The RNA sequences from the 5ʹ UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase – (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5ʹ UTR of  The aminoglycoside antibiotics are among the earliest classes of antibiotics to be discovered and are used clinically to treat severe bacterial infections. They target the A site in the decoding region of the 30S ribosomal subunit and cause mistranslation of mRNA in the translocation process of bacterial translation . Aminoglint1 1), recombination site (attI 1) for integrase binding and the insertion of resistance gene cassettes (that contain an attC recombination site and a resistance gene), and two appropriately orientated (divergent) promoters, one promoter (Pint) drives the integrase gene and the other (Pc) transcribes the cassette genes [Historically, integrons were first discovered through the spread of antibiotic resistance. As mobile genetic elements, integrons accumulate antibiotic-resistance genes through site-specific recombination. The wide-spread application of antibiotics in the clinic created an environment where integron-mediated site-specific recombination on resistance plasmids led to the rapid acquisition of resistance genes and the proliferation of resistance plasmids (reviewed in ). Five ite genes ,9. Site-te genes ,10.attC site and the attI1 site ensures that cassettes are inserted in the correct orientation for expression from the integron Pc promoter [Asymmetric cassette recombination between the bottom strands of the promoter ,12. Exprpromoter . In the promoter ,15 and Spromoter .acc/aad gene expression. Reporter assays showed that specific aminoglycosides can induce expression of a reporter gene mediated by the riboswitch RNA. Direct binding between the aminoglycosides and the riboswitch RNA could be measured by Surface Plasmon Resonance (SPR). Chemical probing experiments and gel electrophoretic mobility assays further proved that aminoglycoside binding to the riboswitch RNA induced a structural transition in the RNA. A specific cross-link between the aminoglycoside sisomicin and the RNA was detected. Overall, the aminoglycosides that induced reporter gene expression displayed higher affinity for the leader RNA by SPR, its binding also caused a structural transition in the RNA. The induction of reporter gene expression through riboswitch binding to aminoglycosides can also take place in the presence of resistant ribosomes. For the published aac riboswitch of P. fluorescens, a model was put forward based on results from chemical probing data and mutational analysis; the RNA has tandem ribosome-binding sites (SD1 and SD2) and in the absence of the aminoglycosides, SD2 (GGAG) is sequestered by an anti-SD sequence (CUUC) which prevents ribosomal access; binding of aminoglycosides causes a structural change and migration of the anti-SD to base pair with SD1 (GGAG) thus making SD2 available for translation of the resistance gene . Subseqansition .aac/aad gene expression leading to aminoglycoside resistance. At the DNA level, the initial acquisition of the aminoglycoside resistance is mediated by class 1 integrons on plasmids carrying the aac/aad gene. The DNA sequence corresponding to the aminoglycoside-sensing riboswitch RNA therefore overlaps with the conserved attI1 site of the class 1 integrons. The relationship between the integron DNA sequence and the coincident riboswitch at the 5ʹ UTR of aac (aminoglycoside acetyltransferases) gene was investigated; sequence analysis of clinical aminoglycoside resistant strains and biochemical evidence showed that site-specific recombination mediated by the integron preserves the function of the aminoglycoside-sensing riboswitch of aac genes [aad (aminoglycoside adenyltransferases) genes. Here we have analyzed RNA sequences from the 5ʹ UTR of aad genes from pathogenic strains that are the end product of site-specific DNA recombination by class 1 integrons. These new sequences were analyzed by reporter assays and MicroScale Thermophoresis (MST). Covariance analysis data revealed an aminoglycoside-sensing riboswitch mechanism that was preserved at the DNA level by the selection of integron-mediated site-specific recombinants. This study reveals a correlation between the class 1 integron and an aminoglycoside-sensing RNA for aad genes and also further confirms the original discovery of aminoglycoside-sensing riboswitch.At the RNA level, the aminoglycoside-sensing riboswitch induces ac genes . In thisSuperscript III reverse transcriptase, ribonucleotide triphosphate (rNTP), RNase inhibitor, DNase I, fluorescein-5-thiosemicarbazide (FTSC) were purchased from Invitrogen. T7 RNA polymerase was purified in-house. Aminoglycoside antibiotics and oligonucleotide primers were purchased from Sangon Biotech. All other reagents were purchased from Sigma-Aldrich unless otherwise stated.3 sequences [aadB related sequences were input into CMfinder to produce local multiple sequence alignments of the structurally conserved motifs, that contained the motifs previously established by chemical probing [The published 75nt minimal aminoglycoside riboswitch RNA sequence was used to blast search the NCBI database (Nucleotide collection (nr/nt)) for more homologous sequences using default settings with an upper limit of 5*10equences . Duplica probing ,37. The  probing  using in probing . RNA str probing  and Adobaac/aad-lacZα as described before [The new riboswitch candidate sequences were cloned into the reporter plasmid pGEX-leaderRNA-d before . Agar did before  and milld before  were per2. The RNA was annealed by heating at 95°C for 2 minutes followed by slowly cooling down to room temperature. MST experiments were conducted in triplicate on a Monolith NT.115 system (NanoTemper Technologies) and data analysis was performed by MO.Affinity analysis software [The riboswitch RNAs were prepared by in vitro transcription using T7 RNA polymerase. Purified RNA was labeled with FTSC as previously described . The FTSsoftware ,43.aac/aad RNA was confirmed experimentally as the minimal functional unit [aad) and 41 acetyltransferases (aac) as shown in aac genes showed that integron-driven site-specific recombination preserves the functional riboswitch RNAs that can induce expression of aac genes in the presence of aminoglycosides [aad and aac genes is at the 3ʹ end and reflects the different origins of the resistance cassettes . The riboswitch RNAs are organized such that they retain the 5ʹ portion of the attI1 site that is highly conserved and includes the first ribosome-binding site (SD1). The 3ʹend of the RNA, adjacent to the aad coding sequence shows greater sequence variation  into thaac/aad-lacZα was previously used for functional analysis of the riboswitch RNA of aac/aad in vivo. In the plasmid, the sequence of the 75nt aac riboswitch RNA in P. fluorescens was placed under the control of the IPTG-inducible tac promoter (Ptac) and upstream of a β-galactosidase  reporter gene. Certain aminoglycosides induced reporter gene expression through the riboswitch RNA as measured by Miller assays in solution and agar diffusion assays on plates. Specific point mutations in the riboswitch RNA of P. fluorescens or unrelated UTR RNAs of cat-86, ermCL or ghoS genes were shown to cause a loss of reporter gene induction [aad riboswitch RNA sequences were identified. To compare the function of these aad riboswitch RNAs with the published riboswitch RNA, six riboswitch RNA sequences were chosen (narJ) [aac/aad-lacZα. The reporter plasmid was transformed into the E. coli strain JM109 and β-gal activity was assessed in the presence of aminoglycoside antibiotics by agar diffusion assays and Miller assays. In agar diffusion assays, on addition of IPTG, RNA is synthesized, and in the presence of the 4–6 deoxystreptamine aminoglycosides sisomicin, gentamicin, kanamycin B (KanB), amikacin or tobramycin, zones of reporter gene expression were observed for all six riboswitch RNAs, in contrast, only faint levels of reporter gene expression could be detected for neamine and ribostamycin, or paromomycin . Miller assays showed clear 4–6 deoxystreptamine aminoglycoside dependent induction of reporter gene expression, for all six sequences and only minimal levels of expression for neamine, ribostamycin and paromomycin . The results from the agar diffusion assays and the Miller assays are clearly consistent. In the absence of IPTG no reporter gene expression was observed for aad-4 with the antibiotics   as contromomycin  for the omomycin  for aad-ibiotics ). For thycosides ) in contaac riboswitch RNA [aac riboswitch sequences can distinguish between different aminoglycosides for optimal levels of reporter gene induction [aad-1-6 riboswitch RNAs, the highest induction of β-gal was observed with amikacin. In the presence of amikacin, aad-1, aad-4, and aad-6 mediate the induction of reporter gene expression by up to 4–5 fold, which is higher than that in the originally published riboswitch sequence, and comparable to the most recently analyzed sequences [aad-2 and aad-5 were also capable of inducing reporter gene expression to similar levels to the published aac riboswitch [aad and aac [Initially, of the aminoglycosides, sisomicin gave the highest induction of reporter gene expression in the itch RNA , and thinduction . Here foequences , both aaboswitch   [aad riboswitch sequences, MST was used to measure aminoglycoside RNA binding. The riboswitch RNA aad-4 was prepared and labeled with fluorescein-5-thiosemicarbazide as described before [µM) and ribostamycin (40 µM) and the 4–5 deoxystreptamine paromomycin (10 µM) have much weaker affinities although their binding is also consistent with the formation of a 1:1 complex  ,36 and td before . The mead before . On titr complex ). There pression ).Figure aac riboswitches: in the absence of the antibiotic, the ribosome-binding site (SD2) of the riboswitch sequence was sequestered by a complementary anti-SD sequence, which blocked ribosome binding for translation; upon antibiotic binding a structural transition occurred leading to anti-SD base-paired with SD1 and free SD2 for ribosome binding/translation of the resistance gene [aad riboswitch RNAs differ from the aac riboswitch RNA at the 3ʹend where the regulatory elements lie  all share covariance structures and the potential for a structural transition. The first gene cassettes of the type 1 integrons can exploit lower frequency start codons and the aad genes are characterized by the presence of a low-frequency UUG initiation codon located 4–5 nt downstream of a consensus ribosome-binding site (SD2) sequence (AGGU) [aad riboswitch sequences, although the SD2 (AGGU) and anti-SD2 (UUCU) sequence were variable, without the aminoglycosides SD2 is sequestered by the anti-SD2 sequence and, upon aminoglycoside binding, can potentially undergo a structural transition in which the anti-SD2 sequence pairs with SD1 and consequently makes SD2 available for ribosome binding. The sequences at SD2 and anti-SD2 of the riboswitch RNA are products of selective site-specific recombination by the integron so that bacteria survive in an antibiotic-rich environment through acquiring aminoglycoside resistance by activation of the aad gene. In the situation where recombination takes place in the absence of selection; the RNA sequences may fail to function as riboswitches and consequently inefficient translation of the aad RNAs may cause sensitivity to the antibiotic resulting in strain death. We note that the anti-SD2 sequence in the aad riboswitch is located immediately after the UUG initiation codon. In contrast, anti-SD2 sequence in the aac riboswitch is found before the AUG initiation codon. Nevertheless, in both cases integron-driven recombination selects viable riboswitch sequences and delineates essential features of the riboswitch.A model was proposed for the regulatory mechanism of the nce gene ,36,47 (Fents