Although testicular germ cell tumor (TGCT) overall is highly curable, patients may experience late effects after treatment. An increased understanding of the mechanisms behind the development of TGCT may pave the way for better outcome for patients. To elucidate molecular changes prior to TGCT diagnosis we sequenced small RNAs in serum from 69 patients who were later diagnosed with TGCT and 111 matched controls. The deep RNA profiles, with on average 18 million sequences per sample, comprised of nine classes of RNA, including microRNA. We found that circulating RNA signals differed significantly between cases and controls regardless of time to diagnosis. Different levels of TSIX related to X-chromosome inactivation and TEX101 involved in spermatozoa production are among the interesting findings. The RNA signals differed between seminoma and non-seminoma TGCT subtypes, with seminoma cases showing lower levels of RNAs and non-seminoma cases showing higher levels of RNAs, compared with controls. The differentially expressed RNAs were typically associated with cancer related pathways. Our results indicate that circulating RNA profiles change during TGCT development according to histology and may be useful for early detection of this tumor type. Testicular germ cell tumor (TGCT) is diagnosed in 1% of men worldwide and is the most common malignancy in males between 20 and 39 years of age. The incidence rates are still rising , 5. The TGCT is a highly curable disease since the introduction of cisplatin-based chemotherapy with a 5-year survival rate of above 95% , 13. Howin situ (GCNIS), the markers include OCT3/4, PLAP, CD117, and SALL 4 , piwi-interacting RNA (piRNA), and long non-coding RNA (lncRNA), regulate gene expression on transcriptional and post-transcriptional levels and have been shown to be present in serum . The difmiR-302/367 and miR-371-373 has been found in serum . JSB is a population-based cancer research biobank containing serum samples from 318 628 individuals collected from 1972 to 2004 , 35. ThePrevious studies have shown the feasibility of using long-term archived serum samples for RNA analyses and the variability of the RNA levels , 31, 36.We extracted RNAs from 400 μl serum using phenol-chloroform phase separation and the miRNeasy Serum/Plasma kit  using a QIAcube (Qiagen). NEBNext® Small RNA Library Prep Set for Illumina  was used for small RNA-seq library preparation. RNA molecules from 17 to 47 nt in length were selected. We sequenced 12 samples per lane of a HiSeq 2500 (Illumina). Additional information is available in our previous study .Total number of reads generated was 3.5 billion with an average sampling depth of 18.4 million raw reads. AdapterRemoval v2.1.7  was usedoptmatch R package (github.com/markmfredrickson/optmatch) allowed us to find optimally matched sets of controls (2 fold change (log2fc) as biologically meaningful and are counted in the summary bar charts. Venn diagrams, produced with the R package , represent common and unique differentially expressed RNA trends among the categories.The controls  for eachcontrols , 31, tercontrols  was usedKyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using kegga function from the limma R package. The inputs included mRNA, miRNA and isomiR targets extracted from miRDB (v5.0) predictions  with a cTo exploit the full statistical power of the dataset when identifying RNAs that differ in cases and controls regardless of other factors, we first compared all cases and controls. To identify histology (determined by ICD-O (3rd revision) codes) specific RNA signals, we compared the RNA levels of seminoma with those of the matched controls, and similarly for non-seminomas and matched controls. To identify RNA signals according to the time between blood draw and diagnosis we divided prediagnostic time into four discrete time intervals, 0–2, 2–5, 5–8, and 8–10 years. We selected these intervals to optimize resolution on time to diagnosis while still having sufficient statistical power. To make the time windows comparable with respect to statistical power, the cut points were chosen to secure the same number of cases and controls, and similar proportions of histologies.n = 79) against matched controls (n = 111), we analyzed 4231 RNAs that passed our inclusion criteria. Of these, we identified 818 RNAs that were differentially expressed with a p-adjusted value ≤0.05, and 88 of these had a log2 fold change (log2fc) outside the range {−1,1}   , 50. Theer (BTB) . Furtherer (BTB) , 53. GCNer (BTB) , 55 as ter (BTB) , 57. FurIn this study we found that differentially expressed levels of mRNAs were related to both testis development and known cancer pathways, as well as the lncRNA, TSIX which exhibited increased levels in TGCT patients. The role of TSIX in X-chromosome inactivation (XCI) has been widely reported –60, acti2fc with RAB21, an oncogene involved in mediating endocytosis and connected to the tumorigenesis and vesicle transport mechanisms of the other Rab proteins  project, and HPA shows RNA-seq tissue data with high protein-coding transcripts per million (pTPM) counts of AP000295.1 in the blood constituents, granulocytes and monocytes . AP00029Circulating miRNAs have been identified as both prognostic and diagnostic markers in biliary tract cancers (BTC), and this has led to earlier diagnosis and less invasive procedure of BTC than previously used techniques . The cirPathways enrichment analysis showed overall many associated cancer related pathways across the 10-years period prior to TGCT diagnosis. The time frames 2–5 and 5–8 years prior to diagnosis showed the highest numbers of enriched pathways, many of which were cancer related. A possible explanation for the absence of enriched pathways in the 0–2 year time interval, could be an overall increase of all RNA types in serum as the tumor develops, this would effectively mask cancer pathways from being detected