for the discovery of small non-coding RNAs. Following a long-standing approach, reads shorter than 16 nucleotides (nt) are removed from the small RNA sequencing libraries or datasets. The serendipitous discovery of an eukaryotic 12 nt-long RNA species capable of modulating the microRNA from which they derive prompted us to challenge this dogma and, by expanding the window of RNA sizes down to 8 nt, to confirm the existence of functional very small RNAs (vsRNAs <16 nt). Here we report the detailed profiling of vsRNAs in  Largely superior than low-resolution techniques , which do not provide a global view of the transcriptome , RNA seqsRNAs do not originate solely from intergenic regions (IGRs) . NumerouThe Gram-negative bacterial OMVs are first and foremost an efficient secretion and delivery system which contain various biologically active molecules such as outer-membrane proteins, lipopolysaccharide (LPS), periplasmic and cytoplasmic proteins as well as nucleic acids (DNA and RNA) . The OMVE. coli RNA contained in OMVs. They found that the majority of the sequenced reads are shorter than 40 nucleotides (nt) in length and mainly derive from tRNAs (tRFs).The standardization of sequencing allowed further exploration of bacterial extracellular RNAs, especially those potentially encapsulated in OMVs, which are known, among other intracellular functions, to regulate microbial interactions  and facilitate pathogenesis . GhosalE. coli    OMVs con El Tor)  and a 24ruginosa . These fruginosa . We receruginosa .Under the premise that they are degradation products or too small to have biological functions or relevance, RNAs shorter than 16 nucleotides (nt) are systematically excluded from small RNA library construction, bioinformatics analyses and datasets . NeverthEscherichia coli and their OMVs in the 8 to 30-nt RNA size window, together with five other bacterial strains, including 4 gram-negative  and one gram-positive strain (S. aureus HG001). We report that bacteria and their OMVs contain large amounts of heterogenous very small RNAs (vsRNAs) shorter than 16 nt. Our results support the presence of thermodynamically stable and potentially functional tRFs, and the loading of selective vsRNAs in E. coli OMVs.In the present study, we used small RNA-seq to analyze the sRNA profile of E. coli K12 MG1655. Bacteria were grown at 30, 37, or 44°C on solid Luria-Bertani (LB) agar plates, in liquid LB medium, in AYE or in BHI media at 250 RPM.Bacterial strains and growth conditions used in this study are indicated in E. coli strain K-12 MG1655 as described previously with modifications (OMVs were isolated from the reference ications  or with E. coli cells were grown in 120 ml of LB (in 1 L flask) to an OD600nm of ∼0.5, upon which bacteria were pelleted by centrifugation at 10,000 × g for 10 min at 4°C and discarded. The supernatant was then successively filtered through a 0.45 and 0.2 µm-pore size VacuCap™ filters , and an inoculum was taken to confirm the absence of bacteria on LB plates. Thereafter, OMVs were pelleted by ultracentrifugation and washed twice with PBS at ∼200,000 × g for 2 h at 4°C in a Thermo Scientific Sorvall™ WX+ Ultracentrifuge equipped with the T-1250 Fixed Angle Rotor. After removing the supernatant, OMVs were resuspended in 200 μl sterile PBS.Briefly, Isolation of OMVs by the Exobacteria™ Kit uses precipitation-free gravity column system. Based on the same approach, 30 ml of pre-cleared (centrifuged and filtered as above) bacterial culture were used to harvest OMVs following the manufacturer’s instructions. Using this method, bacterial OMVs were eluted with 1.5 ml of elution buffer.Purified OMVs were subjected to quality control (QC) and characterization, including LB plating to ensure lack of bacterial contamination, Coomassie blue-stained protein gel, transmission electron micrograph (TEM), and dynamic light scattering (Zetasizer). The pellet was stored at −80°C before being used for downstream applications. Complementary experiments consisting of RNase A treatment of OMVs were performed to verify the interiorization of their sRNAs content.E. coli MG1655 and fractions of intermediate stages (supernatant filtrate 0.45 or 0.22 µm) were charged on a 7.5% Mini-PROTEAN® TGX™ Precast Protein Gel  with the recommended running buffer (Tris-Glycine buffer). The gel was then soaked for 5 min with shaking in 0.5% Coomassie Blue G-250 . The excess staining was removed with the destaining solution (40% methanol and 10% acetic acid) to allow visualization of proteins as blue bands on a clear background.Following dilution in 6X SDS protein loading buffer, 5 µg of OMVs (protein content), total protein extracts from ® electron microscope 1230  operating at an acceleration voltage of 80 kV.Different OMV dilutions were analyzed by the Microscopy Platform of the Institut de biologie intégrative et des systèmes, Université Laval, Quebec City, QC, Canada. OMVs were treated as described previously  and obseA Zetasizer Nano-ZS  light-scattering measurement system was used to determine the hydrodynamic size of OMVs. 100 µl of different OMV samples were loaded in a UV cuvette micro (BRAND), and the particle size was measured (three averaged measurements) at 4°C.Total RNA from bacterial strains was extracted using the hot phenol procedure  or the RThe experimental workflow and the flowchart of data analysis are detailed in 260 using a NanoDrop ND-1000 instrument, and the OD260/OD280 ratio calculated for quality control. RNA integrity and gDNA contamination were tested by denaturing agarose gel electrophoresis. The results are summarized in RNA concentration was determined by ODFor each biological condition, an RNA sample was prepared by pooling equivalent amounts of total RNA isolated from 2 or 3 biological replicate samples. In order to obtain the most representative data, we opted for a pooling strategy so to minimize the influence of interindividual variability. Previous studies have shown it to be a valid alternative to biological replicates at much reduced cost for large-scale gene expression approaches .in situ and finally sequenced for 51 cycles on Illumina HiSeq according to the manufacturer’s instruction.Total RNA was shipped on dry ice to the sequencing platform of Arraystar Inc. . Total RNA of each sample was used to prepare the sRNA sequencing library which included the following steps: 1) 3′ adapter ligation with T4 RNA ligase 2 (truncated); 2) 5′ adapter ligation with T4 RNA ligase; 3) cDNA synthesis with RT primer; 4) PCR amplification; 5) extraction and purification of ∼130–150 bp PCR amplified fragments from the PAGE gel. After the completed libraries were quantified with Agilent 2,100 Bioanalyzer, the DNA fragments in the libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cells, amplified Raw sequences were generated as clean reads from Illumina NextSeq by real-time base calling and quality filtering.Subsequently, the 3′ adapter sequence was trimmed from the clean reads and the reads with lengths shorter than 8 nt were discarded. As the 5′ adapter was also used as the sequencing primer site, the 5′ adapter sequence is not present in the sequencing reads.http://www.novocraft.com). sRNA read counts were normalized as tag counts per million  were aligned to the corresponding genome database  using no million . SequencE. coli MG1655 model organism and 5 other bacterial strains . These bacterial small RNA libraries unveiled the existence of an average of seven million adapter-trimmed reads . The vsRNAs (8–15 nt) accounted for about half of the reads in three strains , a quarter in L. pneumophila and only 13% in the two Pseudomonas  as a second stage of normalization . Indeed,ve 16 nt . Further. aureus .(8–15 nt)] and the other dataset encompassed RNAs of 16–30 nt in length [RNAs(16–30 nt)]. Biotype classification was made based on the specifications of Ensembl genome database  sequences of the analyzed bacterial strains ((16–30 nt) dataset, this proportion went up to 45% for Pseudomonas and even reached 95% in other bacterial strains, including E. coli (Reads that map to the genome but do not match known RNA biotypes (“other”) represented ∼30% of the RNA strains . In the  E. coli .