lie ). Despitansition ). We foue (AGGU) ,15. In taac riboswitch caused a structural transition such that the SD2 sequence became more accessible for translation. The A18 position of the aac riboswitch was identified as part of the aminoglycoside binding site by chemical probing and cross-linking experiments [aad riboswitch was made and its activity with aminoglycosides examined by agar diffusion and Miller assays . The sir assays ,g) suggeThere is a precedent for the translational regulation of ribosomal antibiotic-resistance gene expression through antibiotic-RNA-ribosome interactions. For the erythromycin resistance methytransferase, specific interactions between the amino acids of the nascent peptide and the peptide exit tunnel lead to ribosome stalling ,48. Howeaad at the RNA level was investigated. The aminoglycoside-sensing riboswitch is linked with the class 1 integrons [attI). Blast searches of the aminoglycoside riboswitch identified new sequences from enduring aminoglycoside resistant pathogen strains, which are the end product of site-specific recombination by class 1 integrons. Analysis of the top six aad aminoglycoside riboswitch sequences revealed considerable sequence variation compared to that of aac; in particular the aad and aac riboswitches differ at the 3ʹend. Even single point mutations in the aac riboswitch can impair its function [aad sequences, the RNAs function as riboswitches in reporter assays (Miller assay and plate assays). Specific aminoglycoside-RNA binding was measured by MST  at the DNA level and its associated aminoglycoside riboswitch in the 5ʹUTR of ntegrons ,36,45 anfunction , but desd by MST ,b, and Sibiotics . The indaac riboswitch RNA, each RNA sequence showed distinctive discrimination between different aminoglycosides in the reporter assay. For instance, among the aminoglycosides gentamicin-induced the highest levels of reporter gene expression for the aac-6 RNA, but sisomicin gave the highest reporter gene expression for aac-4 RNA and the optimal fold changes (~2-5 fold induction) were comparable to those observed in a clinical setting [aac riboswitch RNA, amikacin induces optimal reporter gene expression for each of the aad riboswitch RNAs that were investigated. The reporter assay allowed comparison of the aac and aad riboswitch RNAs in identical genetic backgrounds; the only difference in the assay is the different riboswitch RNAs. Both of the 5ʹUTR RNAs of the aac and aad genes have been preserved as functional riboswitches by the integron-mediated recombination.For the  setting ,49. Hereaac and aad riboswitch RNAs are probably due to specific interactions between the individual riboswitch RNA and the particular aminoglycoside. We have found that for each of the three RNAs in this and in previous studies [The polycationic, flexible nature of the aminoglycosides might be expected to lead to nonspecific binding to polyanionic RNA. However, the differences in selectivity observed in both reporter assays and in equilibrium binding experiments ,36 FiguFigure 3 studies ,36, the Genetic recombination is an important evolutionary driving force that plays a key role in the generation of genetic diversity. In pathogenic bacteria the exposure to antibiotics has led to the specific acquisition of antibiotic-resistance genes through integron-mediated site-specific recombination. This flexible mechanism of gene transfer is an efficient survival response to the challenge of new and evolving classes of antibiotics. Aminoglycoside resistance emerged soon after their introduction to the clinic toward the middle of the last century ,50. The Click here for additional data file.


Colon adenocarcinoma (COAD) is a malignant and lethal tumor in digestive system and distance metastasis lead to poor prognosis. The metastasis-specific ceRNAs (competitive endogenous RNAs) and tumor-infiltrating immune cells might associate with tumor prognosis and distance metastasis. Nonetheless, few studies have concentrated on ceRNAs and Immune cells in COAD.The gene expression profile and clinical information of COAD were downloaded from TCGA and divided into two groups: primary tumors with or without distance metastasis. We applied comprehensive bioinformatics methods to analyze differential expression genes (DEGs) related to metastasis and establish the ceRNA networks. The Cox analysis and Lasso regression were utilized to screen the pivotal genes and prevent overfitting. Based on them, the prognosis prediction nomograms were established. The cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm was then applied to screen significant tumor immune-infiltrating cells associated with COAD metastasis and established another prognosis prediction model. Ultimately, co-expression analysis was applied to explore the relationship between key genes in ceRNA networks and significant immune cells. Multiple databases and preliminary clinical specimen validation were used to test the expressions of key biomarkers at the cellular and tissue levels.R = −0.200, P < 0.001), Macrophages M0 and hsa-miR-125b-5p  and Macrophages M0 and FAS . Multidimensional validation and preliminary clinical specimen validation also supported the results.We explored 1 significantly differentially expressed lncRNA, 1 significantly differentially expressed miRNA, 8 survival-related immune-infiltrating cells, 5 immune cells associated with distance metastasis. Besides, 3 pairs of important biomarkers associated with COAD metastasis were also identified: T cells follicular helper and hsa-miR-125b-5p  and tumor-infiltrating immune cells (T cells follicular helper and Macrophages M0) might related to distance metastasis and prognosis of COAD. The nomograms could assist scientific and medical researchers in clinical management. Colon adenocarcinoma (COAD) ranks the third and fourth place in the rankings of cancer incidence and mortality all over the world, respectively . More thThe ceRNA network postulates that any RNA transcript harboring microRNA (miRNA)-response elements can sequester miRNAs from other targets, thereby regulating their expression and coordinating biological processes . PreviouIn the present study, we set up a ceRNA network based on COAD metastasis related ceRNAs, which were identified by gene expression profiling from the TCGA database. In addition, we evaluated the ratios of tumor-infiltrating immune cells associated with COAD metastasis by using the “cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT)” algorithm. Depended on the differentially expressed immune cells and ceRNAs types, nomograms were constructed to predict COAD metastasis and prognosis. Furthermore, the correlation analysis was performed to explore the metastasis mechanism of COAD. The constructed nomograms and the identified regulatory mechanism might provide new insights for the prediction and treatment of COAD metastasis in clinic.1) database, RNA profiles including HTseq-count and fragments per kilobase of exon per million reads mapped (FPKM) of 459 primary COADs were downloaded. The experimental group and the control group in this study were 91 primary COADs with distant metastasis and 368 primary COADs without distant metastasis. We also retrieved the demographic information , survival endpoint  and stage and histological type of primary and new tumors of each patient. When non-colon cancer specific expression genes  had been filtered, the edgeR method was utilized to identify differentially expressed RNAs including lncRNAs, miRNAs, and mRNAs. The downregulated or upregulated gene was, respectively, defined with a false discovery rate (FDR) P value < 0.05, the log(fold-change) > 1.0 or < −1.0.This study was endorsed by the Ethics Committee of the Shanghai Tenth People’s Hospital affiliated to Tongji University. From the cancer genome atlas . After that, prominent results shown in hypergeometric testing and correlation analysis by miRNAs which regulated both lncRNAs and mRNAs were singled out to construct the ceRNA network with Cytoscape v.3.5.1  were applied. Finally, Kaplan–Meier survival analysis was used to validate each biomarker.P < 0.05 were deemed to be worthy of further analysis. It had significant differences in the pro-portion between primary tumors with and without distance metastasis by finding the immune-infiltrating cells with the Wilcoxon rank-sum test.The CIBERSORT algorithm was utilized to assess the 22 kinds of immune cell types in carcinoma COAD. Only if those samples which had a CIBERSORT output of In order to render certain that the multifactor models weren’t overfitting, we integrated all significant cells into the Cox model and then the reduced Cox model was constructed. Applying for Cox proportional hazards model and Kaplan–Meier survival analysis to identify the prognostic value of all tumor-infiltrating immune cells. Then, the calibration curves and receiver operating characteristic curves (ROC) were performed to estimate the accuracy and discrimination of the nomogram. Ultimately, based on Pearson correlation analysis, a co-expressed heatmap was drawn to show the correlation of various immune cells.To minimize bias, multiple databases which included Oncomine , The HumFour-micron sections of formalin-fixed paraffin-embedded (FFPE) tissue blocks from 20 primary COAD samples, including ten diagnosed with metastasis and ten diagnosed without metastasis, were deparaffinized and dehydrated. Following routine rehydration, antigen retrieval, and blocking procedures, the sections were incubated overnight with FAS antibody , NES antibody , CD68 antibody , CD3 antibody  and BCL6 antibody  at 4°C, respectively. Next, all slides were ladeled polymer HRP for 30 min and hematoxylin as a counterstain for 5 min at room temperature. Two pathologists read the pathological sections and determined positive results when the cytoplasm of cancer cells was stained. The percentage scoring of tumor cells was as follows: negative (0), yellowish (1–4), light brown (5–8), and dark brown (9–12). Negative controls had the primary antibody replaced by buffer. Additionally, the non-parametric test and correlation analysis were performed to evaluate the relationship between the percentage scoring of tumor cells and clinical features of patients.TM . Amplification included an initial denaturation step for 30 s at 95°C, followed by 40 cycles of PCR at 95°C for 5 s, and at 60°C for 31 s and a dissociation stage for 15 s at 95°C, 60 s at 60°C and 15 s at 95°C. After the reactions were complete, the cycle threshold (CT) data were determined using fixed threshold settings and the mean CT was determined from triplicate PCRs. A comparative CT method was used to compare each condition to the control reactions. mRNA levels were normalized to U6. The relative amount of gene normalized to control was calculated with the equation 2–ΔΔCT.Quantitative reverse transcriptase-PCR (qRT-PCR) was used to quantitative expression of key miRNAs in the ceRNA network. According to the manufacturer’s instructions, total RNA was extracted from the tissues using TRIzol Reagent (Invitrogen). The OD260/OD280 ratio of total RNA extracted from tissue samples ranged from 1.8 to 2.0. Reverse transcription was performed in a 10 μl reaction volume using M-MLV reverse transcriptase  with 1 μg of RNA. Quantitative real-time PCR was performed using an ABI 7900 Detection System with the SYBR Premix Ex TaqP value < 0.05 was considered to have necessary to statistic. All statistical analysis was put into effect with R version 3.5.1 software   . All these genes made up 4 lncRNA–miRNA pairs and 5 miRNA–mRNA pairs . In the = 0.022) . All thi= 0.022) . Based o= 0.022) . The Las= 0.022) . Both th= 0.022)  were appP = 0.038), B cells naive (P = 0.040), Tregs (P = 0.033), Macrophages M0 (P = 0.034) and Eosinophils (P = 0.028) had significant differences in the immune cell fractions between distance metastasis and primary colon