due to the large amounts of noise from aberrant RNA expression.The presence of PTEN and KRAS genes in our pathway maps , 8, showGenome-wide association studies for TGCT have consistently shown that KITLG (ligand for the receptor tyrosine kinase) is implicated in TGCT susceptibility and with high effect sizes , 76. In A strength of this study is the large and robust RNA dataset. To our knowledge this is the largest study of the RNA profiles from TGCT patients prior to diagnosis also including harmonized confounders collected from health surveys . The timThe sample size is still a limitation, and stratified analyses on histology and time to diagnosis could benefit from an increase in statistical power. Serum samples and survey data were collected over a period of more than 30 years, and lifestyle factors such as smoking and BMI have changed within this time period as well as carcinogens in the environment, such as organochlorine pesticides –80. Casein utero. Stable serum RNAs in the pre-diagnostic period have the potential to be biomarkers for earlier detection. Early diagnosis of TGCT would lead to less use of cisplatin treatment, and thereby reduce long-term adverse effects, such as risk of second primary cancer and cardiovascular disease. Hazard ratios for all secondary cancers after a single cisplatin-based chemotherapy cycle were significantly lower than hazard ratios after two or more cycles  (e) and 9 (2) (j) of the General Data Protection Regulation (GDPR). National legal basis as per the Regulations on population-based health surveys and ethical approval from the Norwegian Regional Committee for Medical and Health Research Ethics (REC) is also required. Requests to access the datasets should be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Norwegian regional committee for medical and health research ethics. The patients/participants provided their written informed consent to participate in this study.TH and TR designed the study. JB and SU performed the analyses of the data. JB, TH, and TR drafted the manuscript. All authors discussed the results, contributed to the writing, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Current bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans‐encoded and based on tRNA‐mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA. Switch‐hitter: A bacterial genetic switch that accepts RNAs, small molecules, and proteins as inputs was developed based on a tRNA‐mimicking structure. The switch is trans‐encoded, which makes manipulation of the target mRNA redundant. It is stable to RNase, shows a strong dynamic response, and exhibits a high level of orthogonality. Synthetic non‐coding RNAs with simple and limited conformational states have been deployed to build RNA switches to control bacterial translation.2, 3 MTo overcome these limitations, we present trans‐encoded genetic switches based on a tRNA‐mimicking structure  of the target mRNA, without disturbing the RBS and the start codon, to block ribosome entry and subsequent translation.lys that binds flanking sites of the RBS  cells. We varied the length of regions A and B in the repressor domain and tested their ability to repress GFP by flow cytometry. Three out of eight d‐TMSIBE switches showed effective GFP repression . These three switches contain the longest A and B domains (>12 nts), likely because the 8 nt transmitter domain needs to dehybridize for the switch to bind the mRNA. We selected the d‐TMSIBE switch with the longest regions of A=10 nt and B=8 nt to obtain the best binding characteristics. We determined the minimum length of the stem in the repressor domain and the stem that connects the repressor domain with the tRNA structure . The need for a minimum stem length is likely due to the stability provided by a tRNA structure in an RNAse environment (see below). The d‐TMSIBE switch maintains its functionality even with a variable loop of 1 nt length.For characterization of the switch, we used a two‐plasmid systemd‐TMSIBE switch in RNase environment, we compared repression of GFP expression by the d‐TMSIBE switch with an RNA oligomer containing only the repressor sequence . The d‐TMSIBE switch repressed GFP expression 21‐fold more effectively than the RNA oligomer, thus demonstrating that the d‐TMSIBE provides stability to the RNA‐based switch in an environment where RNAse is present.Useful switches need to be stable to degradation by RNAses. For example, a trans‐encoded bacterial switch that binds the protein Hfq is stable to RNAse.d‐TMSIBE switch, we simultaneously expressed an anti‐repressor RNA from the d‐TMSIBE switch plasmid . We again used a TMS structure for the anti‐repressor RNA to provide stability. Cells expressing both the anti‐repressor RNA and the d‐TMSIBE switch showed almost the same GFP intensity as in absence of both interacting TMSs  aptamer shows that it has increased stability upon neomycin B binding.[To increase the versatility of input signals, we inserted an aptamer as an additional module into the boswitch,22, 23d‐loop of the TMS switch with a neomycin B aptamer‐IBE, Figure ‐IBE structure can function as an analogue genetic switch against a small‐molecule input signal.We replaced the d‐loop of the TMS . Azide‐modified neomycin B has a very low binding affinity for the kanamycin B aptamer . Indeed, due to the lower affinity of the kanamycin B aptamer for azide‐modified neomycin B, KanB‐TMS‐IBE is unable to control gene expression with azide‐modified neomycin B . Hence, the ligand‐mediated structural changes in the TMS switch depend on the affinity of the ligand for the aptamer component of the switch.To assess whether the ligand‐mediated stabilization of the TMS switch solely depends on the interaction with the corresponding aptamer, we replaced the neomycin B aptamer of NeoB‐TMSd‐TMSIBE switch was designed to target the T7 RNA polymerase gene present in the genome of the E. coli BL21(DE3) cells . As a marker, GFP expression from a plasmid controlled by a T7 promoter should be reduced when the d‐TMSIBE switch represses T7 RNA polymerase translation. Indeed, we observed that the presence of the d‐TMSIBE switch reduced GFP fluorescence . GFP expression is only marginally reduced further when a second d‐TMSIBE switch that targets the +11 to +30 bases upstream of the start codon of the T7 polymerase mRNA is employed simultaneously with the first d‐TMSIBE, thus indicating the excellent performance of the switches.We next asked whether the TMS switches can be deployed to target the bacterial genome. A s Figure , bindinge Figure . Importad‐TMSIBE switch versus the ftsZ gene in the genome, which encodes for the FtsZ protein , we have been able to compensate for the low chemical diversity of input signals of the current RNA switches compared to their protein counterparts.