(16–30 nt) sequences  sequences, except in Pseudomonas PAO1 (19%).The proportion of tRFs varied between 8% and 13% of vsRNAs, and dropped to less than 3% of the RNAPseudomonas species in both datasets. In L. pneumophila, they represented 30% of vsRNAs and only 15% in RNA16–30 nt library. In E. coli, S. typhi and S. aureus, the proportion of rRNAs in both fractions was less than 10%.The rRNA fragments accounted for nearly half of the biotypes of the two E. coli, while in other species they rarely exceeded 1%  was found in eeded 1% .E. coli, S. Typhimurium and that of the two Pseudomonas is similar, RNA profiling and clustering data suggest that vsRNA biotypes are distinct and varied within and across bacterial species.Although the sRNA profile of E. coli K-12 MG1655, isolated its derived OMVs and analyzed their respective content in vsRNAs. Transmission electron microscopy confirmed the presence of OMVs displaying spherical shape with an average diameter of ∼180 nm  and RNAs(16–30 nt) datasets, respectively . Intracellular RNAs and those of OMVs were derived from the same culture and were processed in an identical manner. Heatmap representation of these data illustrate a selective loading of vsRNAs in OMVs with a particular enrichment of RNAs between 13 and 19 nt and, to a lesser extent, RNAs of 23–25 nt in length , the most abundant sequences also included tRFs .Furthermore, previous analysis of bacterial strain DH5α, which belongs to the same clade as the position . In the Ψ) highly conserved among species . S. aureus Pro-tRF exhibited an unstable circular structure  in contrast to its L. pneumophila analog  underlining sequence-specific structure variations.L. pneumophila) and one fragment derived from a mRNA (in OMVs).Sequences from other biotypes  showed mostly unstructured (circular) molecular organization with low minimum free energy  except fTaken together, these findings suggest that bacterial tRFs are produced upon specific processing of tRNAs and have superior thermodynamic stability compared to the other vsRNAs.The enrichment of vsRNAs, particularly tRFs, in OMVs suggests a potential role in intra- and inter-species communications and prompted us to speculate that bacterial tRFs might function as their eukaryotic counterparts or analogous miRNAs. Potential human target mRNAs of these tRFs were identified with DIANA-microT  and BLASp-value: 8.91E-04), BH3 domain binding which is a potent death domain present in Bcl-2 family members , piRNA binding  and MAP kinase kinase kinase activity . The molecular-level activities performed by tRF-targeted gene products were related, among other terms, to metal ion binding , regulation of cellular response to stress , B cell chemotaxis  and regulation of macrophage colony-stimulating factor production .The overrepresented GO terms for Biological Process aspect  were related to biological regulation, cellular and metabolic processes . More spp-value: 8.79E-03), cell body membrane  and RNA-directed RNA polymerase complex .The cellular component in which these targets perform their function were cellular anatomical entities and protein-containing complexes . We founLastly, the protein class terms  associatOverall, the enrichment analyses suggest that tRFs may not target a specific pathway and seem to be capable of assuming various and multiple functions in the hosts.E. coli and Salmonella, to thousands  and RNAs(16−30nt)] displayed heterogeneous and distinct content of RNAs. We noted an over-representation of mischaracterized RNA, which was neither classical ncRNAs nor mRNAs in the 16–30-nt size window in E. coli, S. Typhimurium, L. pneumophila and S. aureus. These unknown non-coding RNAs may correspond to unannotated RNA fragments, 5’/3′UTR or might be part of new genetic elements , were 34.1% and 23%, respectively. Working with the same strain, but focusing on a different window of RNA length (15–50 nt), It should be noted that the use of complex media, such as LB, considerably contaminates the sequencing data. In our study, around 41% of the reads from mponents , making mponents . This shOur study corroborates and expands on previous findings suggesting the presence of tRFs in bacterial OMVs . SimilarTo circumvent these bioinformatics limitations to target identification—the sequence length of vsRNAs may increase their probability of binding RNA targets—we have used heuristic search method combining different tools . The use


With the aging of the global population, accumulating interest is focused on manipulating the fundamental aging‐related signaling pathways to delay the physiological aging process and eventually slow or prevent the appearance or severity of multiple aging‐related diseases. Recently, emerging evidence has shown that RNA modifications, which were historically considered infrastructural features of cellular RNAs, are dynamically regulated across most of the RNA species in cells and thereby critically involved in major biological processes, including cellular senescence and aging. In this review, we summarize the current knowledge about RNA modifications and provide a catalog of RNA modifications on different RNA species, including mRNAs, miRNAs, lncRNA, tRNAs, and rRNAs. Most importantly, we focus on the regulation and roles of these RNA modifications in aging‐related diseases, including neurodegenerative diseases, cardiovascular diseases, cataracts, osteoporosis, and fertility decline. This would be an important step toward a better understanding of fundamental aging mechanisms and thereby facilitating the development of novel diagnostics and therapeutics for aging‐related diseases. RNA modificaitons in aging‐related diseases Molecular hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. In humans, aging can be correlated to increased risks of multiple diseases such as neurodegenerative diseases, cardiovascular diseases, osteoporosis, metabolic dysfunction, defective tissue repair and regeneration, decreased regulation of gut microbes, and cataracts  tail in mature eukaryotic mRNAs, more than 100 distinct chemical modifications have been found to actively regulate mRNA behaviors, including differentially processing, splicing, translation, and decay  groups have been added to the ribose. The 2’‐OMe modification was first demonstrated in bacterial mRNA to affect translation efficiency  conserved across eukaryotes  family of enzymes on both intermolecular and intramolecular double‐stranded RNAs longer than 20 bp. Mammals have 3 ADAR enzymes, ADAR1 and ADAR2 being catalytically active while ADAR3 lacks catalytic activity. Considering both coding and non‐coding transcripts, tens of thousands of A‐to‐I editing sites have been identified in mice and millions have been identified in humans. A‐to‐I editing levels vary across transcripts, tissues, and throughout development ranging from 1 to 100 percent at any given site  that suppress gene expression post‐transcriptionally. Emerging evidence shows that they harbor multiple RNA modifications, which are catalyzed by the same enzymes as other RNA species. These modifications regulate their biogenesis, stability, and base‐pairing with targets. After transcription, the biogenesis of microRNAs starts with the processing of primary microRNAs (pri‐miRNAs) by the microprocessor complex formed by RNA‐binding protein DGCR8 and the type III RNase DROSHA. In 2015, Alarcon et al. discovered that in mammalian cells, METTL3 methylates pri‐miRNAs and marks them for recognition and processing by DGCR8, promoting miRNA maturation. Another methylation modification, “internal m7G,” catalyzed by METTL1, has been found to occur on pri‐miRNA and affects precursor‐miRNA processing. Notably, precursor‐miRNA is a double‐stranded RNA specie, making them potential substrates for adenosine deaminases acting on RNA (ADAR) enzymes. As miRNA's function is dependent on their base pairing with the target mRNA, A‐to‐I editing in miRNAs may modulate their target specificity, resulting in decreased suppressing efficiency of one or more downstream target genes. Other modifications such as m5C and pseudouridine can also affect the binding of miRNAs to targets  through base pairing and it guides the modifying enzymes to the modification sites; Box C/D snoRNAs guide 2’‐OMe and Box H/ACA snoRNAs guide pseudouridylation , Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, and frontotemporal degeneration. Accumulating evidence has indicated that age‐related neurodegenerative diseases result from various reasons. Among them, epigenetic changes especially RNA modification that could have serious implications in this aspect should be further explored.3.1AD was first described by Alois Alzheimer in 1906. It is one of the most damaging aging‐related neurodegenerative diseases, the prevalence of which increases as the global population ages , it is found that the expression level of METTL3 was significantly reduced along with m6A levels in 5xFAD mice when compared with control mice , m7G, and 2’‐O‐methylguanosine, were increased while N2, N2,7‐trimethylguanosine , and N2,N2‐dimethylguanosine were dramatically decreased in the 15‐25 nt RNA fraction from the cortex of AD brains. RNA‐seq analysis revealed that most of these fractions are miRNAs. Interestingly, in the 30–40‐nt small RNA fraction, Cm, 2’‐O‐methyluridine (Um), and m7G modifications showed higher levels compared with controls whereas m1G, m2,2,7G, and pseudouridine modifications were reduced Figure . By usin3.2ALS is one of the most common age‐related neurodegenerative diseases characterized by