adenocarcinoma (P = 0.030), Plasma cells (P = 0.019), T cells CD8 (P = 0.035), Dendritic cells activated (P = 0.048), Dendritic cells resting (P = 0.048), Macrophages M1 (P = 0.010), Mast cells resting (P = 0.043), and Mast cells activated (P = 0.025) had obvious differences in the immune cell fractions between distance metastasis and the recurrence of COAD  , Macrophages M0 fraction , dendritic cells activated (N stage: P = 0.009) and monocyte  all showed significant correlation with clinic features  Macrophages M0 and hsa-miR-125b-5p  and Macrophages M0 and FAS  and these could describe the significant relationship between the key biomarkers and distance metastasis COAD.Firstly, an initial Cox regression model with all significant tumor immune cells was constructed . As the < 0.001) . The co-< 0.001) . T cellsfeatures . FinallyP = 0.005; CD68 in lymph nodes, P < 0.001; CD3 in all tissue, P = 0.027; CD3 in lymph nodes, P = 0.001; BCL6, P = 0.010; NES, P = 0.002; FAS, P = 0.005) (R = −0.500), CD68 and NES (R = −0.650) shown co-expression trends (The results of IHC (20 primary COAD samples including 10 with metastasis and 10 without metastasis) suggested that proteins of FAS, NES, CD3, CD68, and BCL6 were significantly different in COADs diagnosed with metastasis and without metastasis . The res= 0.005) . The exp= 0.005) . Addition trends .P = 4.98E-6) was lowly expressed in primary COAD compared to normal colon and BCL6 , CXCL13 , CD3E  and MME  showed obvious difference between primary COAD and normal colon in the Oncomine  and BCL6  was significantly different between COAD and normal colon in GEPIA (P = 4.623E-6) and hsa-miR-125b-5p (P = 1.102E-9) were significantly related to tumor purity and tumor stage of COAD (P = 4.536E-5), CD3D (P = 1.511E-13) and CD3E (P = 4.536E-5) in LinkedOmics  and NES  had significant metastasis-free survival (MFS) with patients (P < 0.001). And the ROC curve (AUC = 0.698) illustrated acceptable discrimination of the multivariate Cox regression model  and T cells follicular helper  significantly. Meanwhile, FAS was associated with macrophages M0  significantly. We could deduce that the three pairs and their relevant mechanisms would play important roles in the prediction and cure of COAD metastasis.Our study found differently expressed ceRNAs and marked tumor-infiltrating immune cells between COAD metastasis group and non-metastasis group firstly. Then, two prediction nomograms were drawn based on the selected ceRNAs and tumor-infiltrating immune cells. And AUC values of the two nomograms might be used to estimate the clinical diagnosis of COAD metastasis. In addition, we found that metastasis-related ceRNAs of hsa-miR-125b-5p and FAS and immune cells of Macrophages M0 and T cells follicular helper could predict prognosis effectively. At last, the correlation analysis showed that hsa-miR-125b-5p was associated with macrophages M0  receptor superfamily, mediates the apoptosis process . One stuIn our study, some restrictions which should be acknowledged do inevitably exist. First of all, the amount of data we collect from publicly available databases is indeed limited, which means that the clinical pathology parameters analyzed in our study were not completely all-inclusive and might bring about potential errors or partialities. In addition, we didn’t consider the heterogeneity of the immune microenvironment which is allied to the immune infiltration location. Last but not the least, the biggest problem in this study is the lack of experiments on the direct interaction mechanisms of ceRNA and cellular communication . However, in order to diminish the bias, multiple databases were also utilized to uncover the expression of key biomarkers at gene expression, protein expression, tissue and cellular levels and it illustrated that the key biomarkers were regulated upward in normal COAD tissues and cell lines significantly and normal colon tissue didn’t express these proteins –S8. SincBasing on the ceRNA network and tumor-infiltrating immune cells we studied, we constructed two nomograms, in order to predict survival and metastasis of COAD patients. The utility was further attested with their high AUC values. What’s more, the prediction nomograms might provide a good deal of useful comprehensive clinical information for doctors, so as to improve individual management for COAD patients. Moreover, our study inferred that the mechanism of hsa-miR-125b-5p. FAS and Macrophages M0 might be important in COAD prognosis and while T cells follicular helper, Macrophages M0 and hsa-miR-125b-5p, Macrophages M0 and FAS played significant parts of the distance metastasis of COAD.https://portal.gdc.cancer.gov), Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) .Publicly available datasets were analyzed in this study. This data can be found here: The code and datasets generated and/or analyzed during the current study are available in the The studies involving human participants were reviewed and approved by the Ethics Committee of the Shanghai Tenth People’s Hospital affiliated to Tongji University. The patients/participants provided their written informed consent to participate in this study.ZC, RH, WF, TM, ZH, QW, and HQ: conception/design and data analysis and interpretation. TM, ZH, QW, and HQ: provision of study material. ZC, RH, WF, JL, GJ, JH, WS, HY, WW, TM, ZH, QW, and HQ: collection and/or assembly of data, manuscript writing, and final approval of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AluJ and was activated by a C > T mutation optimizing the 5ʹ splice site. Using a combination of SHAPE, DMS and enzymatic probing, we show that the disease-causing mutation disrupted a conserved AluJ stem that evolved from helix 3.3 (or 5b) of 7SL RNA, liberating a primordial GC 5ʹ splice site from the paired conformation for interactions with the spliceosome. The mutation also reduced flexibility of conserved residues in adjacent exon-derived loops of the central Alu hairpin, revealing a cross-talk between traditional and auxilliary splicing motifs that evolved from opposite termini of 7SL RNA and were approximated by Watson-Crick base-pairing already in organisms without spliceosomal introns. We also identify existing Alu exons activated by the same RNA rearrangement. Collectively, these results provide valuable TE exon models for studying formation and kinetics of pre-mRNA building blocks required for splice-site selection and will be useful for fine-tuning auxilliary splicing motifs and exon and intron size constraints that govern aberrant splice-site activation.Transposed elements (TEs) have dramatically shaped evolution of the exon-intron structure and significantly contributed to morbidity, but how recent TE invasions into older TEs cooperate in generating new coding sequences is poorly understood. Employing an updated repository of new exon-intron boundaries induced by pathogenic mutations, termed DBASS, here we identify novel TE clusters that facilitated exon selection. To explore the extent to which such TE exons maintain RNA secondary structure of their progenitors, we carried out structural studies with a composite exon that was derived from a long terminal repeat (LTR78) and  Intron recision . Apart frecision . These precision , howeverAlu elements, have been major contributors to TE exonization during evolution and in human genetic disease [Alus contain a number of decoy splice-site motifs that are readily recognized by the spliceosome, generating a substantive pool of low-inclusion exons that are likely to play an important regulatory role in quantitative gene control by targeting RNAs for nonsense-mediated decay [Mutations or variants anywhere in exons or introns can alter splice-site selection and lead to genetic disease . The mos disease . Alus coed decay ,17,18. Hin silico prediction of mutations that affect pre-mRNA processing is unsatisfactory, despite a growing number of predictive algorithms, totalling to over a hundred to date. Their poor performance stems from our limited understanding of how exactly the spliceosome selects authentic splice sites in a large excess of very similar pre-mRNA motifs that are never used, termed decoy splice sites. Splicing enhancers and silencers represent the weakest link in the puzzle: despite numerous and systematic attempts to define these motifs [ab initio methods. A key obstacle is our limited insight into co- and post-transcriptional pre-mRNA folding and its dynamics that may expose, hide, approximate or separate residues involved in interactions with numerous spliceosome components [Alus from 7SL RNA [Over the last decades, it has become increasingly apparent that e motifs ,19,20, emponents ,22. Despmponents , our knomponents . Because 7SL RNA , diseaseF8 gene altered accessibility of splicing regulatory motifs in the AluJ-derived portion of the exon and of the 5’ss itself. The mutation disrupted a conserved AluJ stem that evolved from the central helix of 7SL RNA, liberating the optimized 5’ss from the highly conserved double-stranded conformation for interactions with spliceosomal components. The stem harbours a primordial GC 5’ss that was base-paired together with prospective exonic splicing regulatory motifs in the opposite strand for ~2 billion years. We also identify existing Alu exons activated by the same RNA rearrangement.The aim of our present study was to identify new mutation-induced TE exons activated in genetic disease. We have found new exonized TE clusters, defined here as iterative genomic invasions of new TEs into existing TEs. We have selected one such composite exon for RNA structural probing. We show that a point mutation activating a new 5’ss in the de novo’ sites) [ex vivo minigene studies were also included because they usually recapitulate in vivo splicing defects with high accuracy [We first updated previously developed databases of aberrant 3ʹ and 5’ss, termed DBASS3 and DBASS5 . They se’ sites) ,32. Alth’ sites) ,10,32. R’ sites) . The seaaccuracy . Exon skhttp://www.ncbi.nlm.nih.gov/Genbank) [http://www.ensembl.org) [Qualifying reports of aberrant splice sites were verified against reference sequences from GenBank (Genbank)  and Ensembl.org) . In-housmbl.org)  and any mbl.org) , as descmbl.org) ,10.Sequences surrounding validated aberrant splice sites were used as an input to search for TEs with a crossmatch search engine of RepeatMasker (v. 3.0), employing its highest sensitivity option . TEs aliF8 pseudoexon, we synthesized wild-type (WT) and mutated (F8 c.5998 + 530 C > T) RNAs for structural probing. Probe templates were prepared using nested PCR with outer primers  and probe- and mutation-specific inner primers . The linkers allow the reverse transcriptase (RT) to become fully processive prior to reaching the region of structural interest and also prevent non-templated primer extension products from masking structural information [To support computational predictions ,40 of prormation . The for2. The samples were incubated at 37 °C for 45 min. 2-methylnicotinic acid imidazolide (NAI) or dimethyl sulphate (DMS) were added to a final concentration of 100 mM and allowed to react with the RNA for 5 min at 37 °C (NAI) or for 4 min at room temperature (DMS). The reaction was quenched with a freshly prepared dithiothreitol at a final concentration of 0.2 M (NAI) or 0.5 M (DMS) and mixed thoroughly. The reactions were immediately loaded on to the RNA Clean&Concentrator™-5 (ZYMO Research). RNAs were eluted in 10 µl of RNase-free double-distilled water and 6 µl of purified RNA was mixed with 1 µl of a 5 µM solution of the Cy5-labelled universal primer (5ʹ-GAA CCG GAC CGA AGC CCG). The samples were heated at 75 °C for 3 min. Two µl of 5x RT reaction buffer were added to each sample to a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 5 mM dithiothreitol. The reaction was incubated at 35 °C for 5 min, which was followed by the addition