The parasitic flatworm Opisthorchis felineus is one of the causative agents of opisthorchiasis in humans.Recently, we assembled the O. felineus genome, but the correct genome annotation by means of standard methods was hampered by the presence of spliced leader trans-splicing (SLTS). As a result of SLTS, the original 5’-end(outron) of the transcripts is replaced by a short spliced leader sequence donated from a specialized SL RNA. SLTSis involved in the RNA processing of more than half of O. felineus genes, making it hard to determine the structureof outrons and bona fide transcription start sites of the corresponding genes and operons, being based solely onmRNA-seq data. In the current study, we tested various experimental approaches for identifying the sequences ofoutrons in O. felineus using massive parallel sequencing. Two of them were developed by us for targeted sequencing of already processed branched outrons. One was based on sequence-specific reverse transcription from theSL intron toward the 5’-end of the Y-branched outron. The other used outron hybridization with an immobilizedsingle-stranded DNA probe complementary to the SL intron. Additionally, two approaches to the sequencing ofrRNA-depleted total RNA were used, allowing the identification of a wider range of transcripts compared to mRNAseq. One is based on the enzymatic elimination of overrepresented cDNAs, the other utilizes exonucleolytic degradation of uncapped RNA by Terminator enzyme. By using the outron-targeting methods, we were not able toobtain the enrichment of RNA preparations by processed outrons, which is most likely indicative of a rapid turnoverof these trans-splicing intermediate products. Of the two rRNA depletion methods, a method based on the enzymatic normalization of cDNA (Zymo-Seq RiboFree) showed high efficiency. Compared to mRNA-seq, it provides anapproximately twofold increase in the fraction of reads originating from outrons and introns. The results suggestthat unprocessed nascent transcripts are the main source of outron sequences in the RNA pool of O. felineus. Opisthorchis felineus is a representative of parasitic flatworms (Trematoda: Opisthorchiidae), which has a complexlife cycle with two intermediate hosts and the mammaliandefinitive host, including humans . Opisthorchiasis caused by parasitism of this fluke in the bile ducts ofthe human liver has a chronic course and leads to a numberof serious concomitant disorders of the hepatobiliary system,including cholangitis, cholecystitis, pancreatitis, and is also arisk factor for the development of cholangiocarcinoma . According to WHO study, more than 1 millionpeople estimated to be infected with O. felineus, with thelargest focus of opisthorchiasis located in the Ob-Irtysh basin,Russia, with the incidence in some regions of the Tomsk andTyumen regions up to 60 % One of the necessary steps to study the biology of thisflatworm, and the corresponding development of moleculargenetic approaches to the diagnostics and pharmacotherapy ofopisthorchiasis, was to obtain a reference genome assemblyand its annotation . However, the existinggene annotation based on polyA-mRNA-seq data, which iscommonly used for this task, has significant drawbacks due toone of the features of RNA processing in flatworms – splicedleader trans-splicing (SLTS). During SLTS, the exons of twoindependent transcripts are fused: a specialized short cappedspliced leader RNA (SL RNA) carrying a 5′-splice site, andone or the other pre-RNA with a corresponding 3′-site . At the same time, the original 5′-region of thepre-RNA, together with the SL RNA intron attached to itsbranchpoint through the 2′–5′-phosphodiester bond, is excised. It is assumedthat this Y-branched product (Y-outron) undergoes rapid debranching and degradation . Thus, in the polyA-mRNA-seq data, thesequences of excised outrons are practically absent, and withthem, localization of the actual transcriptional start sites andpromoters of the corresponding genes, as well as informationon the operonic organization of gene groups, becomes inaccessible. It should be noted that due to SLTS, a large groupof experimental methods for identifying transcription startsbased on selective sequencing of the capped 5′-ends of RNAsis also ineffective. According to our estimates, products of more than half ofall genes in the O. felineus genome undergo trans-splicing . Thus, the correct annotation of such genesrequires alternative high-throughput sequencing methods for large-scale identification of outrons. Most often, to describegene outrons, researchers combine information on transcription initiation sites obtained by such methods as GRO-seq,GRO-cap, ChIP-seq to RNA polymerase II, with data on SLTSsites detected by mRNA-seq . In this way, the region of the genome from the startof transcription to the 3′-site of the SLTS is accepted as anoutron. It should be noted that most of the mentioned methodsrequire a large amount of starting biological material and, inthe case