progressive weakness and muscle atrophy, causing damage to upper and lower motor neurons  site of GluA2, a subunit in the majority of AMPA receptors in the adult brain, and changes the glutamine  to an arginine (edited to CIG and translated as CGG) within the ion pore of GluA2, which is indispensable for normal AMPA receptor function  and stroke  . These findings underscore the significance of this novel mechanism of cardiac hypertrophy. It is well known that leptin directly induces cardiomyocyte hypertrophy. Recently, Gan et al. demonstrated that the expression levels of FTO were upregulated after leptin treatment in cultured myocytes, suggesting its potential role in mediating the hypertrophic response to leptin , was significantly increased in TAC‐induced hypertrophic mouse hearts. CHAPIR directly interacted with METTL3 which suppresses the m6A modification of Parp10 mRNAs thereby increasing Parp10 protein levels and promoting NFATC4‐induced pathological cardiac hypertrophy , NOP10, NHP2, and GAR1. Histological analysis has revealed that in Dkc16Aging‐related osteoporosis is characterized by low bone mass and over‐accumulation of fatty tissue in the bone marrow environment that increases the risk of fracture  were generated. These mice showed a significant decrease in bone volume and trabecular number at 30 weeks of age. Furthermore, the results of static and dynamic histomorphometric analyses showed that the bone formation rate in mutant mice was decreased by 66% with bone marrow adipocyte number per bone marrow area being increased when compared with controls. FTO functioned through demethylating and then enhancing the stability of the mRNAs of Hspa1a and other genes that can protect cells from genotoxic damage (Zhang et al., Oc KO mice manifest the phenotype consistent with age‐related bone loss. miR‐149‐3p was found to directly target FTO mRNA and modulate the adipogenic differentiation of bone marrow‐derived mesenchymal stem cells (Li et al., FTO is a key regulator associated with adipogenesis, and the complete depletion of FTO in mice results in postnatal growth retardation. FTO knockout mice have not only a significantly shorter body length over the lifetime but also a much lower bone mineral density (Gao et al., 7Age‐related fertility decline is inevitable and irreversible, especially for female reproductive potential. Several demographic and epidemiological studies have long recognized that female fertility declines with age, most notably the decline in ovarian function Leridon, . The mecAll m6A modifications of the granulosa cells of aged human ovaries were measured in order to investigate the relationship between ovarian aging and m6A modification. It was found the level of m6A modifications was significantly increased and the expression level of FTO was downregulated. m6A sequencing showed that increased m6A in the 3′UTR of FOS mRNAs resulted in reinforcing the stability of FOS mRNAs (Jiang et al., 8In conclusion, the field of epitranscriptomics has emerged rapidly in recent years and RNA modifications have been emerging as a new focus and novel therapeutical targets against aging‐related diseases. In this review, we have summarized RNA epitranscriptomic regulation and the mechanisms involved in aging‐related diseases. We found that RNA modification is involved in many diseases that are aging‐related, and plays an essential role in impacting mRNA stability, translation, and control of protein levels of key genes that are involved in pertinent disease‐associated pathways Table . CollectZ.J., D.S., L.H., Y.G., and G.L. conceived the manuscript. Z.J., D.S., L.H., S.S., and L.Z. drafted the manuscript, made the figures, and summarized the Tables. HI.L., P.G., G.V., Y.G., and G.L. finalized the manuscript, figures, and Tables. L.H. and Y.G. significantly contributed to the manuscript revision.The authors declare that they have no conflict of interest.


In this study, we identify RNA-dependent proteins in A549 lung cancer cells in a proteome-wide unbiased screen to elucidate the ribonucleoprotein complexes present in cancer cells. This dataset will help to understand and further dissect the role of RNA and RNA-dependent proteins in lung cancer. It is presented in the user-friendly R-DeeP 2.0 database which provides detailed information about RNA-dependent proteins and their properties in A549 along with data from HeLa cells to the scientific community.Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells. In recent years, the non-coding part of the genome has gained significant attention due to its crucial role in normal developmental processes and diseases . AlthougMiRNAs are short ncRNAs generated by the RNases Drosha and Dicer, which function as regulators of gene expression in complex with an Argonaute protein . Severallet-7 associates with the reduced post-operative survival of lung cancer patients  ATP. Autoradiography revealed the RNA binding to the immunoprecipitated protein. The immunoprecipitated RNA–protein complexes were exposed to different concentrations of RNase which gradually decreased the size of the bound RNA. Consequently, the size of the complex shifted downwards on the blot with increasing amounts of RNase due to the partial degradation and size reduction in the RNA verifying its identity as RNA. At the highest RNase concentration (1:5 dilution), this smear, the RNA signal, was most intense at the height of the DOCK5 protein, while a more dispersed smear toward higher molecular weights was observed at lower RNase concentrations (increasing dilutions: 1:50 and 1:500), proving that DOCK5 was an RNA binding protein (Dedicator of Cytokinesis 5 (DOCK5) belongs to the DOCK family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho GTPases such as Rac and Cdc42 . DOCK5 p protein D. As a c protein E.The “EnguLfment and cell MOtility” or ELMO family of proteins comprises three members, ELMO1, ELMO2 and ELMO3, that are evolutionarily conserved and are essential for engulfment and cell motility. They are one family of well-known interactors of the DOCK proteins ,56. InteThe BRCA1-A complex subunit ABRAXAS1, also called ABRA1, CCDC98 or FAM175A, is one of the subunits of the BRCA1-A complex involved in the DNA damage repair pathway . It is ahttps://R-DeeP2.dkfz.de (accessed on 11 January 2022) for public use. R-DeeP 2.0 contains the analyzed proteome-wide mass spectrometry data of proteins after sucrose density gradient fractionation in presence of RNA molecules and after RNase treatment in human HeLa S3 (previous dataset) [The R-DeeP 2.0 database  is availdataset)  and A549dataset) . In addiGiven the functional importance and complexity of RNA and RNA–protein interactions, several methods were developed to identify RBPs ,39,42,60The R-DeeP screen in A549 cells found 1189 RNA-dependent proteins, while 1894 proteins previously linked to RNA in at least one out of 43 human proteome-wide studies did not show significant RNA dependence. One reason for this could be the loss of weak interactions during long centrifugation times or during cellular lysate preparation using detergents . NotablyInterestingly, we noticed that DOCK5 shifted to its monomeric size after RNase treatment, while ELMO2 and ABRAXAS1 remained in a complex larger than their respective monomeric sizes. This illustrates that proteins could be completely released from an RNA-dependent complex (DOCK5) or that they could remain in a complex via protein–protein interactions such as oligomerization of the protein.Out of the 3743 proteins detected, we quantified 1525 left shifts and 241 right shifts. Left shifts toward lower molecular weights indicate the loss of an interaction partner in the absence of RNA. Given the established functions of RNAs as protein interactors, scaffolds or docking platforms for proteins ,61 as weAs any method using cellular extracts, artificial interactions could be possible between proteins that do not necessarily take place in a cell . HoweverFrom the group of newly identified RNA-dependent proteins, we have exemplarily validated the RNA dependence of three proteins. Using the iCLIP2 technique, we further showed the direct RNA binding of DOCK5 and ABRAXAS1, while no suitable antibody for immunoprecipitation was found for the third protein ELMO2. Notably, these proteins are all involved in cancer progression ,57,59. MFurthermore, the R-DeeP 2.0 database contains the complete datasets of RNA-dependent proteins from both HeLa S3 and A549 cells . It alloIn summary, this R-DeeP screen provides a comprehensive landscape of RNA-dependent proteins in lung cancer cells and identifies novel potential RBP candidates as well as offers an advanced database for RNA-dependent proteins combined with orthogonal data on the proteins and protein complexes. It will stimulate the RNA and RBP research fields with the potential to impact lung cancer research.