of 0.5 µl of dNTPs (10 mM) and 0.5 µl of Superscript III RT (200 U/µl), and a 15-min incubation at 50 °C. Next, 0.5 µl of 2 M NaOH was mixed with each RT reaction and samples were heated at 95 °C for 15 min to degrade RNA and denature RT. The reaction was then mixed with an equal volume of 2x stop solution, containing 95% deionized formamide, 20 mM Tris (pH 7.5), 20 mM EDTA (pH 8.0) and Orange G (Abcam) for tracking. The samples were heated at 95 °C for 5 min and the RT products were size-fractionated on 8.3 M urea-8% polyacrylamide gels at the constant power of 65 W for 3–5 hrs. Gel images were collected with a Typhoon PhosphorImager 9210 and individual bands were quantified using ImageQuant 8.2. The nucleotide identity of RT stops was determined from dideoxy-sequencing lanes run in parallel. DMS and NAI signals were normalized to the fully extended product [probability of unpaired) values were computed as described [F8 pseudoexon and 100-nt flanking intron sequences as an input. PU values predict single-stranded conformation of auxiliary splicing motifs and were defined previously [Validated transcripts were denatured at 95 °C for 90 seconds and cooled to 4 °C. An equal volume of a 2x reaction buffer was added to 10 pmol of each RNA probe to a final concentration of 100 mM KCl, 40 mM HEPES pH 7.5) and 0.5 mM MgCl product  or using product . Signals product ,44,45. T.5 and 0.For enzymatic probing, we digested the same probes with RNAse A (Ambion), which cleaves single-stranded pyrimidines. The reactions were incubated in a final volume of 100 μl at room temperature for 3 min. A control aliquot of RNA without RNase A was processed simultaneously with digested samples. Reactions were stopped by adding SDS (0.5%) and proteinase K (200 μg/μl) and incubated at 55°C for 1 hr. The cleaved RNA was purified using TRI-Reagent and 200 µg of each probe was reversed transcribed as described above. Signals from digested products were quantified using ImageQuant 8.2 and normalized to the full-length signal.de novo or cryptic splice sites that were activated by disease-causing mutations in ~390 genes. Full DBASS data are freely available at http://www.dbass.org.uk or http://dbass.soton.ac.uk, with direct links to DBASS3 or DBASS5 at http://dbass3.soton.ac.uk and http://dbass5.soton.ac.uk. DBASS3 currently holds 381 aberrant 3’ss in 193 genes that were causally associated with ~200 distinct human phenotypes. DBASS5 provides details of 693 aberrant 5’ss that were activated in 283 disease genes and were responsible for ~290 recognizable phenotypes.DBASS3 and DBASS5 show sequences of 1,074 experimentally verified de novo splice sites were more frequent among aberrant 3’ss than among aberrant 5’ss (2 test). We attribute this bias mainly to the accumulation of de novo 3’ss upstream of authentic counterparts in extended 3’ss motifs that are located in the AG-dinucleotide exclusion zone and define the first splicing step, ie. PPT and BPS [de novo 3’ss in PPT upstream of authentic counterparts [Breakdown of the updated DBASS data showed that  and BPS . In additerparts  is offse2 test), indicating that multiple aberrant 3ʹ and 5’ss are induced by disease-causing mutations with approximately equal frequencies.The fraction of multiple aberrant 5’ss activated by a single pathogenic mutation was ~13% . These cases are exemplified by reports of aberrant 3’ss in IVD [IDS [GCK [top right). Such introns may lack 3’ss consensus motifs, are recognized by intron definition rather than exon definition, and may require cross-intron bridging interactions [bottom): a third of strong cryptic 5’ss activated in introns had small upstream exons, as exemplified by intronic 5’ss reported in ATM [COL7A1 [COL1A1 [cf. bottom left and right).Employing a sample of 92 pairs of weak-authentic and strong-cryptic splice sites, we explored if their activation was constrained by the length of adjacent introns or exons (Table S1). The breakdown of DBASS entries showed that introns with over a fifth of such strong cryptic 3’ss were flanked by small (≤100 nt) exons downstream  but was longer than the median of human exons that were skipped as a result of disease gene mutations instead of activating aberrant sites (111 nt) [To further explore the importance of size limits, we computed median exon sizes for strong-aberrant 3ʹ or 5’ss activated in introns. The median of their adjacent exons was somewhat shorter than the median size of human internal exons  .in silico prediction tools.Together, these data demonstrate intron and exon size constraints for about a fifth of strong-cryptic counterparts of weak-authentic sites and highlight the importance of incorporating size limits into P< 10−6). We also found more TEs in the former than the latter pseudoexons . Alignmmophilia , which dCOL4A5 pseudoexon, including the predicted BPS, was derived from a DNA transposon (MER58A) that was inserted into a more ancient long interspersed element (LINE1 or L1). The L1 copy contributed the full 5’ss consensus and the 3ʹ end of the pseudoexon (F8 pseudoxon [Alu exonization as a result of a downstream mutation outside this element.eudoexon ). As witseudoxon , the milseudoxon . The synseudoxon , also wiAluJ exon for structural probing of F8 transcripts representing the splicing-proficient mutant and splicing-deficient WT  [AluJ stem instead of the 7SL RNA moiety of the SRP S-domain (AluJ exonizations.We selected the LTR78/tant RNA . Both re 7SL RNA  and the luJ exon . The two (BC200) . Both ad (BC200) , revealeS-domain . The triS-domain  and is mS-domain , suggestF8 transcripts revealed that the mutated cytosine was in the middle of AluJ-derived helical structure that evolved from helix 3.3 . The destabilization was supported by alternative secondary structures . This finding suggests that the optimized 5’ss was released from the paired conformation in H3.3 to a more flexible, possibly unpaired conformation. The rearrangement may also stabilize a hairpin capped by an apical tetraloop GAGG  of toop GAGG . The samAlu-derived exons, we inspected the database of exonized TEs [Alus in the human transcriptome, 110 were found in the sense orientation and 22 of them carried AluJ-derived fragments [F8 exon and 7SL RNA revealed at least five existing AluJ exons that used identical decoy 5’ss . In addition, analysis of splicing regulatory hexamers around their F8 triloop homologs confirmed that they have largely enhancer activities, with a median ESRseq score of 0.41 (Table S3). Homologs of the CCA triloop in exons derived from younger Alu families were usually CGA .To explore if the same decoy GC 5’ss was used by existing ized TEs . Among ~ragments . Comparion in F8 ). We thede novo 5’ss of the LTR78/AluJ exon in F8 altered accessibility of a composite splicing regulatory motif ~35-45 nt upstream and of the 5’ss itself. The mutation destabilized the AluJ ortholog of 7SL RNA H3.3  and possibly the entire central stem. The H3.3 disruption would free the highly conserved decoy 5’ss from the double-stranded conformation to allow base-pairing with the U1 small nuclear RNA, a critical interaction for selection of human 5’ss by U1 small nuclear ribonucleoproteins [Alus, generating almost a fifth (~19%) of sense Alus exons in at least 20 other human genes .Taken together, the C > U mutation creating a The impact of DNA variants or mutations on RNA processing has been notoriously difficult to accurately predict from the sequence alone without structural information. By providing large sets of verified aberrant transcripts induced by recent mutations, the updated DBASS offers an opportunity to better understand structural consequences of Nature’s own human experiments, i.e. pathogenic mutations causally associated with verified splicing defects. Our data support the notion that recently exonized TEs and their TE partners have a more predictable folding landscape than average coding sequences. Their identification thus provides a useful resource for future pre-mRNA structural studies, ultimately leading to more accurate predictions.in silico tools. Apart from size thresholds discussed for DBASS now contains genomic coordinates and can be better integrated into currently available predictive algorithms, including variant interpreters for clinical use, such as ClinVar . DBASS3 Alus [TEs have been exapted as coding and regulatory sequences in many host genes . For exaAlus .F8 exon was contingent on the presence of both LTR78 and AluJ copies  is not used in vivo, as excluded by RT-PCR [Alus via new 5’ss [F8 are base-paired in the conserved 7SL RNA-derived stem Alu domain of SRP RNA moiety is conserved in eukaryotes and archaea, but not in all eubacteria [Alu domain that maintained the highly conserved fold to date. Our comparison with published structures of the Alu domain RNAs [F8 was present already in archaeon M. jannaschii (CAU/GCCCAC). The paired configuration of the primordial 5’ss consensus can even be traced back to some eubacteria, such as B. subtillis, where the Alu domain is stabilized by prokaryote-specific 7SL RNA extensions that make interacting proteins dispensable [The bacteria . Bacteribacteria ,89. Thisbacteria ,89. Assubacteria , the C >ain RNAs ,91 reveapensable ,91.F8 AluJ with 7SL RNA and existing Alu exons suggests that release of decoy 5’ss from H3.3 took place not only in AluJ exons, but also in free left Alu monomers and younger subfamilies  also points to the importance of their 3’ss and cross-exon motifs. Finally, apart from the left arms of sense Alu, a homologous decoy 5’ss was likely used for exonization of their right arm, as exemplified by AluS in TBL1Y , althouF8 AluJ could not sustain a swap of the central GC base pair for the wobble GU pair  flips back onto the highly flexible 5ʹ portion upon binding of the SRP9/14 heterodimer [Alu domain fold [Alu-like RNAs, including BC200 [Alu domain folds back up to 180 degrees to contact the 5ʹ portion and SRP9, progressing into the closed Alu RNP conformation [Alu RNA 5ʹ domain remains flexible [The erodimer . The hetain fold  and probng BC200 . It firsng BC200 ,100. In ormation . Interesflexible .Alu elements appear to fold independently, each maintaining the overall cruciform 7SL RNA structure [Alu dimers seem to provide a much more efficient substrate for splice-site activation than expected for Alu monomers alone, arguing for a thus far unexplained synergistic effect [Alu exons; they were activated more frequently through 3’ss with PPTs derived from antisense polyA tails [Alus do not enjoy a luxury of PPT-driven support of antisense copies [F8 probes is supported by stable stem(s) consisting of 7 contiguous base pairs (in vivo, at least for a limited time after transcription. In the SRP RNA, however, the GAGG motif is a part of the 3-way RNA junction (F8 AluJ copy would open up the structure, potentially precluding SRP9/14 binding and formation of the closed conformation. Future studies should also characterize interactions affected by the C > U mutation in H3.3 orthologs in more detail, both with protein and RNA trans-acting factors, and address how exactly the 5’ss and auxilliary motifs upstream cooperate when no longer held together by base-pairing.The left and right arms of tructure . Howeverc effect . The antyA tails , rather e copies ; to exone copies  or LTR (se pairs . Such lojunction . Speculaie. organisms that lack spliceosomal introns.In conclusion, we report that a single-nucleotide substitution unleashed a decoy GC 5’ss motif that was hidden by Watson-Crick base-pairing in the central stem of 7SL RNA ~2 billion years before the 5’ss could become activated in the primate central stem progeny and cause haemophilia. The paired conformation of decoy GC 5’ss and its primordial exon repressor sequences in the opposite strand and 7SL RNA terminus can be traced back into secondary structures of archaea and eubacteria, Click here for additional data file.


Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast  Saccharomyces cerevisiae (yeast) and human are the best-studied examples in this class. Unlike T7 RNAP, the mitochondrial RNAPs depend on transcription factors for promoter-specific transcription, a known feature of multisubunit RNAPs. The yeast mitochondrial RNAP, Rpo41, requires Mitochondrial Transcription Factor 1 (Mtf1) to initiate promoter-specific transcription  that are distinct from nuclear RNAPs and homologous to single-subunit bacteriophage T7/T3 RNAP . The mitcription ,3 and thcription .Mtf1 and TFB2M are evolutionarily related to RNA methyltransferases, but as transcription initiation factors, they facilitate promoter-specific transcription initiation ,6–9. BotB-reader of the initiation factors of bacterial and nuclear RNAPs, respectively, which are present in a similar location in the path of the initially growing RNA transcript  with T7 RNAP bound to a 7-bp RNA–DNA hybrid (PDB: 2E3E)  shows thanscript ,18. The anscript . The bacHerein, we have used ensemble and single-molecule fluorescence-based methods to investigate the role of the C-terminal tail region of Mtf1 and TFB2M. Contrary to previous studies , we showThe expression and purification of Rpo41, Mtf1, and C-tail deletion mutants were carried out as reported previously . The expDNA oligodeoxynucleotides were custom synthesized with biotin and amino modifications and purified by HPLC . DNAs were labeled with Cy3 or Cy5 NHS ester fluorophores at the amine groups by standard methods  and unreacted dyes were removed by ethanol precipitation. Pairs of single-stranded DNAs were mixed in 1:1 ratio, annealed at 95°C for 1 min, and cooled over an hour to room temperature to make the duplex DNA molecules.robs) were recorded with excitation at 555 nm and emission at 580 nm. The robs versus protein concentration plots were fit to Equation (Kd).Fluorescence anisotropy measurements were carried out on Fluoro-Max-4 spectrofluorometer  at 25°C. TAMRA fluorophore-labeled promoter DNA (5 nM with TAMRA label on the 5′ end of template strand) was titrated with 1:1.2 ratio of Rpo41 and Mtf1 in reaction buffer A . Anisotropy values (The fluorescence intensity (excitation at 315 nm and emission 380 nm) of 2AP labeled promoter (200 nM) in reaction buffer A was recorded before and after the addition of 400 nM Rpo41 and 400 nM Mtf1-WT or C-tail mutants in succession. After subtracting background fluorescence from buffer alone, the contribution of Mtf1-WT or mutants in promoter melting was calculated as fold change over the fluorescence of DNA alone .Kd of the initiating nucleotides .Increasing concentrations of initiating nucleotides were added to a complex of position -1 modified 2AP promoter DNAs (200 nM), Rpo41 (400 nM), and Mtf1-WT or Mtf1-Δ20 (400 nM) at 25°C in reaction buffer A. Fluorescence emission at 380 nm after excitation at 315 nm was recorded. With the human mitochondrial transcription proteins, we used 100 nM LSP or HSP1 promoter modified with 2AP at the –1 position, 200 nM each of TFAM, POLRMT, and TFB2M in reaction buffer B  labeled with 2AP at position −4 was incubated with Rpo41 (400 nM) at 25°C in reaction buffer A. Mtf1-WT, or Mtf1-Δ12 (200 nM) was added and transcription was initiated by adding a mixture of NTPs (100 μM) and 3′dNTP (250 μM) to stall complexes at positions +7 to +10. The time course of 2AP fluorescence intensity change was measured as described above. The fluorescence intensities were normalized for comparison.32P]ATP. The 2-mer synthesis was measured at increasing concentrations of ATP (0-4000 μM) or an equimolar mixture of ATP and GTP. Reactions were stopped after 15 min using 400 mM EDTA and formamide dye . Samples were heated to 95°C for 2 min, chilled on ice, and the RNA products were resolved on 24% sequencing gel containing 4 M urea. The gel was exposed to a phosphor screen overnight and scanned on a Typhoon 9410 PhosphorImager instrument (Amersham Biosciences). The free ATP and RNA bands were quantified using ImageQuant and molar amounts of RNA synthesized were calculated according to Equation   .We used a home-built total internal reflection fluorescence (TIRF) microscope described previously  for measEFRET  =  (IA − 0.08  ×  ID)/(ID + IA), where ID and IA are the background-subtracted intensities of donor and acceptor dyes, respectively. Acceptor dyes were briefly excited at the beginning and end of each movie to exclude traces lacking acceptor dyes from further analysis. Each FRET histogram was built from >50 movies by selecting the traces with a single pair of Cy3 and Cy5 dyes and representing each trace by EFRET averaged over five frames. Hidden Markov analysis was performed by using ebFRET software developed by Gonzalez group  of Mtf1 from the C-terminus Figure , B and ps Figure , D. We aart site .in vitro transcription reaction profile on the promoter fragments consists of an initial distributive phase of abortive products from 2-mer to 7-mer in progressively decreasing amounts, followed by a processive elongation phase when the RNA is elongated to the runoff length  consistent with published results  fluorescence-based assay to measure DNA melting with the C-tail deletion mutants Figure  25). Th. Th25). vice versa. Therefore, the efficiency of template strand alignment in the active site can be assessed by quantifying the binding affinity of the initiating nucleotides. The fluorescence intensity of 2AP at position –1 is sensitive to the incoming initiating nucleotides  but shows an 8-fold weaker affinity for the Mtf1-Δ20 complex (500 μM Kd)  Figure . Similarkcat, Km and the catalytic efficiency (kcat/Km) of 2-mer RNA synthesis. We found that C-tail deletion affects both the Km and kcat of 2-mer synthesis (kcat of 2-mer synthesis by ∼2-fold (kcat of 0.4 s−1 and 0.25 s−1 for Mtf1-WT and Δ20) and increased the ATP Km by 2-fold (Km of 165 μM with Mtf1-WT and 364 μM with Mtf1-Δ20)  Figure , E. Thus) Figure . This ex7 mutant .Km of ATP and GTP due to 20 aa C-tail deletion and 4-fold lower kcat of 2-mer synthesis  of RNA priming. The data shows that increasing C-tail deletion leads to an increasing defect in the catalytic efficiency of A-ladder synthesis on the LSP  state and shorter lifetime relative to the complex with Mtf1-WT Figure , F. The Keq of 1.5 vs. 6 for Mtf1-Δ12 vs. Mtf1-WT), which indicates reduced stability of the scrunched conformation with Mtf1-Δ12. Thus, ensemble and single-molecule FRET measurements consistently suggest that the C-tail of Mtf1 is necessary for stabilizing the scrunched/bent conformation of the +2 initiation complex with the template productively aligned in the active site for RNA priming.To uncover the dynamic behavior of the +2 initiation complex, we performed a hidden Markov analysis of the FRET time traces, assuming three hidden states, representing closed, open, and scrunched states Figure . The traEFRET ∼ 0.6 to 0.75)   than that at position +6. On the other hand, the FRET level decreased (EFRET ∼ 0.35), which is close to the mid-FRET level observed at position 0 (EFRET ∼ 0.38), whereas the higher FRET level in the Mtf1-Δ12 was a minor population . By con) Figure . Upon wan Figure .Single-molecule FRET traces of Mtf1-Δ12 at position +3 exhibited fast fluctuations in the high FRET range, suggesting that the broad FRET distribution in +3 stalled complexes is due to rapid conformational changes, similar to the observations at positions +1 and +2 Figure . Single-A hallmark of transition from initiation to elongation is the release of the promoter contacts and upstream bubble collapse. Transition to elongation typically occurs between 8–12 nt RNA synthesis ,35. The Next, we carried out transcription reactions on DNA Templates I and II using NTP mixes as in the above single-molecule FRET measurements and analyzed the RNA products on polyacrylamide gels with single-nucleotide resolution Figure . With MtWhen the initiation complex transitions to the elongation complex, the initially melted −4 to −1 bases reanneal and this is referred to as the upstream bubble collapse. This process can be monitored in real-time using promoter DNA labeled with 2AP at position −4. The upstream bubble collapse results in the decrease of 2AP fluorescence intensity, and the kinetics provides a measure of the efficiency or speed of bubble collapse at each stalling position Figure . TranscrThe human and yeast mitochondrial RNAPs rely on transcription initiation factors, Mtf1 and TFB2M, respectively, for promoter-specific transcription initiation. These initiation factors are well known for their essential roles in promoter melting wherein the factors stabilize the open complex by trapping the non-template strand in the nucleic acid binding pocket ,7,10,13.To investigate the role of Mtf1 C-tail interactions in steps beyond template strand alignment and 2-mer RNA synthesis, we employed a combination of single-molecule FRET and ensemble 2AP based bubble collapse assays. Our recent single-molecule FRET studies characterized each step of the transcription initiation pathway and transition into elongation by Rpo41 and Mtf1-WT . These sFigure Although previous studies suggested that the C-tail region is important for promoter melting , direct After 2-mer synthesis, the RNAP continues to unwind the downstream DNA to bring the template DNA into the active site for RNA elongation. The RNAP maintains upstream promoter contacts throughout initiation; therefore, the initially melted template DNA and the newly unwound non-template DNA gets scrunched. Evidence for the DNA scrunching mechanism has been presented for T7 RNAP and multisubunit RNAPs ,32,37–39In addition to DNA scrunching, we find that the C-tail region is important for the timely transition into elongation. Typically, transition from initiation to elongation occurs after 8-mer synthesis ,35, whicCritical insight into the trigger mechanism for the transition into elongation was obtained from our observation that Mtf1 C-tail deletion mutants produce low amounts of abortive RNA products. These results are consistent with the presence of the Mtf1 C-tail in the path of the nascent RNA and suggest that steric clashes between the C-tail and RNA–DNA hybrid result in abortive synthesis when the RNA–DNA hybrid is short and energetically weak. However, as the RNA–DNA hybrid gets longer and more stable, the clashes reorganize the C-tail region, and after 8-mer synthesis, the C-tail region may eventually dissociate from the active site weakening upstream promoter contacts. Thus, C-tail region ejection may trigger the release of upstream promoter contacts and initiate the transition into elongation. The timing of upstream promoter release could be controlled by the relative strengths of the C-tail region interactions in the active site and the stability of the RNA–DNA hybrid. Such a mechanism assures that transition from initiation to elongation occurs only when the RNA–DNA hybrid reaches a stable length to support RNA elongation. A similar mechanism has been proposed for the bacterial RNAP with the σ3.2 region clashing with the RNA–DNA hybrid .Lacking steric clashes in Mtf1-Δ12 complexes, the upstream promoter contacts remain stable until the RNA–DNA hybrid reaches 10-bp in length when we observe upstream bubble collapse in this mutant. Perhaps by the time the RNA–DNA hybrid is 10-bp long, the 5′-end of the RNA transcript naturally begins to thread into the RNA channel, which triggers upstream promoter release in the Mtf1-Δ12 complexes. Unassisted by the trigger from the full-length C-tail region, the transition process is inefficient and leads to a higher accumulation of 8–12 mer RNAs in the transcription reactions with the Mtf1 C-tail deletion mutants Figure .Overall, our findings provide detailed insights into the mechanism of transcription by the human and yeast mitochondrial RNAPs while revealing new and specific roles of the flexible C-tail regions of the initiation factors. The similarity in the functions of the C-tail and σ3.2 finger or B-reader of multisubunit RNAP factors is remarkable. These elements are evolutionarily unrelated, but in the initiation complex, they are located in a similar position near the active site between the melted DNA strands and performing the same functions during initiation ,19. The gkaa040_Supplemental_FileClick here for additional data file.