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease. RNA–protein interactions are the nodes in a complex regulatory network that controls the fate of each RNA expressed in a cell. On the one hand, recent advances in RNA sequencing (RNA-seq) and computational techniques have dramatically expanded the repertoire of cellular RNAs, realizing that 80–90% of the eukaryotic genome is transcribed into RNA. Thereby, only 1–2% of the genome serves as a template for protein synthesis, while the majority of the genome is pervasively transcribed into non-coding RNAs, a large fraction of them as long non-coding RNAs (lncRNAs), which are defined as transcripts of more than 200 nucleotides without evident protein coding functionality ,4,5. TheA starting point to explore a cell’s RNA–protein interaction network architecture is given by a systematic analysis of interactors at particular nodes or points of control. This can be approached from two angles—either the protein- or RNA-centered—that in principle provide a complementary view. Technically, the “protein-centric” approach relies on the ability to purify a particular RBP or accommodated complex thereof, followed by identification of the bound RNAs with DNA microarrays or high-throughput sequencing. Therefore, RBPs are usually isolated from cellular extracts with specific antibodies or via affinity purification of epitope tagged proteins. Such RBP immunoprecipitation followed by microarray or sequencing analysis of interacting RNAs (RIP-Chip/RIP-seq) was used early on for the recovery of native RNA–protein complexes under physiological conditions ,12,13; wSaccharomyces cerevisiae using oligonucleotide deoxythymidine (dT) beads to identify interacting proteins with MS [In contrast to the rapidly progressing “protein-centric” approaches, the establishment of global “RNA-centric” approaches has lagged behind for some time. Thereby, a distinction can be made between (i) the identification of proteins binding to an entire population of RNAs, such as polyadenylated (poly(A)) RNA, or total RNA; and (ii) defining all proteins/RNA that interact with a specific RNA expressed in a certain cell-type or tissue. The first is aimed at gaining an unbiased identification of RBPs in a cell or organism. Early attempts used protein microarrays or recovered poly(A) RNAs from budding yeast  with MS ,22. Whil with MS ,22. The  with MS ,24,25,26 with MS ,10). Bes with MS , a varie with MS , phenol- with MS , and pro with MS , as well with MS ,32. Furt with MS ,34,35. F with MS ,37.Conversely, the systematic investigations of factors that interact with a particular RNA, which remained notoriously challenging because most RNAs have considerably low expression levels in cells, are much less well explored. Many laboratories currently implement more simple in vitro approaches, where for instance, an in vitro transcribed (IVT) “bait” RNA is coupled to beads and incubated with cell extracts to pull-down interacting proteins . This apXIST) lncRNA, where one copy in the nucleus accomplishes X-chromosome inactivation in females [In principle, one can distinguish between in vitro approaches, where the “bait” RNA is usually coupled to beads and incubated with extracts for pull-down of associated proteins; and in vivo approaches, where RNA–protein interactions are stabilized in cells and then RNAs captured from cell lysates. Essentially, most biochemical procedures for isolation of specific RNAs can be applied in vitro and in vivo with modifications and either involve tagging the RNA of interest with an affinity aptamer and recovery with a high-affinity ligand coupled to beads, or the capture of native RNAs with modified ASOs . While t females . Thus, a females ,36,37. I females ,41). FinOne commonly used concept for purification of RNP complexes involves the addition of an RNA aptamer tag to the RNA of interest, which permits capturing the tagged RNA with a high-affinity ligand . A varieLEF1) mRNA was used as RNA target to unravel proteins associated with IRES function. SILAC-based proteomics identified about half the number of proteins under denaturing conditions (326 proteins) as compared to native conditions (535 proteins), suggesting that denaturing conditions removed indirect interactors and nonspecific binding factors. Using a non-IRES containing mRNA as control, 36 proteins were eventually identified as IRES binding proteins, which included known IRES binding factors but also novel interactors [A second class of aptamers includes short RNA hairpins that interact specifically with proteins, such as the coat proteins from the R17/MS2 bacteriophage , bacterieractors .Bacillus subtilis, termed BASU, is expressed as a fusion with the lamdaN peptide that specifically interacts with BoxB stem loops. BASU is therefore tethered to the RNA-binding motif, thereby biotinylating proximal proteins within 10 nm upon growing of cells in media supplemented with biotin. The biotinylated proteins are then recovered with streptavidin-coupled beads under stringent conditions [Noteworthy, an alternative approach to directly label the proteins interacting with a tagged RNA motif has been introduced recently . RaPID . This iNEAT1 and MALAT1 lncRNAs [Recognizing their potential, the methods were further advanced and combined with MS analysis to identify interacting proteins. At first, in a proof-of-concept study, CHART-MS uncovered proteins bound to human  lncRNAs . Therefo lncRNAs . A largeXIST lncRNA interactome in different cell-types [XIST lncRNA was induced and isolated with bound proteins from nuclear extracts derived from 200–800 million UV crosslinked SILAC-labeled mouse embryonic stem cells (mESCs) using an array of long (90-mer) biotinylated DNA oligonucleotides covering the entire transcript [Serratia marcescens), 10 proteins were identified that reproducibly associated with XIST RNA over background levels determined from U1 small nuclear RNA (snRNA) control affinity isolations with quantitative MS. Follow-up studies using siRNA-mediated knockdown of the interacting proteins showed that three proteins were required for transcriptional silencing  and that interaction of XIST with SHARP promotes the recruitment of other factors like SMRT and HDAC3 that deacetylate histones and thereby exclude access of polymerease II to the X-chromosome [At the same time, three independent studies used a “tiling” approach to investigate the ll-types ,67,68. AU1 and U2 snRNAs, identifying 418 and 370 interacting proteins from HeLa cells, respectively; including proteins involved in splicing and pre-mRNA biogenesis, indicating the suitability of the approach. While only one oligo was used to capture these snRNAs, XIST was recovered from engineered mESCs with 43 20-mer DNA probes bearing a biotin triethyleneglycol (TEG) moiety at the 3′end, annealing along the entire mouse XIST. RNPs were liberated from beads by gentle biotin-elution to minimize contamination from nonspecific binders and samples subjected to label-free liquid chromatography (LC)-MS/MS analysis [U1, U2, U3) affinity isolations) with known roles in chromatin modification, nuclear matrix, and RNA remodeling pathways; among them 14 proteins previously known to interact with NEAT1 and MALAT1 lncRNAs. Further functional characterization of interactors suggested that hnRNPK, a well-known RBP, participates in XIST-mediated gene silencing but not in localization, while SPEN is required for gene silencing. Collectively, the study concluded that XIST engages with proteins in a modular and developmentally controlled fashion to coordinate chromatin spreading and silencing. Noteworthy, the ChIRP-MS approach was used later to identify interactors of MALAT1 [MALAT1 as compared to U1 and probe-free control isolates. Additionally, follow-up studies validated the results and suggested that MALAT1 binds to and, as a consequence, inactivates the prometastatic transcription factor TEAD, preventing the associating with its coactivator YAP and target gene promoters [Substantially more proteins were identified with ChIRP-MS, possibly due to the implementation of chemical crosslinking of cells with 3% formaldehyde for a relatively long period (30 min) . Initialanalysis . In totaf MALAT1 . Consideromoters .XIST from UV-irradiated female mouse fibroblast expressing physiological levels of XIST RNA [XIST-specific complexes recovered with nine complementary 25-mer DNA oligonucleotides spaced across the entire 17 kb long RNA. Essentially, MS analysis revealed 80 proteins that were at least 3-fold enriched with XIST RNA over control samples obtained by recovery of abundant cytoplasmic and nuclear RNAs . While some previously known interactors for XIST were identified, the analysis revealed new proteins, many of them part of epigenetic complexes, such as cohesion complex proteins, histone modifiers, chromatin remodeling factors, topoisomerases, nucleoskeletal and matrix proteins.A third approach, termed ‘identification of direct RNA interacting proteins’ (iDRiP) was used to isolate the proteins interacting with XIST RNA . Here, nMALAT1, NEAT1, and NORAD from prostate cancer PC3 cell lysates. As compared to the “tiling” approaches, only 2–3 biotinylated capture oligonucleotides complementary to each lncRNA were mixed for capturing target RNA–protein complexes from formaldehyde crosslinked PC3 cell lysates with reasonable efficiency (up to 35%). Importantly, HyPR implements specific displacement oligonucleotides to release captured complexes from beads in a so-called ‘toehold-mediated release strategy’, which could increase selectivity towards particular RNA–protein complexes. As a result, 127, 94, and 415 interacting proteins were identified with label-free LC-MS/MS from MALAT1, NEAT1, and NORAD affinity isolates. Besides previously known interactions that served as an internal validation step, many novel interactors with functions related to documented features of the associated lncRNAs were discovered, including histone modifiers and transcriptional regulators. All interactomes were enriched for RBPs: while nuclear MALAT1 and NEAT1 were enriched for nuclear proteins, NORAD, a cytoplasmic lncRNA, was enriched for cytoplasmic proteins, confirming selectivity of underlying interactors in regard of the cellular compartmentalization. Interestingly, 20 proteins interacting with MALAT1 were previously identified with CHART-MS [More recently, Spiniello and colleagues introduced hybridization purification of RNA–protein complexes followed by mass spectrometry (HyPR-MS), which enables the analysis of interactomes of multiple RNAs in a single experiment . In thisCHART-MS , corroboXIST, where shearing could break the RNA during preparation, thus making aptamer tags or using only a few oligos placed in particular locations less attractive. Nevertheless, the “tiling approach” bears limitation for analysis of distinct transcript isoforms or association of particular regions of interest within a given