Glycine max Sugars Will Eventually be Exported Transporters (GmSWEETs), including paralogs GmSWEET29 and GmSWEET34 (AtSWEET2 homologs) that were consistently upregulated across all ten genotypes in each of the western locations over three years. GmSWEET29 and GmSWEET34 are likely candidates underlying the lower seed protein content of western soybeans. GmSWEET20 (AtSWEET12 homolog) was downregulated in the western locations and may also play a role in lower seed protein content. These findings are valuable for improving soybean agriculture in western growing regions, establishing more strategic and efficient agricultural practices.Over the past two decades soybeans grown in western Canada have persistently had lower seed protein than those grown in eastern Canada. To understand the discrepancy in seed protein content between eastern- and western-grown soybeans, RNA-seq and differential expression analysis have been investigated. Ten soybean genotypes, ranging from low to high in seed protein content, were grown in four locations across eastern (Ottawa) and western  Canada. Differential expression analysis revealed 34 differentially expressed genes encoding  The global population is expected to approach 10 billion over the next three decades; the growing demands for crop production to provide food security are driving an urgent need to improve agricultural practices worldwide . Yield, Glycine max (L.) Merr.] is a valuable crop with many agricultural uses, including human food and animal feed. It is a leading source of oil and protein as its seeds have approximately 20% oil, 40% protein, 35% carbohydrates, and 5% ashes .,37.Glymar) SWEET . AtSWEET content . Next stGlyma.15G049200), an AtSWEET15 homolog, was not DE between eastern- and western-grown soybeans in our data. GmSWEET39 underlies an important soybean protein QTL, in which a 2 bp CC deletion was found to be associated with high seed oil and low seed protein content [GmSWEET39 ( content . GmSWEET content . This pr content . Patil e content  identifiO. sativa L.) and maize (Z. mays), hexose transporter SWEET4 mutations to OsSWEET4 and ZmSWEET4c respectively, lead to defective seed filling in both species, suggesting SWEET4 is important for increased import of sugars into endosperms [SWEETs have been recently identified to play a role in yield and biomass accumulation ,58. Exprdosperms . Low condosperms ,54. A redosperms . With thHeterodera glycines), was recently discovered in Manitoba, only a few years after its presence in eastern Ontario was discovered [The environmental conditions in Ottawa include more precipitation, higher humidity, and warmer temperatures, favoring soybean growth over the more arid environments in the west , Morden (Manitoba), Brandon (Manitoba), and Saskatoon (Saskatchewan) (SF1A). Ottawa  represents the eastern growing region of Canada, and Morden , Brandon , and Saskatoon  represent the western soybean growing regions of Canada. All ten lines were planted with four replicates in each location over four years from 2018 to 2021, with the exception of Saskatoon in 2018 and Brandon in 2020. Each trial was carried out using 4 × 5 rectangular lattices, including four replicates per line per site. Each plot was planted at a rate of 50 seeds per mSampling of each line was done from three different replicates at each location. Young trifoliate leaves were collected from otherwise-heathy looking plants during R5 . Leaf tiSamples were crushed directly in their respective microfuge tubes using a sterile nuclease-free mortar and liquid nitrogen. The RNA extraction was carried out using the SPLIT total mRNA extraction kit ; however the RNA samples from 2020 were extracted by the Génome Québec, McGill University . RNA quality was assessed using a Nanodrop™ 2000 Spectrophotometer , TapeSation 4200 RNA ScreenTape  and 2100 Bioanalyzer  at Génome Québec . Line 1 samples from 2021 did not pass RNA QC and were not included in DE analysis. The E0 mix of the Spike-in RNA variants (SIRVs)  were integrated with the RNA samples as controls to monitor and compare parameters including sensitivity and quantification. Reverse transcription was carried out by the Canadian Centre for Computational Genomics .Paired-end sequence reads were assessed for RNA-seq quality control (QC) using dupRadar  . GmSWEET20 and GmSWEET12 (AtSWEET12 homologs) are downregulated in soybeans grown in the west. These findings are valuable for future research to increase soybean protein in western Canada. It would be of scientific interest to genetically modify expression of GmSWEET29 and GmSWEET34 in soybean in such a way that the sugar transport channel is disrupted to determine whether protein content, yield, or both would be affected.


RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. No circRNA was detectable from the PAN region. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with eight exons and seven introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF57 inhibits splicing of both K15 introns 1 and 2. ORF70/K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70/K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70/K3 locus, we further confirmed ORF57 regulation of ORF70/K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70/K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection. Like other herpesviruses, the expression of KSHV genes is in a tightly regulated manner during viral infection. The contribution of RNA splicing in the regulation of KSHV gene expression was extensively investigated in this report. We found that approximately one-third of all viral genes express spliced transcripts of which many undergo RNA alternative splicing. We identified a subset of viral RNA splicing events susceptible to the regulation of viral RNA-binding protein ORF57. This led to the discovery of ORF57-mediated regulation of the bicistronic ORF70/K3 RNA splicing and production of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3. Such regulation allows timely but mutually exclusive expression of these two viral proteins to execute their unique roles in immune evasion and viral genome replication during the KSHV lytic cycle. Thus, these data provide new insights into KSHV biology. Finally, our study represents a blueprint for RNA splicing studies of other pathogens in infected cells. Kaposi’s sarcoma-associated herpesvirus  is considered as an etiological cause of several malignancies, including Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease –3. KSHV RNA splicing plays an essential role in eukaryotic gene expression and almost all human pre-mRNAs are spliced. RNA splicing has been observed in transcripts of numerous viruses, including herpesviruses ,13. HoweSplicing of viral RNAs is mediated by host splicing machinery and thus subjects to regulation by host factors, including snRNA components of the cellular spliceosome and host splicing factors. The contribution of a viral factor(s) to RNA splicing was a long time suspected but not systematically studied. We previously showed that KSHV ORF57, a viral RNA-binding protein, acts as a viral splicing factor to promote the splicing of several KSHV immediate-early and early transcripts, including ORF50 and K8 . To promThe introduction of next-generation sequencing has provided an opportunity to study RNA splicing events across the entire transcriptome. This study aims to provide an unbiased splicing-focused analysis of KSHV transcripts based on RNA-seq analysis of primary effusion lymphoma (PEL) BCBL-1 cells, a patient-derived KSHV-transformed B-cell line . The mapFig 1A). Interestingly, for an unknown reason, all selected single-cell WT clones transfected with the empty Cas9 vector express a much lower amount of ORF57 protein when compared to the parental BCBL-1 cells but still support KSHV lytic replication . By mapping the sequence libraries to a host GRCh38 (hg38)/BCBL-1 KSHV (GenBank Acc. No. HQ404500) chimeric genome using STAR aligner [S1 Table). Surprisingly, there was only a slight expression decrease  for PAN RNA from the 57KO #6 cells to WT B4 cells (S1 Table), despite that PAN RNA has been identified as a sensitive downstream target of ORF57 [Fig 1D). We showed a ~73 (68–78)-fold reduction of ORF59 and only ~7 (4–10)-fold reduction of abundant PAN RNA in the 57KO #6 cells when compared with the WT B4 cells. Data suggest an unknown compensatory mechanism for PAN RNA expression in the absence of ORF57 in the course of long-term selection of single-cell clones.To perform a comprehensive genome-wide analysis of KSHV RNA splicing and its regulation by ORF57, we carried out RNA-seq of total RNA isolated from BCBL-1 single-cell clones carrying a wild-type  or ORF57 knock-out  KSHV genome 24 h after induction of viral lytic replication with valproic acid (VA24)  lication ,22 and t aligner , we foun aligner  . Among those viral RNA splice junctions, 145 were commonly detected in both WT and 57KO cells. However, many