of a small size of individuals, is suitable mainly fororganisms cultured in the laboratory. There are few knownexamples when it was possible to detect outrons directly byRNA sequencing methods. Thus, in one of the studies onC. elegans, growth of individuals at low temperature led tothe accumulation of unprocessed transcripts, which madeit possible to detect the 5′-ends of outrons by SAGE of thenuclear fraction of RNA .On non-model organisms, including trematodes, RNA-seq“SLTrapping” methods have been successfully used for directmassive detection of trans-spliced mRNAs, based on selection for the universal 5′-sequence of processed transcripts,the SL exon . Sinceall Y-outrons similarly contain the universal 3′-sequence ofthe SL intron, it seems attractive to use this property for selective identification of outrons and symmetric confirmationof SLTS sites. In addition, direct identification of Y-outronswould serve as direct evidence of the generally acceptedmechanism of SLTS on a large sample of genes.In the current study, to identify O. felineus outrons, weevaluated two candidate approaches based on targeted enrichment for Y-outrons, in one case, by sequence-specific reversetranscription primed from the SL intron towards the 5′-end ofthe outron, in the other – by hybridization of Y-outrons withan immobilized single-stranded DNA probe complementaryto the SL-intron. In addition, two approaches to sequencingof rRNA-depleted total RNA that do not use hybridizationprobes were used as an alternative: one that use rRNA cleavageby Terminator exonuclease, and the commercial Zymo-SeqRiboFree kit based on enzymatic normalization of cDNA. Theproportion of sequences containing outrons in these librariesshould certainly be higher than in standard polyA-mRNA-seqlibraries.Biomaterial. Adult O. felineus worms were isolated fromthe bile ducts of golden hamsters Mesocricetus auratus after3–4 months after infection with metacercariae obtained fromthe tissues of naturally infected fish (Leuciscus idus) from the Ob river. The worms were washed in saline buffer and usedimmediately or frozen and stored at –80 °C.RNA isolation. RNA was isolated from fresh or frozenO. felineus samples using PureZOL  according to the manufacturer’s protocol. Precipitation of RNA wascarried out by adding an equal volume of isopropanol and 5 μgof linear polyacrylamide (LPA) as a co-precipitant; the resulting solution was left overnight at –20 °C. The precipitate wasdissolved in bidistilled water, and additional purification ofsamples was carried out using an Aurum total RNA mini kit, including a stage of treatment with DNase I.The quality and amount of isolated RNA was assessed usinga NanoDrop 2000 spectrophotometerTerminator exonucleolytic cleavage. Total RNA wastreated with Terminator enzyme  according to the manufacturer’sprotocol. The reaction mixture contained 500 ng of total RNA,1 μl of RiboLock , 2 μl of10× buffer A, 1 U of Terminator exonuclease. The reactionvolume was brought up to 20 μl with bidistilled water. Thesample was incubated for 60 min at 30 °C, the reaction wasstopped by the addition of 1 μl of 100 mM EDTA. The products of the reaction were purified using Agencourt RNACleanXP beads . The decrease in theamount of ribosomal RNA was checked on an Agilent 2100Bioanalyzer using an RNA 6000 Pico chip.Isolation of RNA fraction enriched in Y-outrons (SLiBC). A biotinylated ssDNA probe ([biotin-TEG]-5′-GGCTAGCCAAATAATTCATCCGACCATAGGCCGGAGTCGATTCTT-3′) was immobilized on magnetic beads  in accordancewith the manufacturer’s protocol, monitoring the approximatecorrespondence of the amount of bound probe to the declaredbinding capacity (~200 pmol/mg). 1 μg of RNA in a buffer forhybridization , 0.5 mMEDTA, 0.1 % Tween-20) was added to the magnetic beadscovered with a DNAprobe and incubated with constant stirringaccording to the following protocol: 75 °C for 2 minutes, thenthe temperature was lowered by 1 °C/min to 55 °C and fixedfor 15 min at 55 °C. The particles were washed three timeswith warm (50 °C) buffer. 10 μl of water was added to theparticles and heated to 94 °C for 5 min. The RNA-containingsolution was collected in a clean tube and the reverse transcription reaction was carried out immediately. For the reversetranscription reaction, 200 U RevertAid Reverse Transcriptase and random hexamers wereused. The reaction was carried out under the following conditions: 5 min at 25 °C, 60 min at 42 °C, and 5 min at 70 °C.The resulting cDNA was used to prepare the libraryReverse transcription reaction with sequence-specificprimers (SLi-RT). The thermostable Maxima H Minus Reverse Transcriptase  was usedfor the reverse transcription reaction. The reaction mixturecontaining 300 ng RNA, 2 pmol of a specific primer to theSL-RNA intron ,1 μl 10 mM dNTP, 4 μl 5 M betaine, was incubated for2 min at 75 °C, then the temperature was lowered at a rateof 2 °C/min to 55 °C; 20 U RiboLock RNase Inhibitor and100 U Maxima H Minus Reverse Transcriptase were addedand the mixture