RNA, which could benefit from using fewer oligos specifically designed to capture certain transcript isoforms. Finally, the use of multiple oligos substantially increases the probability for cross-hybridization with unrelated RNAs. Nevertheless, these studies revealed new facets for understanding the function and molecular mechanisms of XIST and other lncRNAs, and the reader is referred to a recent review further highlighting these developments [In conclusion, these studies showed the applicability of ASOs for capturing even low expressed lncRNAs. Nevertheless, the different approaches revealed only limited overlap of identified proteins, indicating that the screen is not exhausted yet and further technical improvement may be required. The “tiling” approach may be particularly beneficial for capture of very long RNAs such as the 17 kb long lopments .HCV RNA through hybridization with the subgenomic HCV (−) RNA in MH14 host cells (derived from Huh7 hepatoma cells) [Similarly, ASO-based approaches were used to identify interaction partners of viral RNAs, which could provide information about key RBPs that could contribute to viral replication and proliferation. An early study used 39 nts long biotinylated DNA molecules that annealed with synthetic 3′ nontranslated region (3′NTR) of the hepatitis C virus (HCV) (+) genome as bait to capture interacting cellular proteins from hepatocyte extracts . A totala cells) . This ina cells) . NeverthThiouracil cross-linking mass spectrometry (TUX-MS) applied a modification of the RIC procedure to capture poly(A) RNAs using immobilized oligo(dT) to identify proteins bound to the polyadenylated poliovirus (PV) RNA genome in infected cells . TherebyDENV RNA is not polyadenylated, the common strategy for capture of cellular polyadenylated RNAs was modified. Essentially, 10 DNA oligos (between 17 and 30 nts in lengths) containing a TEG spacer between the biotin moiety and the DNA at the 3′ end were used to recover the viral RNA from UV-crosslinked samples using streptavidin beads under stringent conditions. Similar to CHART-MS, selection of best ASOs for recovery of viral RNAs was adjusted through the testing of several antisense DNA oligos in RNase H mapping assays for their suitability to anneal with viral RNA [DENV RNA in vivo applying stringent selection criteria (20-fold above control isolates). Finally, siRNA mediated gene silencing followed by analysis of viral replication showed that at least half of the tested proteins likely play a role in virus replication [DENV RNA genome was used to capture the viral RNA from total RNA [A “tiling” ASO approach was further used to purify dengue virus (DENV) RNPs from infected hepatocytes Huh7 cells ,85. Sinclication . Of noteotal RNA . It woulHIV-1 RNA was apparently sufficient to fairly-well recover the viral replication-deficient RNA from cell extracts (35% efficiency). MS analysis identified 189 proteins that were enriched with HIV-1 RNA as compared to scrambled control oligo isolates , among them 90 proteins that were previously known to impact HIV replication. Furthermore, siRNA-mediated knockdown of several proteins revealed changes in HIV expression, suggesting biological implications in HIV life-cycle [Finally, the above-mentioned HyPR-MS approach was also applied to enrich spliced full-length HIV RNA–protein complexes preserved in vivo by formaldehyde crosslinking to identify interactors from infected human cells . Only onfe-cycle . Thus, tIn conclusion, several ASO-based approaches have been successfully applied to recover viral RNAs from infected cells. While some studies used multiple ASOs for recovery of the RNA, this may not be necessarily required; and UV-crosslinking generally disclosed less interaction partners compared to chemical crosslinking procedures as could be expected.To date, only a few studies have attempted to recover selected native mRNAs with bound proteins/RNAs. One of the first attempts used biotinylated DNA oligonucleotides to isolate mRNAs from formaldehyde crosslinked cell extracts to detect microRNAs that could modulate the mRNA’s expression . While tp27/CDKN1B 3′UTRs in cisplatin treated cells [p27 mRNA upon cisplatin (CP) treatment, and knockdown of KHSRP enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment [To analyze the proteins interacting with particular mRNAs in vivo, we have recently introduced a tandem RNA isolation procedure (TRIP) to capture selected cytoplasmic mRNAs ,88. Esseed cells . While 5reatment . NoteworDrosophila Sex-lethal (Sxl) binding motifs in vitro, and for exploring the repertoire of proteins binding to 18S and 28S ribosomal RNA (rRNA) from UV-crosslinked Hela cells in vivo [Like TRIP, ‘specific RNP capture’ also implements nuclease resistant modified ASOs that have increased affinity towards RNA targets and reduces background binding . In part in vivo . While rc-myc mRNA, coding for one of the best studied oncogenes, was captured from formaldehyde crosslinked K562 cells [c-myc transcript, while a scrambled oligo was used as control. In total, 229 proteins were found to be associated with c-myc compared to control samples (≥5-fold), including several RBPs previously known to interact with the mRNA. It also revealed novel interactors and comparison with published CLIP/RIP data strongly suggested those being bona fide interactors [gld-1 mRNA from the nematode Caenorhabditis elegans [gld-1 in C. elegans. In addition, small RNA sequencing recovered known and biologically relevant miRNA interactors, revealing miR-84 as specific regulatory of gld-1 transcripts. In conclusion, vIPR seems to be well-suited for in vivo applications; however, the implementation of many DNA oligonucleotides needs careful evaluation for each transcript because of potential cross-hybridization. Furthermore, the direct capture of RNP complexes with streptavidin beads from extracts may recover prominent biotin binding