identified viral RNA splice junctions were supported with only a few reads and were not consistently detected in all samples within individual groups, indicating they represent rare, low-frequency RNA splicing events (see details in S2 Table). Therefore, we next focused only on more prevalent viral RNA splice junctions supported by at least 10 or more (≥10) splice reads. Even though applying this threshold led to ~5-fold reduction of the number of viral RNA splice junctions to 57 in the cells with the WT genome and 70 in the cell with the 57KO genome, the splice reads assigned to these junctions counted for 98% of all identified viral splice reads, indicating that these RNA splice junctions represent most of the viral RNA splicing events  with 54 in the WT and 64 in the 57KO consistently being detected in all four samples in each group (S2 Table). As expected, the highly prevalent splice junctions were found in viral RNAs from the WT to 57KO genome, with 50 viral RNA splice junctions commonly detected in the viral RNAs expressed from both genomes. The remaining 7 viral RNA splice junctions passed the minimal 10 reads threshold only in the WT and the other 20 only in the 57KO genome .As our previous study showed high reliability in qualitative and quantitative analysis of genome-wide RNA splicing of mouse papillomavirus (MmuPV-1)  by usingFig 2C). We also observed several noticeable differences between RNA splicing from the WT to the 57KO genome . To determine the biological relevance of the detected viral splicing events, all highly prevalent splice junctions were mapped to each annotated KSHV transcripts with previously mapped or predicted transcriptional start sites (TSS) and polyadenylation cleavage sites [Mapping the obtained splice junctions to the reference KSHV genome revealed a landscape of viral RNA splicing with clusters of split genes being separated by the genomic regions lacking splice sites , confirming the previous estimates that ~30% of KSHV genes are undergoing RNA splicing when expressed [Figs 4). We also identified several novel RNA splicing events in the genes that were not found to be spliced in previous studies. These include splice junctions in the ORF2 and K4.1/4.2 loci . Despite their relatively low prevalence, the RT-PCR results support the newly detected introns in the KSHV WT genome in BCBL-1 cells . Other examples of novel splice junctions detected, validated, and functionally annotated in our study are the junctions between K5 encoding membrane-based E3-ubiquitin ligase and K6 encoding viral cytokine vMIP-I [Fig 5A). The existence of these splice junctions suggests the expression of not only monocistronic K6 and K5 transcripts but also K6/K5 bicistronic transcripts, which are further processed by RNA splicing. The alternative splicing in both K5 and K6 monocistronic RNAs occurs in their 5′ UTRs and is mediated by several alternative 5′ ss and single 3′ ss right upstream of their corresponding coding regions . Interestingly, expression of several upstream ORFs (uORF) was detected in both 5′ UTRs [Fig 5A). The existence of these RNA splicing events was confirmed by RT-PCR . Together these data reveal a comprehensive profile of KSHV genome-wide RNA splicing events during KSHV lytic infection.All 57 highly prevalent splice junctions with ≥10 splice reads detected in viral RNAs from the WT genome were named SJ-1 to SJ-57 in the order from the KSHV genome 5′ to 3′ direction and are summarized in xpressed . These ixpressed , but we xpressed ,29. In ce vMIP-I . InteresTables 2). The introns within the splice junctions are all canonical “GU-AG” introns. We found that most viral 5′ ss participate in splicing of a single intron. Only 9 (19%) of all 47 WT 5′ ss use an alternative 3′ ss, with the 5′ ss at nt 69,684 located in the ORF47/48 intergenic region being spliced mostly to three alternative 3′ ss (Table 2). Similarly, only 8 (23%) of all 35 WT 3′ ss are associated with an alternative 5′ ss usage. A striking exception is that the 3′ ss at nt 26,091 upstream of K5 could accept 8 different 5′ ss for various RNA splicing events (Table 2).During RNA splicing, the intron recognition is mediated by binding of U1 snRNP to 5′ ss and U2 snRNP to 3′ ss. The binding affinity of these factors depends on the sequences surrounding the individual splice sites. Thus, analysis of splice site surrounding sequences can be used to determine the strength of each splice site and ultimately the efficiency of intron removal. We found that viral RNA splicing in the WT KSHV genome is mediated by a total of 187 distinct 5′ ss and 138 3′ ss. The identified 57 RNA splice junctions supported by ≥10 splice junction reads in the WT genome were found being mediated by splicing from 47 distinct 5′ ss to 35 3′ ss . While most suboptimal intron 5′ ss contribute to relatively low-frequency splicing, two of these splice sites mediate RNA splicing to express functional K8.1 (SJ-8) and K15 (SJ-54) proteins. Their relative weakness could be overcome by their optimal exon size of the upstream exons  and presumably the stimulatory effect by splicing of adjacent introns (K15) [Table 2B). Interestingly, the only weak 3′ ss with MAX:ENT under 4.0 was mapped at nt 20,263 within the ORF70 coding region and the splicing of this intron in the ORF70 coding region subjects to ORF57 regulation (see our data in the latter text). Another relatively weak 3′ ss (MAX:ENT 4.42) was found in the LANA 5′ UTR at nt 126,373 and could accept a 5′ ss at nt 126,873 (SJ-50) for LANA RNA splicing. Interestingly, the same 5′ ss is also spliced to a much stronger (MAX:ENT 11.34), but distal 3′ ss at nt 122,971 upstream of ORF72 encoding vCyclin (SJ-49) [To understand the contribution of splice sites to KSHV RNA splicing regulation, we analyze their strength by a maximum entropy (MAX:ENT) model based on sequence composition surrounding 5′ ss and 3′ ss . As showns K15) . Like 5′ (SJ-49) . Both RN . Like 5Fig 2B). The additional 20 splice junctions (SJ-58 to SJ-77) with ≥ 10 splice junction reads were found in the 57KO genome, but less than 10 splice junction reads in the WT KSHV genome (Table 3). These RNA splice junctions could be split into two groups based on their mapping. The first group represents splice junctions mapped to viral transcripts with notable splice sites in the WT genome , but these splice sites could be alternatively spliced to another splice site in the 57KO genome, thus creating a new splice junction enriched in the 57KO genome. This group of viral RNA transcripts includes the alternatively spliced RNAs from ORF50/K8/K8.1 (SJ-61 to -63), ALT RNA (SJ-66 and -67), K14/ORF74 (SJ-68 and -69), ORF70/K3 (SJ-70), K5/K6 (SJ-71), and ORF45/46/47/48 (SJ-72 and -73) . These splice junction reads could be readily detected and verified by RT-PCR and sequencing . It is worth noting that most of these RNA splice junctions could also be detected from the WT genome with less than 10 splice junction reads (Table 3), but the SJ-68 was found only from the 57KO genome. Although detected in the expression of both 57KO and WT genomes, the spliced PAN RNA was just a tiny fraction among the abundant PAN transcripts. We identified total 54 RNA splice junction reads in the WT cells out of total 3,307,738 reads mapped to the PAN locus . Similarly, we identified total 86 splice junction reads out of total 2,057,347 reads mapped to PAN in the 57KO cells. Thus, the splice junction reads from the PAN region represent only 0.0016% of the WT and 0.0042% of the 57KO total PAN reads. Nevertheless, these data indicate that loss of ORF57 expression led to alternative splicing of KSHV RNAs by activation of cryptic splice site usage in the intron-containing and/or intronless viral transcripts.Similarly, we analyzed the biological significance of viral splice junctions detected in viral RNAs from the 57KO genome in BCBL-1 cells. As mentioned above, we identified 70 viral splice junctions with ≥ 10 splice junction reads from the 57KO genome, of which 50 were also observed from the WT KSHV genome . Although the cause of this discrepancy remains to be understood, we were assuming that the remaining expression of PAN RNA from the 57KO genome might compensate ORF57 loss by sponging SRSF3 in the 57KO cells. As expected, we found that overexpression of PAN RNA could promote K8 intron 2 splicing in HEK293T cells . Additional reduction of SRSF3 RNA and protein levels due to virus-host shut off during lytic replication  may further reduce the dependence of K8 splicing on ORF57, especially in the later stage of KSHV infection.We previously identified ORF57 protein as a viral splicing factor promoting splicing of K8 intron 2 to express functional K8α  by interFigs 6). However, we also detected numerous long-range splicing events crossing over the large parts of the virus genome both from the WT and 57KO genomes . To better define these splicing events, we first extracted all splice junction