RNA helicases are known from their ability to bind and unwind double-stranded RNA initiating RNA processing events. These evolutionary conserved RNA binding proteins are broadly expressed in a variety of tissues; however, we can distinguish those, which represent tissue-specific expression pattern and play unique roles in certain cell lineages. For instance, some RNA helicases mediate transcriptomic changes triggering cell differentiation which results in specification and establishment of germline in a developing embryo. Others act as safeguards responsible for maintenance of DNA integrity in germ cell. In this article, we focus on selected DEAD/DEAH-box RNA helicases involved in germline development and spermatogenesis presenting their diverse functions and implications for male fertility. The large family of RNA helicases orchestrates gene expression by contributing to RNA metabolism during a whole life span of these molecules. These enzymes are classified as a vast group of highly conserved proteins characterized by their roles in rearrangement of RNA secondary structure and facilitation of RNA-protein interactions in an ATP-dependent manner. The ability of these enzymes to unwind RNA initiates cascade of posttranscriptional regulation events such as splicing, translation initiation and mRNA decay  comprised of subfamilies divided based on protein primary sequence variation. In humans, among SF2 group, there are two main subfamilies distinguished: the DEAD-box (DDX) helicases containing Asp-Glu-Ala-Asp (DEAD) motif represented by 42 members and DEAH-box (DHX motif: Asp-Glu-Ala-His) helicases with 16 members . In humans, DDX3Y is expressed in germ cells at the premeiotic stage of spermatogenesis and mutations in this gene are associated with Sertoli cell-only (SCO) syndrome leading to azoospermia and infertility  and a significant progression in differentiation of AZFa-deleted human-induced pluripotent stem cells (iPSCs) into germ cell-like cells post restoration of DDX3Y protein. Presence of DDX3Y evoked higher expression of transcriptional and translational regulator genes such as DDX21, SF3A1, RPRD2, and a group of zinc-finger proteins, which might suggest an initiation of global changes in transcriptome activating germ cell differentiation. Taken together, these findings emphasize a significant contribution of DDX3Y helicase in the germ cell’s fate determination.Gene encoding DEAD-Box Helicase 3 Y-linked protein (DDX3Y or DBY) is located in azoospermia factor (AZF) Y-chromosomal locus which is associated with male fertility. The data presented by Kotov et al.  indicateDDX25 gene found in azoospermic men results in phosphorylation disruption of the helicase, which interrupts chromatin remodeling and contributes to germ cells apoptosis. This finding underlines a significant influence of DDX25 protein on spermatogenesis completion  also described as gonadotropin-regulated testicular RNA helicase (GRTH) is present in meiotic spermatocytes, round spermatids, and Leydig cells and its expression is controlled by hormonal stimulation via gonadotropin/androgen regulation. DDX25 is a multifunctional protein, which facilities several molecular events crucial for spermatogenesis completion. DDX25 binds to various mRNAs and determines their fate by forming ribonucleoprotein complexes, mediating export of transcripts from nucleus to cytoplasmic chromatoid bodies of spermatids for their further translation during spermatogenesis  and late germ cell markers  and increases the percentage of cells positive for postmeiotic marker (ACROSIN) after differentiation towards germ cell-like cells. This leads to the conclusion that VASA plays significant role in promotion of meiotic progression in human germ cells derived from pluripotent stem cells. Studies of male patients with azoospermia or oligozoospermia imply that hypermethylation status of VASA promoter may lead to maturation arrest phenotype in testis and results in infertility . This protein comprises DEAH-box domain responsible for ATP-dependent RNA unwinding and Tudor domain which recognizes methylated arginine and lysine residues and mediates macromolecular complex assembly. In mice, TDRD9 helicase forms complex with MIWI2 protein which is involved in piRNA-mediated LINE-1 retrotransposons silencing. The experimental mouse model with knock-in allele comprising mutations within MOV10L1 gene were identified in infertile men and associated with spermatocyte maturation arrest resulting in azoospermia  is involved in genetic stability maintenance in germ cells. In mice, homologous protein is shown to be a key regulator of piRNA-directed LINE-1 retrotransposon silencing in male germline (Frost et al. RNA helicases are evolutionary conserved proteins present in a broad range of species. Growing number of evidence emphasizes the fact that the role of these proteins in developmental processes is crucial. RNA helicases play distinct roles in promoting germline establishment and maintenance and gametogenesis. They act as safeguards of germline DNA stability and influence changes in transcriptome by contributing to meiotic progression and sperm maturation. Importance of these proteins is supported by data indicating that several mutations in RNA helicases genes are associated with male infertility. Relevance in spermatogenesis combined with mutations occurrence among male population implies that RNA helicases may serve as potential genetic markers improving diagnostics of infertile men. Despite the demonstrated significance in male gamete formation, the exact mechanisms, functions, and pathways involving RNA helicases remain to be elucidated and demand further investigation.


To explain the increased transport of nutrients and metabolites and to control the movement of drug molecules through the transporters to the cancer cells, it is important to understand the exact mechanism of their structure and activity, as well as their biological and physical characteristics. We propose a computational model that reproduces the functionality of membrane transporters by quantifying the flow of substrates through the cell membrane. The model identifies the force induced by conformational changes of the transporter due to hydrolysis of ATP, in ABC transporters, or by an electrochemical gradient of ions, in secondary transporters. The transport rate is computed by averaging the velocity generated by the force along the paths followed by the substrates. The results obtained are in accordance with the experiments. The model provides an overall framework for analyzing the membrane transport proteins that regulate the flows of ions, nutrients and other molecules across the cell membranes, and their activities. Cancer cells synthesize increased amount of fatty acids and protein building blocks to successfully divide and metastasize. To support their metabolism cancer cells require increased supply of metabolic substrates and nutrients. Plasma membrane transporters secure import of a wide range of substrates into the cytoplasm. Consistently, increased expression of several transporting proteins has been correlated to the increased metabolic activity of cancer cells and poor disease prognosis Natecz, .There are two types of transporters, ATP biding cassette (ABC) and secondary active transporters. Secondary active transporters carry substrates across the plasma membrane using electrochemical gradient of ions. They bind the substrate on one side of the membrane followed by a conformational change allowing to release the substrate on the other side of the membrane s−1  a Gaussian random quantity of mean zero and correlation < η(t)η(t′)>=δ(t−t′), T the temperature, kB the Boltzmann constant and kBT units  of the channel changes from a maximum value hmax to a minimum value hmin evolving in time asTo study how changes in conformation of the transporter affect the velocity of the substrate, we must model its geometry. A simple yet representative form is that of a conical shaped region of length L that oscillates periodically in time with a frequency ω , given in Equation (1), in time and over an ensemble of identical transporters. The average velocity is thus obtained asMeasurements of translocation rates are performed over a time interval long enough to comprise many time periods where < ... > means average over an ensemble of realizations or initial states of the system.v(t) >, we integrate Equation (1) using a stochastic Velocity-Verlet algorithm . Once the particle reaches the exit of the transporter into the intracellular medium, we calculate the time average fμ typically takes values in the range (10−1 − 1) , therefore To calculate < fμ, reveals the occurrence of an amplification of the velocity of the substrates at a certain value of the oscillation frequency and therefore shows that transporters work under optimal transport conditions. The peaks become more pronounced when fμ increases or equivalently the rates are higher when the concentration of substrates increases, as observed for example in experiments for LeuT transporters seg−1. Our model also provides values of the rates in ion channels. In this case, one would expect that since particles are lighter they move faster. Typical values of 3 − 104)μm2/seg, therefore Γ takes values between (105 − 106)seg−1. These model predictions are in good agreement with data found in the literature for transport rates of slow and fast protein channels in membranes  or by a electrochemical gradient of ions (secondary transporters) give rise to a force that makes the substrates able to overcome the potential barrier generated by the constrictions in order to pass to the other side of the membrane. Such a force results from the variation of the space that the substrates have to move within the transporter when its configuration changes, which generates an entropy gradient.Transport rates are computed by following a simulation protocol in which we first obtain the time average of the velocity of the substrates which is subsequently averaged over a set of initial conditions of the position of a substrate. Our model reproduces experimental values of the rates for different ABC and secondary transporters and shows that they depend on substrate concentration, in accordance with data reported in recent experiments (Fitzgerald et al., L. The rates found scale with the combination of these quantities: How readily substrates cross the membrane depends on the frequency of oscillation ω, which regulates the entropic force and therefore the changes in the conformation of the transporter, on the size of the substrates which is involved in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors have made a substantial contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dear Editor,1 with over 84,000 infections in China and over 3,000,000 infections outside China worldwide . The whole genome of SARS-CoV-2 was sequenced and then released to the public on January 5th, 20202, which served as the basis for nucleic acid-based diagnostics such as reverse transcription–polymerase chain reaction (RT–PCR)4. Since human-to-human transmission has been confirmed6, field-deployable detection methods against SARS-CoV-2 are highly