proteins depending on the type of cells/tissues.Two recent studies have successfully implemented short unmodified DNA oligonucleotides to capture mRNAs from cells. Applying HyPR-MS, 62 cells . Therefoeractors . Convers elegans . The stuNORAD identified 51 candidate proteins, six of those were previously reported to interact with NORAD, including proteins involved in splicing and mRNA processing. Furthermore, the approach was also used to capture the p21 mRNA interactome, showing adaptability for this application to different types of RNA. Likewise, CRISPR-assisted RNA–protein interaction detection method (CARPID) leverages CRISPR-CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific lncRNAs in the native cellular context [XIST as well as other lncRNAs such as DANCR and MALAT1. Essentially, the method uses two different guide RNAs (gRNAs) to tether cleavage incompatible CRISPR fused to the biotin ligase BASU to the RNA target, which then biotinylates proteins in close proximity. The study found 73 XIST-interacting proteins that were significantly enriched in at least one set of gRNAs over controls, with 23 proteins discovered with at least two different sets of gRNAs.The recently developed CRISPR-based RNA targeting systems provide a new option to directly label RBPs in living cells without the need for crosslinking procedures ,90. For The CRISPR based systems allow the labeling of proteins that interact with (or are in close proximity to) a specific RNA fragment in living cells, alleviating some risk of RNA degradation through processing of samples i.e., extract preparation for biochemical purifications. Furthermore, and in particular with CRUIS, the RNAs were left at natural expression levels. Although promising, there are some drawbacks with these techniques. For instance, it is not yet fully clear how well RBPs or indirect interactors are labeled, and what the in-cell spacing range apart from the assembly of the gRNAs may be for detection of interacting proteins. Essentially, the proximity ligation approach provides a snapshot of the proteins within a certain distance to the CRISPR-attached ligase without discriminating between direct or indirect interactors with RNA. Furthermore, it is difficult to predict gRNA specificity and off-target effects may apply. Finally, the CRISPR complex is relatively large and upon assembly could affect the local structure of the target RNAs and compete/synergize with interactors. In conclusion, while the in-cell methods provide new and valuable tools to study local RNA–protein interactions in cell-culture models, further studies are needed to learn more about its reliability and the interpretation of the data.As RNA-centered investigations are becoming increasingly popular to characterize the dynamics of the RBPome, methods to study the protein architecture on particular RNAs are now also becoming feasible. Besides a variety of different aptamer-based RNA-capture approaches, substantial progress has been made implementing ASOs, which is the sole approach for isolation of unmodified and in vivo formed RNPs. The “tiling” approaches seem to achieve very efficient recovery of the target RNA (between 70% and 90%) but specificity could be compromised through cross-hybridization and limitations apply for analysis of transcript isoforms. Several studies have also accomplished a fairly good recovery of target RNAs with only one to three ASOs, minimizing potential contamination through off-targets and may be the preferred route for investigation of “shorter” RNAs. While most studies used either way—UV or chemicals—for crosslinking RNA–protein complexes in vivo, only a few side-by-side comparisons have been done . UsuallGenerally, the currently developed methods to investigate in vivo formed RNA–protein interactions may now find firm adaptation for studying the dynamic rearrangement of RNPs on particular RNAs under stress conditions, during development and in disease. Furthermore, studies of the RNA assembled factors across different cellular compartments may become feasible . Finally, as more quantitative data will become accessible, it could pave the way to model regulatory networks for prediction of functional output. Currently, we are just at the starting point for a new journey along the RNA that will certainly lead to many exciting discoveries and fundamental insight into RNA biology, and ultimately lead to a better understanding of their cellular function in health and disease, thereby offering new options for therapeutic intervention.


The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression.Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB; the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality  were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL;  Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related mortality with a five year survival rate of only 8% ,2. The lIn the latest years, transcriptomic signatures have also shown potential for the prediction of prognosis and response to chemotherapy beside mutations ,8. In paEndoscopic ultrasound tissue acquisition (EUS-TA), either with fine needle aspiration (FNA) or biopsy (FNB), represents the method of choice for the diagnosis of most PDAC patients, with a complication rate of less than 1% . The evaThe primary aim of our study was to determine the best method in terms of tissue conservation and needle choice to obtain RNA of a good amount and quality from PDAC samples through EUS-TA. The secondary aim was to investigate the possibility of using EUS-TA derived RNA samples for transcriptome analyses aimed at measuring tissue contamination and at classifying tumor subtypes or aggressiveness.This was a prospective observational study conducted upon approval of the Internal Review Board (IRB BIOGASTRO/2011 updated on 6 November 2017). Patients with a clinical history and radiologic imaging suggestive of non-metastatic PDAC