reads covering an intron size ≥10 kb and had at least two splice reads either from one genome or both WT and 57KO genomes . We found a total of 14 such splice junctions, 7 of them from the WT genome and 10 from the 57KO genome, with 3 found consistently from both genomes in BCBL-1 cells (S2 Table). A small overlap between the WT genome and the 57KO genome suggests a possible role of ORF57 in regulation of generating these RNAs. Most long-range splicing events occur in low frequency with a few (less than 10) splice reads and only two long-range splice junctions derived from ORF48 splicing to ORF25 (SJ-34) and from ORF48 splicing to the second exon of ORF29 (SJ-35) passed ≥10 supporting splice reads threshold (Table 1). All long-range splice junctions were generated using canonical splice sites (S1 Table). To verify selected long-range splicing events, we performed RT-PCR using various sets of flanking primers on total RNA isolated from both WT and 57KO BCBL-1 cells. Sanger sequencing of the obtained RT-PCR products confirmed the mapped splice junctions . Some of these long-range RNA splicing events were also detectable in iSLK/Bac16 and JSC-1 cells with lytic KSHV infection . Together, these data indicate the existence of numerous conserved long-range splicing events in KSHV RNA processing. Although their biological relevance remains to be determined, these long-range spliced RNA isoforms may encode several new proteins  lacking any homology to the known viral proteins worth further investigating in future studies.Most previously described RNA splice junctions represent the highly prevalent splice junctions supported by ≥10 splice junction reads, which were mapped within well-defined transcriptional units (https://sourceforge.net/projects/ciri/) [S3 Table). As shown in Fig 8A, we predicted a total of eight KSHV viral circRNAs, of which 6 were from the WT and 2 from the 57KO genome. Among 6 circRNAs from the WT genome, one mapped to PAN, four to antisense PAN , and one to vIRF4 (K10). Only 2 viral circRNAs were predicted from the 57KO genome, with one mapped to the antisense PAN and the other to ORF57. The supporting back-splicing reads to individual circRNAs were relatively low, ranging from 2 to 9 back-splicing reads from all samples in each group. All viral circRNAs, except WT-circRNA-1, were predicted in low abundance in BCBL-1 cells by Toptan and colleagues [S4 Table). We found that most viral circRNAs  are not supported by the splice sites used for linear RNA splicing, besides WT-circRNA-6 and KO-circRNA-2. As the ORF57 gene is reversely orientated in the 57KO genome [Fig 8B). We noticed that the predicted PAN circRNAs from Toptan’s report were at different positions from the reported PAN circRNAs in Tagawa’s report  [S5 Fig and S5 Table). We found none of them give a reasonable splice site score strong enough to carry out a true RNA splicing reaction . Data suggest that all predicted PAN circRNAs with the current prediction program were unreal.RNA splicing usually occurs from the 5′ to 3′ direction and produces a spliced RNA in linear form. However, RNA splicing may occasionally generate a circular RNA (circRNA) by back-splicing when a downstream 5′ ss splices back to the upstream 3′ ss crossing over the exon(s) . Viral cs/ciri/) ,39  , 36. UngTable 1). It is worth noting that retention of the SJ-43 intron is required to express full-length vIRF4 protein. The 3′ ss of the SJ-43 intron in vIRF4 also functions as an acceptor site for the generation of WT-circRNA-6, whereas the donor site used for WT-circRNA-6 production is in vIRF4 exon 3 and not even used for RNA splicing of linear vIRF4 RNA. As a result, the second intron could be spliced or retained in vIRF4 circRNAs, leading to the generation of two isoforms of circRNA . We observed about two-fold reduction of splice junction reads in BCBL-1 cells with the 57KO genome when compared with the WT. Using different primer sets in RT-PCR and RNase R digestion, we were able to detect and distinguish the linear from circular vIRF4 RNAs and the reduced expression of vIRF4 RNA from the 57KO genome in BCBL-1 cells when compared with the WT genome , where the linear vIRF4 RNA in the 57 KO cells appeared ~80% reduction and accompanied by ~20% reduction of the vIRF4 circRNA. Compared to GAPDH, we also found the linear vIRF4 RNA appeared unexpectedly a little more resistant to RNase R digestion .Interestingly, the predicted WT-circRNA-6 matches the previously identified circRNA from vIRF4 RNA ,37. We iTable 1). Using a minimal 2-fold change cut-off, we identified 21 dysregulated RNA splicing events from the 57KO genome, with 11 being downregulated and 10 being upregulated. As expected, the most downregulated was the splicing of ORF57 first intron (SJ-9) due to the inversed ORF57 orientation in the 57KO genome [Table 1). Together, this study discovered that ORF57 has both stimulatory and suppressive effects on KSHV RNA splicing, which could also be confirmed by RT-PCR . Subsequently, we focused on the altered RNA splicing of KSHV K15 and ORF70/K3, two viral transcripts containing multiple introns and expressed from two different genomic loci without overlapping transcription. Thus, we could examine how ORF57 directly affects alternative RNA splicing of these two different transcripts.To fully comprehend the contribution of ORF57 to KSHV RNA splicing regulation, we also compared the number of splice reads assigned to each splice junction detected from both WT and 57KO genomes . All internal introns are required to express K15 isoforms with a variable number of transmembrane domains via alternative splicing [Table 1). Using the primer sets shown in Fig 9B, RT-PCR confirmed the increased intron 1 and intron 2 splicing in K15 RNA expressed from the 57KO versus the WT genome . Calculated PSI (percentage spliced-in index) for both introns also showed a 2-fold increase of the splicing from the 57KO genome over the WT genome, as shown by RNA-seq analysis (Table 1). Consistently, we observed an increased amount of fully spliced K15 RNA expressed from the 57KO genome in BCBL-1 cells . In contrast, expression of ORF57 from the WT genome led to an increased level of unspliced K15 RNA, accompanied by decreased level of fully spliced K15 RNA . Retention of the intron 1 or intron 2 from K15 RNA splicing in the presence of ORF57 creates a premature termination codon in these isoforms of K15 RNA, presumably triggering the non-sense mediated RNA decay. We also observed a few other K15 introns with alternative RNA splicing in the WT or the 57KO genome, but none passed ≥10 reads threshold (S2 Table). More specifically, in the WT cells, we observed exon 5 skipping and use of an alternative 3′ ss in exon 8, whereas the cells containing the 57KO genome showed the usage of an alternative 5′ ss in exon 1 and an alternative 3′ ss in intron 1. Together, our data indicate that, by regulating K15 RNA splicing, ORF57 might alter the levels of full-length K15 protein and its isoforms during lytic infection.K15, located at the right end of the long unique region of the KSHV genome, encodes a viral membrane protein with a variable number of transmembrane domains ,42. Throsplicing ,42. We fTable 1). It encodes viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3 [Fig 10A). Similar to previous reports, we also observed low-frequent RNA splicing from the intron 1 donor site to the intron 2 acceptor site (SJ-19), resulting in removing a 1228 nt-long intron [Fig 10B).ORF70/K3, a bicistronic RNA with complex alternative splicing, is the second viral RNA of which splicing was notably affected from the 57KO genome . Subsequently, we compared the abundance of individual isoforms by Northern Blot on total RNA extracted from the WT and 57KO genome in BCBL-1 cells with lytic replication to confirm these data. First, we use an antisense oligo probe derived from exon 2 (oVM441) to detect all predicted isoforms except isoform E. As shown in Fig 10C, three major RNA isoforms were detected from the WT genome, corresponding to the predicted RNA isoforms A (2.5 kb), B (2 kb), C (2 kb), and D (1.5 kb), with the 2 kb transcripts being the most prevalent. Two minor RNA species, between ~3–4 kb, are presumably expressed from the upstream promoters as reported [Fig 10C). Since this probe could not differentiate the RNA isoform B from the isoform C due to their similar sizes, we reprobed the same membrane with the oligo probe from the intron 1 (oVM476). We observed two major RNA bands in the size of ~2.0 and 2.5 kb from the WT cells, but not from the 57KO cells, thus confirming their identity as the unspliced isoform A and the single spliced isoform B with retention of the intron 1 in the WT cells . As expected, this probe cannot detect the double spliced isoform D missing both intron 1 and intron 2 .Our RNA-seq analysis revealed a significant shift in ORF70/K3 RNA splicing between the WT and 57KO genomes during KSHV lytic