required 9 introduced by GenDx Biotech, belonging to isothermal nucleic acid amplification techniques that can be carried out at constant temperatures without the need for thermocycles11. In our one-pot multienzyme RT–ERA reaction system, RNase inhibitor, reverse transcriptase, recombinase, polymerase, single-stranded DNA-binding protein, creatine kinase, and nuclease are present to enable the detection of RNA. A fragment of N gene and a fragment of S gene within the entire viral genome were selected for detection  Fig. . ERA is We first introduced exo FRET (fluorescence resonance energy transfer) probe for fluorogenic detection of SARS-CoV-2 N gene Fig. . We founwhole-course encapsulated procedure for exponential amplification from RNA) procedure for “sample-in, results-out” one-pot detection of RNA in a sensitive and reliable way , and let the reaction proceed for additional 26 min at 40 °C. Finally, the reaction product can be simply detected via green fluorescence excited by blue light. During the entire course, the reaction proceeds through four steps: RT, ERA activation, pre-reaction, and final reaction, to give fluorescence readout without uncovering the lid at all. Hence, it greatly reduces any possible contaminations , and ensures the reliability of this approach to detect trace amounts of RNA.We also introduced a WEPEAR  of RNA can be detected . We also demonstrated the detection specificity of these exo probes and primers for SARS-CoV-2 over other highly similar coronaviruses such as SARS-CoV   and S gene (red) in one reaction, and used the real-time PCR machine to record the time courses. After optimization of parameters, we found that 100 nM of each primer and 30 nM of each probe worked well. Both N- and S genes were detected simultaneously with high signal-to-noise ratios Fig. . The resIn order to make the detection approach simpler, or even can be conducted at home, we were motivated to design the so-called nfo-affinity probe system plus lateral flow (LF) strips for RNA detection Fig. . We desiIn the nfo-affinity detection approach, a temperature-controlled water bath or a heating block may still be needed, which are usually inexpensive, but may not be always available. Therefore, we checked that the nfo-affinity detection approach could be performed by simply using a thermos cup that is easily available. By gradually adding hot water into a thermos cup containing room-temperature water with stirring, it is easier than expected to make 40 °C water. Then a control reaction vial (0-pg RNA) and a sample vial (100-pg RNA) were put into the cup via a float followed by covering the thermos cup by a foam ; hence, it facilitates the development of digital RT–RPA for quantitative RNA detection where single-copy sensitivity is requiredSupplementary information


Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of human malignancy. For decades, research into HNSCC invasion and metastasis has been dedicated to the study of protein-coding genes. Along with whole-genome and transcriptome sequencing development, long non-coding RNA (lncRNA) has attracted greater attention. Compelling evidence has proven the critical role of lncRNAs in the occurrence and development of HNSCC by means of epigenetic modifications, regulation of gene transcription, and post-transcription level. More importantly, crosstalk between lncRNAs and microRNAs was recently proven to regulate HNSCC metastasis through EMT modification. Based on these, this review summarizes the critical roles of lncRNAs in HNSCC metastasis and the crosstalk between lncRNAs and microRNAs as well as the detailed regulatory mechanism of the interaction. Thus, a deeper understanding of the lncRNA network in cancer metastasis is finally uncovered in order to provide a rationale and innovative concepts toward new therapeutic strategies for the highly metastatic HNSCC. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of human malignancy and involves carcinoma of several anatomic sites, such as lip, oral cavity, pharynx , and larynx, with an annual incidence of ~500,000 . Even thExperimental and clinical studies have attempted to establish the biological basis of this metastasis cascade. Mountains of evidence highlight the irreplaceable role of long non-coding RNA (lncRNA) in cancer metastasis, including HNSCC , 5. SuchLncRNAs are a heterogenous group of RNAs containing more than 200 nucleotides and recently involved in many biological processes . Their nIn the past few years, there has been a paradigm shift in the understanding of non-coding RNAs (ncRNAs) and their role in cancer biology , 12. Duein vitro experiments demonstrate that, compared with low invasiveness, HOTAIR in the invasive oral squamous cell carcinoma cell lines is upregulated. Moreover, knocking down HOTAIR expression levels globally inhibits cell proliferation, migration, and invasion (LncRNA HOX transcript antisense RNA (HOTAIR) is one of the most well-studied oncogenic lncRNAs, originally characterized as a regulator of the HOX gene family, helping to control cellular identity . The 5′ invasion . Furtherinvasion  and triginvasion .in vitro and in vivo, through WNT/β-catenin activation (In tongue squamous cell carcinoma (TSCC), the expression level of lncRNA urothelial carcinoma antigen 1 (UCA1) is significantly increased and correlated with lymph node metastasis. In addition, the expression of UCA1 in lymph node metastasis is higher than that in the primary tumor. In a cell culture of TSCC, the overexpression of UCA1 promotes cell migration but has little effect on cell proliferation . Furthertivation . ConsequAbnormal expression of lncRNA metastasis–associated lung adenocarcinoma transcript 1 (MALAT1) is reported in multiple human cancers, including prostate cancer , colorecLncRNA H19 is described as participating in the metastasis of various cancers. In TSCC, H19 is demonstrated to be upregulated in the tumor tissue compared with adjacent samples. Furthermore, the expression level of H19 in metastatic tumor is significantly higher than in non-metastatic tumor. Subsequently, H19 is demonstrated to function as ceRNA to sponge let-7a, resulting in HMGA2 enhancement, and finally, increasing the capacity of TSCC invasion and metastasis . H19 is The main regulating factors of EMT include the EMT effect factor, EMT core regulating factor, and EMT induction factor . EMT effin vitro. Meanwhile, both cytoplasm and the nucleus NF-κB/β-catenin axis is significantly triggered after MALAT1 elevation. It is noteworthy that targeting MALAT1 globally inhibits the proliferation capacity of TSCCA-induced xenograft tumor, suggesting MALAT1 as an important prognostic factor of OSCC and a satisfactory target with therapeutic potential. Furthermore, MALAT1 also acts as a transcriptional regulator within the regulation of activating the Wnt/β-catenin signaling pathway (in vitro through activation of β-Catenin/GSK3β/E-cadherin signaling. In addition, animal models show that H19 inhibition significantly impairs tumor progression and lung metastasis (in vivo experimental metastasis model also demonstrates that NKILA inhibits lung metastasis of NOD/SCID mice with TSCC tumors, suggesting NKILA as a potential predictor for OS and distant metastasis in patients with TSCC.Recently, it is confirmed that EMT is also regulated by post transcription factor lncRNA, which plays its role through regulatory effectors, transcription factors, and signal transduction pathways . Unlike  pathway . In addi pathway , indicattastasis . Apart ftastasis . Mechaniin vitro and in TSCC-induced xenograft tumors (During ncRNA crosstalk, on the one hand, the stability of lncRNA can be affected due to coaction with specific miRNA. On the other hand, lncRNA, also known as competitive endogenous RNA, could bind certain miRNAs to isolate the miRNA from its target mRNA, thereby antagonizing miRNA's function . The tumt tumors . Anothert tumors . Meanwhit tumors . Other et tumors  and lncRt tumors , 30 can LncRNA is constitutively deregulated during the progression and development of human cancers and globally suggested as a critical regulator in tumor cell motility. At present, the understanding of lncRNA in HNSCC metastasis remains confused and ambiguous, and there is little information about the function of lncRNA in HNSCC, which needs further research. This review provides a comprehensive study of the expression profile of lncRNA in HNSCC and summarizes the control of ncRNA crosstalk on the EMT process, emphasizing the leading influence of lncRNA crosstalk in the metastasis of HNSCC. Even so, a great deal of work is still urgently required to characterize the complex ncRNA networks that contribute to HNSCC metastasis, and it is necessary to carry out further research to clarify the relationship between lncRNA and miRNA in order to seek better treatment strategies.All authors agreed to the publication of this review.YW and SW contributed to conception, drafting, interpretation, and manuscript revision. XZ and YR contributed to interpretation and manuscript revision. All authors provided final approval and agreed to be accountable for all aspects of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization. Cis-elements unique to each mRNA, even at the isoform level, control the repertoire of RNA binding proteins (RBPs) that they recruit. For example, constitutive or alternative 3′ UTRs of mRNAs can recruit specific RBPs that influence both RNA and protein fate , a detergent-free method that fractionates tissue homogenate across a continuous sucrose gradient by density ultracentrifugation, followed by RNA sequencing and mass spectrometry. We have used this approach to develop a map of the subcellular organization of the transcriptome in mouse liver and find that transcripts encoding proteins involved in similar biological processes display similar sedimentation profiles. These profiles reflect a wide array of cellular compartments and correlate with RBP sedimentation patterns, making predictions about regulatory associations. Global characterization of these profiles is a first step towards the elucidation of how RNA–protein interactions generate and maintain these subcellular compartments.Wild-type FVB female mouse livers were dissected and washed in ice cold PBS. Tissue was placed in a tube containing 0.25 M buffered sucrose solution, 20mM Tris, water supplemented with protease inhibitor cocktail and 10 mM ribonucleoside-vanadyl complex (VRC) as a ribonuclease inhibitor) with 2.8 mm ceramic beads and placed in a bead homogenizer to homogenize tissue. Homogenized tissue was centrifuged at 5000 × g for 10 min to remove nuclei. A Biocomp Gradient Master™ was using to generate an 11 ml 10–50% sucrose gradient (with 10 mM VRC). Homogenate was layered onto the gradient, and components were resolved by ultracentrifugation in an SW41 rotor for 3 h at 30 000 rpm (4°C). Twenty-four 0.5 ml fractions were collected from the gradient using the BioComp Piston Gradient Fractionator™. Fractions were split for RNA and protein extraction. RNA was extracted from each fraction by Direct-zol RNA miniprep kit. Ten equivalents of EDTA (relative to the VRC concentration) were added to each sample in Trizol-reagent