diagnosis were considered for potential enrollment in the study upon informed consent. Patients were naïve to any treatment before the EUS. Exclusion criteria are reported in ®, Zeneca, Germany) using a Pentax therapeutic linear echoendoscope  and Hitachi ultrasound platforms  by expert endoscopists who perform more than 500 EUS procedures a year. The evaluation of the lesion size and features was performed either from the stomach in the case of body and tail lesions or from the duodenal bulb and the second portion in the case of pancreatic head masses. The EUS-TA was performed from the position that could provide the best stability of the scope, the closest position of the lesion to the probe and the best window for puncture in terms of the absence of blood vessel interposition and evaluated with a color Doppler.EUS procedures were performed under deep sedation with an intravenous infusion of Propofol  for diagnostic purposes and the sample was expressed onto a clean glass slide with the reinsertion of the stylet. At least two slide smears were prepared in each case, which were fixed in absolute alcohol and stained with hematoxylin and eosin using standard techniques. An onsite cytopathologist would then examine the slides and provide a real-time impression on the adequacy and nature of the lesion and whether the nature of the cells was benign , suspicious or malignant. The diagnosis was based on pathologic criteria taking into consideration tissue architecture, pleomorphism, hyperchromatic nuclei, high nuclear-to-cytoplasmic ratio and the presence of giant cells. In the case of no adequacy, another pass with the 25G FNA needle was performed and reevaluated. If also the second pass did not retrieve the adequacy, the patient was excluded from this study.We compared three different needles and for all of them the slow-pull technique was employed. The first EUS-TA was always performed with a 25G FNA needle  and then either a pass with a 20G needle  or with a 25G needle . These FNB needles were alternated for each consecutive patient with the slow-pull technique being employed for all cases. Both samples were then expressed in two different vials. The first set of 14 consecutive patients (enrolled between November 2018 and January 2019) had the samples expressed into empty vials positioned in dry ice and then immediately stored at −80 °C. The second set of 11 consecutive patients (enrolled between February 2019 and April 2019) had the samples expressed into a vial containing 600 uL of RNALater  at room temperature for at least 1 h and then stored at −80 °C. The third set of 12 consecutive patients (enrolled between May 2019 and October 2019) had the samples expressed into empty vials positioned in dry ice and then immediately taken to the laboratories where 1000 uL of Trizol  was added to the vial under a chemical hood and then stored at −80 °C. A total of nine experimental conditions were therefore set, matching three different needle types with three different conservation methods. No sample size calculation was performed ahead of the study.In the case of an adequate sample suspicious for malignancy, the patient would undergo another pass with the 25G FNA needle  according to the manufacturer’s protocol. For homogenization, a hand-held battery operated homogenizer VWR Pellet Mixer  was employed, working on the sample while on ice until no floating fragment of the tissue was seen in the vial. The final RNA extracted was diluted in 30 uL of RNAse-free water. RNA quantification and evaluation of integrity was performed using a 2100 Bioanalyzer in combination with the RNA 6000 Pico kit .RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen/(exon inclusion + exon skipping) band intensities. qPCR analysis was carried out using a LightCycler 480 SYBR Green I Master (Roche) and a LightCycler® 96 Real-Time PCR System (Roche) system (Applied Biosystems) according to the manufacturer’s instructions. To quantify the relative changes in gene expression, the 2-ΔΔCq method was used and the reactions were normalized on the endogenous control gene L34.In order to check whether the samples obtained with the above mentioned methods were useful for transcriptome analyses, we planned to employ 20 samples acquired from 10 patients among those characterized by having both FNA and FNB samples with adequate RIN (> 3) and quantity (> 60 ng) to perform real-time quantitative PCR (qPCR) for PRRS1 and COL1 as markers of acinar and stromal contamination, respectively, and GATA6 and ZEB1  as markers of classical subtype and epithelial-to-mesenchymal transition (EMT) and semi-quantitative PCR (sqPCR), respectively, for the splicing analysis of ARHGAP17  see Sup. A totalDescriptive statistics were calculated as means and standard deviation (SD) for normally distributed continuous variables, as means and SD and median and interquartile range (IQR) for continuous variables with skewed distribution and as numbers and percentages for categorical variables.An unpaired T-test was employed for the comparison of normally distributed continuous variables and a Mann–Whitney U test for continuous variables with skewed distribution. One-way analysis of variance (ANOVA) was used to test the difference between the means of gene expression among the different needle types. Correlation analyses were performed calculating the Kendall rank correlation coefficient.p value < 0.05 was considered statistically significant.All calculations were performed using MedCalc version 13 . A Among 1954 EUS performed between November 2018 and October 2019, 37 patients were enrolled see  with a mp < 0.0001) or snap frozen samples  (p = 0.8) while both conservation methods resulted in higher quality RNA compared with samples collected in RNALater  compared with RNALater  a. No sigTo determine whether the initial sample  weight or days between tissue sampling and RNA extraction affected the final RNA concentration or quality, correlation analyses were carried out on samples conserved in Trizol. As shown in In order to establish whether one needle was superior to the others in terms of reducing acinar or stromal contamination, real-time PCR (qPCR) analyses were performed for the expression of PRSS1, the gene encoding trypsin as a marker of acinar cells, and of COL1, the gene encoding collagen 1 as a marker of stromal cells.