infection. In BCBL-1 cells with the WT genome, ORF70/K3 RNA predominantly uses the intron 2 splicing to avoid disruption of the ORF70 ORF, with splicing efficiency of the intron 1 being ~5-fold less than that of the intron 2 (691 intron 1-spliced reads vs 3393 intron 2-spliced reads). On the contrary, in the cells with the 57KO genome, we observed almost equal splicing efficiency for both intron 1 and 2 (1839 intron 1-spliced reads vs 2048 intron 2-spliced reads), indicating a 2.66-fold increase of the intron 1 splicing efficiency but a 1.67-fold decrease of the intron 2 splicing efficiency in the 57KO cells. However, the splicing from the intron 1 5′ ss to the intron 2 3′ ss (SJ-19) appeared only a minimal change between the WT genome (26 reads) and 57KO genome (38 reads) . These data reconfirmed that ORF57 regulates ORF70/K3 RNA splicing in the context of KSHV genome in iSLK/Bac16 cells.To investigate if ORF57-mediated regulation of ORF70/K3 RNA splicing is unique to BCBL-1 cells, we took advantage of inducible iSLK/Bac16 cells carrying a KSHV WT or 57KO genome generated using the same set of gRNAs for the 57KO in BCBL-1 cells . SimilarFig 10E), 53 nts upstream of the nt 20263 3′ ss.KSHV ORF57 is a viral RNA-binding protein that binds to its target RNAs via its disordered N-terminal domain ,52. To dFig 10F, we demonstrated that the anti-ORF57 antibody, but not a non-specific IgG control, efficiently pulled down the ORF70/K3 RNA.To further confirm ORF57 direct binding to ORF70/K3 RNA, we performed RNA-immunoprecipitation (RIP) using an anti-ORF57 antibody to immunoprecipitate the UV cross-linked RNA-protein complexes in the cell lysate prepared from BCBL-1 cells with KSHV lytic infection reactivated by valproic acid (VA) treatment for 24 h. The ORF70/K3 RNA in the RIP complex was RT-PCR amplified using ORF70-specific primers oVM441 and oVM481 spanning over the peak region identified by CLIP-seq. As shown in Fig 10G, the WT cells expressed mainly the B/C isoforms, while the D isoform was the most abundant in the 57KO cells. Most importantly, we found that ectopic ORF57 expression led to partially rescue the isoforms B/C expression from the 57KO genome in a dose-dependent manner. These data provide the strong evidence of ORF57’s role in regulation of ORF70/K3 RNA splicing.Given a profound role of ORF57 in ORF70/K3 splicing, we next tested if restoration of ORF57 expression will rescue the isoform B expression in the 57KO cells. We transfected the iSLK/Bac16 57KO cells with an increased amount of the ORF57-expression vector, followed by the induction of the viral lytic infection by combined butyrate and doxycycline treatment. We used the corresponding amount of empty vector (pcDNA 3.0) as a negative control. The total RNA collected 24 h after induction was analyzed in Northern blot analysis. As expected in Fig 10B. As shown in Fig 10H, all three RNA bands representing isoforms A, B/C, and D were detectable at 12, 24, and 36 h; however, their ratio displaced a substantial change between 12 and 24 h, from an approximate proportion 1.2 to 1 of isoforms D to B/C at 12 h to only 0.4 to 1 at 24 h, in favor of isoform B/C production in BCBL-1 cells with lytic KSHV infection. Consistently, this ORF57-induced isoform switch of ORF70/K3 RNAs was reproducible in iSLK/Bac16 cells with KSHV lytic infection in the other time course studies.K3 was proposed to be a viral immediate early gene , 50 and Fig 11A). The RNA isoforms C and D lacking intron 1 would express a truncated or spliced ORF70 (ORF70SP) in size of 157 aa residues. ORF70SP lacks the central domain between aa 88 to 269, which bears a catalytic site of thymidylate synthase [Fig 11A). Interestingly, the K3 coding region for viral E3-ubiquitin ligase remains intact in all spliced RNA isoforms.Given the bicistronic nature of ORF70/K3 RNA, we next investigated how its RNA splicing regulated by ORF57 affects the expression of individual proteins. As mentioned above, ORF57-sensitive intron 1 lies entirely within the ORF70 coding region. Thus, only the RNA isoforms A and B retaining intron 1 could express a 337-aa full-length ORF70 protein . The ORF70/K3 minigene was then transfected into HEK293T cells in various ratios with a control empty or an ORF57-expressing vector. Twenty-four after transfection, the cells were harvested and blotted with an anti-FLAG antibody to detect ORF70 and an anti-myc antibody for K3 protein detection. As shown in Fig 11C, the minigene in the absence of ORF57 mainly expressed K3 protein and also a small amount of ORF70 protein (lanes 3 and 5). On the contrary, ORF57 co-expression switched from K3 expression to all three isoforms of ORF70 protein, with ORF70FL being the most abundant . The efficiency of the switch was dependent on the ORF57 level and was most prominent at the 2:1 ratio of the minigene plasmid versus the ORF57-expression vector .Due to the lack of available antibodies against ORF70 and K3, we cannot directly measure the effect of observed regulation of ORF70/K3 alternative RNA splicing by ORF57 on the expression of ORF70 and K3 proteins. Therefore, we constructed a minigene reporter by cloning the ORF70/K3 locus into a mammalian expression vector to monitor their expression by introducing two different epitope tags: FLAG-tag on the ORF70 N-terminus and c-myc on the K3 C-terminus , which could be confirmed by both exon 2 (oVM441) and intron 1 (oVM476) probes. In contrast, co-expression of ORF57 was found not only to stabilize all isoform RNAs expressed from the minigenes as reported [We next performed the Northern blot analysis of the corresponding total RNA samples using the same antisense oligoprobes described in reported , 26, butIn this study using RNA-seq analysis combined with bioinformatics and multiple experimental approaches, we performed a comprehensive investigation of viral RNA splicing events associated with KSHV gene expression in BCBL-1 cells carrying a viral WT or a 57KO genome. In addition to detection of all RNA splicing events in the reported gene transcripts ,9,14,30,The patient-derived primary effusion (PEL) cell lines represent naturally infected, transformed B-cells used as a model for studying KSHV biology, with BCBL-1 cells being one of the most widely used due to the absence of EBV coinfection observed in many other PEL cells , 56. HowRNA splicing represents an important step in the processing of both eukaryotic and viral pre-mRNAs and is prevalent in the production of numerous KSHV mRNA transcripts ,10,14,30Although the newly annotated RNA splicing events in the coding regions of ORF2, K4.2, ORF45-47, and K12 may lead to the expression of potentially new protein isoforms, other mapped splicing events occur in the untranslated regions (UTR), such as the 5′ and 3′ UTRs or in the intergenic regions, thus providing an additional layer of regulation of viral gene expression. For example, we observed extensive alternative RNA splicing from the 5′ UTR of viral K5 and K6 transcripts that harbor several upstream ORFs (uORF) or small ORFs (sORF) . The uORKSHV has a common strategy to express a cluster of genes using a gene-specific promoter, but all RNA transcripts transcribed from these different promoters are polyadenylated at a single pA site . The useWe have identified a group of splice junction reads spanning large parts of the viral genome (≥10 kb) across multiple transcriptional units. This long-range RNA splicing happens in both plus-strand RNA transcripts and minus-strand RNA transcripts, predominantly in the same transcription direction but also in the opposite direction of RNA transcripts. Some of these splicing events are consistently detected in different KSHV-infected cells, including JSC-1 and iSLK/Bac16 cells, further supporting their authenticity. The biological relevance of this long-distance RNA splicing remains to be investigated. The coding potential analysis identified several short novel ORFs crossing the splice junction from these transcripts derived by long-distance RNA splicing. Despite their low abundance, the biological relevance of these novel ORF-encoded small proteins also needs to be studied.Although the presented study focuses on profiling KSHV genome-wide RNA splicing of all viral RNA transcripts from RNA 5′ to 3′ direction , recent reports suggested the presence of detectable RNA back-splicing from RNA 3′ to 5′ direction in KSHV transcripts leading to expression of viral circular RNAs –37. UsinOn the contrary, the back-splicing reads detected from vIRF4 (WT-circRNA-6) locus were real and more comparable to the number of linear RNA splice junction reads (ratio 1:5 in the WT cells) and the overall transcription level. Its 5′ end overlaps with the 3′ acceptor site of vIRF4 intron 1 (SJ-43), being spliced to a new 5′ donor site downstream of intron 2. We confirmed the production of vIRF4 circRNA in the WT B4 cells during KSHV lytic infection by RT-PCR using an inverse primer pair next to the