before ethanol was added to remove the ribonucleoside-vanadyl complex. Protein concentrations were measured by the Pierce BCA protein assay kit.The Kapa stranded RNA-Seq with RiboErase kit was used for prepare libraries according to manufacturer's instructions. An equal mass (500 ng) of RNA was used as input to each individual library. Libraries quality was assessed using a BioAnalyzer  and quantified using a Qubit (Life Technologies) prior to pooling for sequencing. Pooled libraries were 75-bp paired-end sequenced on an Illumina Next-Seq 550 v2.o C and then alkylated with 55 mM iodoacetamide for 1 h at 25oC in the dark. Proteins were digested with modified trypsin at an enzyme/substrate ratio of 1:50 in 100 mM ammonium bicarbonate, pH 8.9 at 25oC overnight. Trypsin activity was halted by addition of acetic acid (99.9%) to a final concentration of 5%. Peptides were desalted using C18 SpinTips  and then vacuum centrifuged. Peptide labeling with TMT 10-plex was performed per manufacturer's instructions. Lyophilized samples were dissolved in 70 μl ethanol and 30 μl of 500 mM triethylammonium bicarbonate, pH 8.5, and the TMT reagent was dissolved in 30 μl of anhydrous acetonitrile. The solution containing peptides and TMT reagent was vortexed and incubated at room temperature for 1 h. Samples labeled with the ten different isotopic TMT reagents were combined and concentrated to completion in a vacuum centrifuge.Proteins were reduced with 10 mM dithiothreitol for 1 h at 56m/z, AGC 3e6 and maximum IT 50 ms. The full MS scan was followed by MS/MS for the top 10 precursor ions in each cycle with an NCE of 32 and dynamic exclusion of 30 s. Raw mass spectral data files (.raw) were searched using Proteome Discoverer (Thermo) and Mascot version 2.4.1 (Matrix Science). Mascot search parameters were: 15 ppm mass tolerance for precursor ions; 15 mmu for fragment ion mass tolerance; two missed cleavages of trypsin; fixed modifications were carbamidomethylation of cysteine and TMT 10-plex modification of lysines and peptide N-termini; variable modifications were methionine oxidation.Peptides were separated by reverse phase HPLC (Thermo Easy nLC1000) using a precolumn  and a self-packed 5 μm tip analytical column  over a 140-min gradient before nanoelectrospray using a QExactive mass spectrometer (Thermo). Solvent A was 0.1% formic acid and solvent B was 80% MeCN/0.1% formic acid. The gradient conditions were 0–10% B (0–5 min), 10–30% B (5–105 min), 30–40% B (105–119 min), 40–60% B (119–124 min), 60–100% B (124–126 min), 100% B (126–136 min), 100–0% B (136–138 min), 0% B (138–140 min), and the mass spectrometer was operated in a data-dependent mode. The parameters for the full scan MS were: resolution of 70 000 across 350–2000 Reads were aligned using Spliced Transcripts Alignment to a Reference (STAR) algorithm . RNA-SeqP-values were determined by Fisher's exact test with Bonferroni correction for multiple testing. In Figure P-value according to the HG model and the FDR q-value was corrected using the Benjamini-Hochberg method.Data release from AmiGO 2 version: 2.5.12 was used to determine GO enrichments . Pantheret al.  are reported in the figures and their legends. Two independent, biological replicate gradients were generated from mouse liver. Each replicate was analyzed independently, with ‘gradient 2’ being the replicate used for all main figures. For hierarchical clustering analysis, the SciPy.cluster.hierarchy library was used. All Correlations were calculated using NumPy corrcoef function which returns a Pearson correlation coefficient for variables. Wilcoxon rank-sum tests were used to compute statistical significance.Graphs were generated using Matplotlib version 2.2.2. Statistical Analyses were performed using Python, SciPy 1.1.0 and NumPy 1.14.3 libraries. Statistical parameters, statistical tests, and statistical significance . Overall, gene expression profiles of fractions with similar densities were more highly correlated than fractions with greater density differences Figure . Two indThese clusters were subjected to Gene Ontology (GO) analysis. Of the 76 clusters, 53 showed enrichment for particular cellular compartments (cluster identities and GO results are in R = 0.823), and importantly, a single cloud of points centered along a diagonal  and also the presence of subsets of RNAs lying off the main diagonal  on 4 proteasomal core complex RNAs , a nuclear-encoded mitochondrial RNA (Atp5b), and a signal sequence-containing RNA (Fn1). All proteasome-encoding RNAs and Atp5b exhibited similar ATLAS-Seq profiles, whereas Fn1 exhibited a highly distinct profile that was representative of RNAs encoding secreted proteins Figure . SmiFISHR = 0.93), whereas the RNA and protein for Psmd13, a 26S proteasome subunit protein, were anti-correlated (Pearson's R = −0.88)  Figure . We plot) Figure . This su) Figure . Indeed,) Figure . Thus, aAlthough most RNAs do not co-sediment with the proteins they encode, our previous Gene Ontology analysis of RNA clusters Figure  suggesteWe next investigated whether alternative isoforms from the same gene loci exhibit differential sedimentation patterns across the ATLAS-Seq gradient. Because untranslated regions have known roles in regulating RNA localization, we focused on alternative UTR isoforms for these studies. We considered both alternative first exons  and alternative last exons . We quantitated the proportion of each isoform present in each fraction of the gradient, labeled percent spliced in, or PSI (Ψ) . After lIf the relative abundance of these alternative isoforms is important for cell function, they may contain sequences subject to positive selection through evolution. To determine whether isoforms with differential sedimentation patterns are more phylogenetically conserved, we measured their conservation using PhyloP scores. AFE isoforms showing strong (ΔΨ > 0.5) and moderate (0.5 < ΔΨ < 0.25) differential sedimentation showed similar conservation scores but were more highly conserved than isoforms lacking differential localization (ΔΨ < 0.25) Figure . In contRBPs can control the RNA localization of their targets, but few RBP-RNA pairs have been functionally validated in this context. Because each RNA interacts with many RBPs, the impact of each RBP-RNA interaction may only subtly influence final destination of that RNP. Therefore, analysis of many RNAs showing similar sedimentation patterns may be required to provide the power necessary to identify potentially weak, yet true, significant signals.R = 0.92, Figure R > 0.85); these RNAs encoded proteins enriched for GO Cellular Compartment categories such as ER, golgi, endosome, and vesicles  . The rels Figure . Enriches Figure . Similars Figure . Notablys Figure .R > 0.85) were enriched for specific GO categories, including ER membrane , which contains RNAs encoding proteins in the pICln-Sm protein complex, the U12-type spliceosomal complex, and U2snRNPs , which contains RNAs encoding cilium components  supported by mass spectrometry peptides across our ATLAS-Seq gradient. We correlated their profiles to all ATLAS-Seq RNA clusters  and founs Figure . Consists Figure , is a cos Figure . As anots Figure . Myh9 iss Figure . Taken tWe have used ATLAS-Seq to uncover unexpected relationships between sucrose gradient sedimentation profiles of RNAs encoding proteins involved in similar biological functions. Deep sequencing of RNA transcriptome-wide and mass spectrometry of peptides with high resolution across the gradient facilitated the discovery of these relationships and characterized the presence of cellular microenvironments to which RNAs are sorted. Surprisingly, subtle differences in profile shape can resolve differences in the composition of cellular compartments. These profiles likely reflect not only engagement with large macromolecules such as the ribosome, but also membranes and other structures with distinct physiochemical properties. We observed that these interactions were reflected in the divergence of ribosome footprint profiles from ATLAS-Seq. Future studies directly comparing polysome profiles to ATLAS-Seq or other gradients prepared by diverse detergents, cytoskeletal disruptors, or other agents might further elucidate how various interactions drive sedimentation profiles.Distinct microenvironments in the cell arising from these interactions—the sum of weak attractive and repulsive forces between biomolecules—may create the appropriate settings for translation, sorting, decay, and other cellular processes. Although these specialized environments are sometimes membrane-bound organelles, our observations suggest they may also reflect membrane-less organelles in the cytoplasm such as the proteasome, sites of spliceosome component assembly, or even RNP granules. These RNP granules could contain single mRNAs bound to multiple RBPs, or perhaps supra-molecular assemblies in which multiple RNPs are linked via protein–protein, protein–RNA or even RNA–RNA interactions. Thus, there exists spatial organization among thousands of RNAs revealed by physical separation across a density gradient. The observations here provide a blueprint for how RNAs might map to specific subcellular microenvironments in liver cells and provide insights into higher scale organization of the transcriptome.Interestingly, correlations of RNA to their encoded proteins revealed that most RNA-protein counterparts are not co-localized, but that some are, most notably those encoding membrane and secreted proteins. In these cases, the co-localization may reflect co-translational insertion into specific lumenal compartments. However, both RNAs with and without signal peptide sequences often co-sedimented, suggesting that there may be additional signals within RNA that influence their localization. Notably, proteins encoded by co-sedimenting RNAs also tend to co-sediment, suggesting regulatory mechanisms that bridge the subcellular localization of each molecule. This has been previously observed for specific mRNAs at the isoform level; for example, localization of some proteins has been shown to be directly influenced by 3′ UTRs of their mRNAs via association with membraneless organelles such as TIGER domains . This istrans-factors associated with these microenvironments.A key goal in studies of RNA sorting and localization is to identify RNA elements and RBPs that might define subcellular localization of RNAs and locally translated proteins. Only a small fraction of putative RBPs have been functionally characterized, but co-sedimentation of RBPs and RNA targets may reveal functional interactions. In summary, high resolution subcellular fractionation on a transcriptome-wide scale can provide important insights into the regulation of higher order, subcellular compartmentalization of mRNAs by revealing groups of RNAs that co-segregate within the cell and implicating post-transcriptional processes and http://ericwanglab.com/atlas.php.Raw sequencing reads for all samples are available through the NCBI via GEO Accession GSE140630. An interactive browser that allows users to explore profiles of transcripts, proteins, and ATLAS-Seq clusters can be found at gkaa334_Supplemental_FilesClick here for additional data file.