®: 0.25, 25G Acquire®: 0.04, 25G Slimline®: 0.03; p = 0.16) and stromal cells  , three were conserved in RNALater (out of the 22 RNALater samples = 13.6%) and 18 in Trizol (out of 24 Trizol samples = 75%). Of the 28 adequate samples, 20 were FNA/FNB matched samples from 10 patients and were therefore employed for qPCR. This analysis indicated that a 20G FNB needle resulted in a higher, despite not significant, mean contamination from acinar (20G ProCore = 0.48) .p = 0.08) (see p < 0.0001).The endodermal transcription factor GATA6 was recently proposed as a marker of the classical PDAC subtype and correlated with better prognosis in patients ,13,14. B.08) see . To confIt was recently reported that the recurrent mutations in TP53 lead to widespread changes in alternative splicing in PDAC. In particular, the inclusion of a polyC-rich exon in the GTPase GAP17 (ARHGAP17 gene) was shown to cooperate with a mutant KRAS protein in the activation of the downstream pathway . To deteDespite the remarkable efforts in the context of translational and clinical research in the past years, the five year survival rate of PDAC patients remains around 8% and has not changed noteworthily in the recent decades . TreatmeTranscriptional features of tumoral cells have shown great potential for the stratification of patients in several human cancers. In the context of PDAC, two main subtypes have been described on the basis of differential gene expression signatures: the classical and the basal-like. Classical PDAC is characterized by a better prognosis and a response to Folfirinox; the basal-like PDAC is characterized by a worse prognosis and a possible response to gemcitabine-based therapy or targeted therapy . Despiten = 45) resulted in being adequate for nanostring analysis and the RNA quality was not reported. In another study, RNA extraction was performed from 40 snap frozen samples acquired through a standard 22G needle, achieving a high quantity of RNA (1 µg) with the quality assessed by agarose gel electrophoresis and spectrophotometry but not quantitatively reported [RNA extraction from pancreatic tissue is cumbersome and more difficult compared with DNA for the abundance of RNAse in the pancreatic gland that leads to a fast RNA degradation after sample acquisition. Furthermore, PDAC is characterized by a low percentage of tumoral cells for the abundance of stroma and the acquisition through EUS can lead to contamination due to the passage of the needle through the gastric or duodenal wall and through healthy pancreatic tissue . To datereported . These sAmong the three types of needles evaluated  no single one resulted in a significant superiority in terms of the quantity or quality of RNA retrieved. As for contamination, there was an insignificant higher amount of both acinar and stromal contamination (based on PRSS1 and COL1 gene expression) with the use of the 20G ProCore FNB needle . We can Recently, numerous studies have reported a superiority of the Franseen needle in terms of the adequacy of the tumoral specimen for diagnostic purposes ,32. Neven = 10). This result suggests that combining the evaluation of the expression levels of relatively few genes from EUS-TA derived samples may help predict the prognosis of non-resectable PDAC patients.Recently, the importance of the expression of GATA6 has been highlighted in clinical studies on surgical specimens and in silico. GATA6 is a regulator of pancreatic development and activates epithelial differentiation ,33. NotaRecent studies have clearly shown that splicing is generally deregulated in human cancers including PDAC  and thatThe present study has some strengths: it is the first study to specifically evaluate prospectively the combination of three different conservation methods and of three different needles for the acquisition of RNA from PDAC samples; the RNA retrieved was of high quantity and good quality and was successfully used to perform qPCR to determine contamination from acinar and stromal cells, to evaluate the expression of prognostic markers such as GATA6 and ZEB1 and also to determine splicing events. The patients were from similar stages and the prognosis of all patients was prospectively recorded for a correlation with biomarkers.The study carries the intrinsic limitations of a pilot one, which aimed at testing experimental conditions and feasibility such as the relatively small sample, the lack of a head-to-head comparison of all three needles and conservation methods as this was not ethically acceptable and the execution of qPCR only on a set of patients. The PRSS1 and COL1 gene expression evaluation on EUS-acquired normal pancreatic tissue for comparison was also not carried out, as this was not ethically acceptable. Moreover, the ratio between GATA6/ZEB1 is only speculative and not validated as also the overall survival was partial and not definite. Lastly, the mutational status of P53 was not investigated in the patients undergoing the GAP17 splice event evaluation.In conclusion, in our study EUS-acquired samples of PDAC conserved in Trizol resulted in a significantly higher concentration and sufficient integrity of RNA and hence usable to perform qPCR for tissue and prognostic biomarkers including the evaluation of splicing events. Further studies on a larger set of patients and of RNA sequencing on EUS-acquired samples are needed in order to evaluate its feasibility and investigate additional prognostic markers, valid also for locally advanced diseases.