back-splice sites. Interestingly, the production of this circRNA was related to ORF57 expression. We found that the reduced expression of vIRF4 circRNA was correlated with the reduction of linear vIRF4 RNA splicing in the 57KO #6 cells. However, the active role (s) of ORF57 in the biogenesis of viral vIRF4 circRNA remains unknown.cis-elements in interaction with host splicing factors, etc. [The regulation of KSHV splicing is poorly understood. Viral transcripts are spliced by cellular RNA splicing machinery and thus subject to similar regulatory mechanisms as host RNAs, including splice site selections, RNA rs, etc. . We prevrs, etc. . ORF57 rrs, etc. . HoweverTwo most remarkable effects of ORF57 on KSHV RNA splicing were identified for RNA processing of monocistronic K15 and bicistronic ORF70/K3 RNA transcripts. K15 gene comprises eight exons and seven introns  and encoBicistronic ORF70/K3 RNA transcript encodes viral thymidylate synthase (ORF70) and viral membrane-bound E3-ubiquitin ligase (K3), each being expressed from an individual ORF separated by a 480-nt long intergenic region. In accordance with previous reports, we detected multiple spliced isoforms of ORF70/K3 RNA generated by alternative splicing of two major introns, with a suboptimal intron 1 in the ORF70 coding region and a strong intron 2 within the intergenic region. We found that the intron 1 is alternatively spliced and ORF57 strongly inhibits its splicing. The intron 2 is a constitutive intron not susceptible to ORF57 regulation. ORF57 was found to bind the intron 1 region right upstream of its weak 3′ ss, consistent with ORF57 binding to the K8 suboptimal intron 2 . The depFig 11E. In the absence of ORF57, K3 expressed from the double spliced bicistronic ORF70/K3 RNA as a viral immediate-early protein functions as an E3-ubiquitin ligase which promotes KSHV immune evasion by degrading host MHC-I proteins to establish productive viral infection [Functionally, the intron 1 retention in the presence of ORF57 is necessary for the expression of ORF70 during viral lytic infection, but the double spliced RNA isoform D is responsible for K3 protein production. Using an ORF70/K3 minigene, we confirmed that the double spliced RNA isoform D expresses K3 protein, while the intron 1 retention in the ORF70 ORF region mediated by ORF57 led to the expression of ORF70 protein. The biological significance of KSHV ORF57-mediated regulation of the expression of ORF70 and K3 is highlighted in nfection . The risnfection . As reponfection ,70. Nevenfection ,72.In conclusion, we have identified KSHV ORF57 as a viral splicing factor in the global regulation of KSHV genome expression and RNA splicing. The ORF57-mediated splicing regulation profoundly affects the expression of affected genes as shown in the expression of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3 during the KSHV life cycle, thus providing further insight into KSHV biology.KSHV-positive BCBL-1  and JSC-Total RNA from 4 replicates of BCBL-1 WT clone B4 and 57KO clone #6 cells were isolated by TriPure Reagent (Roche) 24 h after induction with VA. The genomic DNA was removed using RNeasy Mini Kit (Qiagen) on-column DNase-treatment. Total RNA-seq libraries were prepared with TruSeq Stranded Total RNA Library Kit and subjected to pair-end sequencing using HiSeq3000/4000 chemistry and 2 × 150 nts modality. Reads were trimmed with CutAdapt v.1.18 and mapped to human GRCh38 (hg38)/KSHV (GenBank Acc. No. HQ404500) chimeric genome using STAR aligner v.2.7.0f. . The cha2 UV light of wavelength 254 nm. The cross-linked cells were lysed in the radioimmunoprecipitation assay buffer  and the obtained cell extract was used for immunoprecipitation with protein A-agarose beads (Millipore-Sigma) coated with the rabbit anti-ORF57 antibody [To isolate ORF57 bound RNAs, we performed a RIP assay as previously described . Brieflyantibody  or a conS6 Table. The reactions without reverse transcriptase were used as the negative controls. The RT-PCR products were separated in agarose gel, purified, and the splicing junctions were verified by Sanger sequencing. For RT-qPCR, the DNase-treated RNAs were converted to cDNAs by SuperScript First-Strand Synthesis System (Thermo Fisher Scientific) using random hexamers as recommended. The resulting cDNAs were used as templates in qPCR reactions containing TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) and custom PrimeTime qPCR assay (IDT) for KSHV PAN and ORF59 RNAs [t method.Before RT, the total RNA isolated by TriPure Reagent (Roche) was treated with Turbo DNase (Thermo Fisher Scientific). The specific products were amplified using two-step RT-PCR using random hexamers and primers listed in the F59 RNAs ,26. The S6 Table) end-labeled with T4-PNK (New England Biolabs) using 32P-γ-ATP (Perkin Elmer). Washed membranes were exposed to a phosphor imaging screen and scanned by GE Typhoon TRIO Imager . Obtained images were processed by Image Lab software (BioRad).Total RNAs isolated by TriPure reagent were separated along with Millennium RNA marker (Thermo Fisher Scientific) in formaldehyde-containing 1% agarose gel in 1 × MOPS buffer. After transfer, the membranes were hybridized overnight with antisense oligo probes (S6 Table for details) on BCBL-1 total DNA allowing its in-frame cloning into pCMV-FLAG-myc-22 (Sigma-Aldrich) using KpnI and NotI restriction sites. All plasmid transfections were performed by LipoD293 In Vitro DNA transfection reagent (SignaGen Laboratories) as recommended.The construction of K8 splicing reporter pST3, containing KSHV K8β cDNA, was described in . The plaTotal proteins, prepared by cells direct lysis in SDS protein sample buffer, were separated in 4–12% Bis-TRIS NuPAGE gels (Thermo Fisher Scientific) in 1 × MES buffer and transferred onto a nitrocellulose membrane. The in-house produced anti-ORF57 polyclonal antibody was used to ORF57 expression . The anthttps://sourceforge.net/projects/ciri/) [S6 Table.KSHV circRNAs were predicted by CIRI2 pipeline (s/ciri/) ,39 with s/ciri/) . For cirS1 FigFig 4). Numbers in parenthesis are the RNA splice junction reads detected from the cells with KSHV latent (-VA)/lytic (+VA) infection. See primer’s information in Figs S6 Table.Total RNA from BCBL-1 cells without (VA-) or with (VA+) virus lytic replication was used as an RT-PCR template. SJ-43+42 means a RT-PCR product from the double RNA splicing first from the splice junction SJ-43 and then SJ-42 (see (TIF)Click here for additional data file.S2 FigFigs 4). Numbers in parenthesis are the viral RNA splice junction reads detected from the WT/57KO cells during KSHV lytic (+VA) infection. See primer’s information in Figs S6 Table.Total RNA from the WT and 57KO KSHV genome containing BCBL-1 cells treated with valproic acid (VA) for 24 h to induce KSHV lytic replication was used for RT-PCR to detect viral RNA splicing events from the 57KO (A) and WT KSHV genome (B) or splicing events with altered splicing efficiency from the WT to 57KO KSHV genomes (C). SJ-61+6 means a RT-PCR product from the double RNA splicing first from the splice junction SJ-61 and then SJ-6, and SJ-17+21 first from SJ-17 and then SJ-21 (see (TIF)Click here for additional data file.S3 Fig(A) HEK293T cells were co-transfected with 300 ng of K8β cDNA plasmid (pST3) . The sam(TIF)Click here for additional data file.S4 FigFig 7B were predicted using an ORF prediction program  with an AUG initiation codon ORF encoding at least 50 aa or more from all three frames. The nucleotide positions and amino acid sequences of the newly identified ORFs are shown below.(A) The conservation of long-range KSHV splicing events in JSC-1 and iSLK/Bac16 cells. Total RNA from JSC-1 cells treated with the sodium butyrate (Bu) for 24 h or from iSLK/Bac16 cells with the KSHV WT or 57KO genome treated with sodium butyrate/doxycycline (Bu/Dox) for 48 h was used in RT-PCR with indicated primers. The reaction without reverse transcriptase (RT-) was used as a negative control. *, non-specific RT-PCR products. (B) Novel transcripts generated by long-range RNA splicing identified in KSHV lytic infection. The annotated KSHV ORFs are shown in orange and blue. The novel ORFs (green arrows) spanning the long-range splice junctions (^) shown in (TIF)Click here for additional data file.S5 Fig(A) Diagrams of predicted circRNAs from the sense (+) or antisense (-) RNA transcripts expressed from the KSHV genome and their relations to the corresponding splice sites (ss). (B) The KSHV K7/PAN RNA locus in Bac36/BCBL-1 reference genome with the nt position and orientation of each circRNA predicted in this study (WT-circRNA-1-5) and other three reports –37. The (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableThe splice site strength was determined by MAX:ENT algorithm .(XLSX)Click here for additional data file.S5 TableThe splice sites strength was determined by MAX:ENT algorithm .(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.