incubated for 30 min at 55 °C and for 5 min at 85 °C to inactivate the reaction. The resulting cDNA wasused to prepare the library.1. Enrichment was calculated by the ΔΔCt methodas the change in the difference of outron from exon betweencontrol and selected cDNA. The control was cDNA obtainedby reverse transcription of total RNA primed from randomhexamers. In the case of SLi-RT, the selected cDNA was obtained by reverse transcribing the total RNA from the SLi_r1primer, and in the case of SLi-BC, by converting the enrichedRNA fraction using random hexamers.Evaluation of Y-outron enrichment of SLi-RT andSLi-BC cDNA samples using real-time PCR (RT-PCR). Toassess the enrichment for Y-outrons during the elaboration ofthe SLi-BC and SLi-RT protocols, RT-PCR with primers tothe outron and exons  of the mmce gene was carried out http://vavilov.elpub.ru/jour/manager/files/SupplErshov_engl.pdf Supplementary Materials 1–3 are available in the online version of the paper:PCR was carried out in a 20 μl reaction mixture containing 0.25 mM dNTP, 2 μL 10X PCR buffer B+EVAGreen,2.5 mM MgCl2, 10 pmol of each primer, 2 μl cDNA sample,0.3 U/μl SynTaq DNA polymerase with antibodies inhibiting polymerase activity . The reaction wascarried out with preheating to 95 °C for 5 minutes followedby 39 amplification cycles including denaturation at 95 °Cfor 15 seconds, primer annealing and elongation at 60 °C for20 seconds. Melting curves were acquired within the temperature range 65 to 95 °C. All reactions were performed intwo technical replicates.Library preparation. To prepare RNA-seq libraries afterTerminator treatment, NEBNext Ultra II Directional RNALibrary Prep Kit for Illumina  was used according to the standard protocol. To prepare libraries from cDNAsamples obtained by the SLi-BC and SLi-RT protocols,NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used, starting from the stage of the second strandcDNA synthesis. Fragmentation of the cDNA was performedusing the dsDNA Fragmentase enzyme simultaneously withthe repair of the DNA ends. Fragments of the required lengthwere selected using Agencourt AMPure XP beads, after whichseveral cycles of library amplification were performed.rRNA-depleted RNA-seq libraries were prepared usingthe Zymo-Seq RiboFree Total RNA Library Prep Kit  according to the standard protocol. 500 ng oftotal RNA was used in the reaction; depletion after renaturationwas carried out for 60 min.The size and quantity of the resulting libraries was determined using an Agilent 2100 Bioanalyzer .The resulting libraries were sequenced in paired-end mode(2×250 bp) on the Illumina MiSeq platform . Raw sequencing data hasbeen deposited in the open-access repository Zenodo . Previously published mRNA-seq data used in the studyare available in the NCBI repository (PRJNA257351).Computational processing of sequencing data. Sequencing data in the FASTQ format was processed using the Cutadapt software  to remove adapter sequencesand mapped to the O. felineus reference genome (GenBank IDGCA_004794785.1) using the STAR aligner . The annotation of genomic elements was constructedusing the corresponding gene annotation and previouslyobtained genome-wide data on positions of trans-splicingsites  using in-house Perl scripts. Theassembly of the genomic rRNA repeat was carried out manually using the data of genomic DNA sequencing . The boundaries of rRNA genes in the genome weredetermined using the Rfam database (https://rfam.xfam.org)and the RNAmmer tool . Read countsin the defined genomic intervals were computed using thefeatureCounts tool from the Subread package . Statistical processing and graphical data visualizationwere carried out in the R environment.Description of the approachesAt the moment, we have not been able to find any reference inthe literature to experimental methods for the direct massiveidentification of Y-outrons in the transcriptome. Upon examination of the previously obtained O. felineus mRNA-seq data, we found that the 5′-regions upstreamof the trans-splicing site  of many geneshave the same weak read coverage as their introns ,despite the depletion of non-coding and unprocessed transcripts as a result of polyA selection. In total, about 0.15 %of mRNA-seq reads were mapped to outronic regions (5 kbupstream of the site) of trans-spliced transcripts (on averagewith 0.8-fold coverage). The source of such outronic readscan be: (1) unprocessed nascent pre-RNA, (2) Y-outrons –intermediate Y-branched byproducts of trans-splicing, as wellas (3) products of their further debranching, which lack thecovalently bound SL-intronic arm . We assumedthat the amount of Y-outrons (2) in the total RNA pool couldpotentially be sufficient for its isolation by targeted enrichment methods.The possibility of targeting Y-outrons is due to the presence of a universal sequence – an SL-RNA intronic arm,covalently bound at a branchpoint to outron of pre-RNA . Using this property, we have developed two differentapproaches to direct selection of Y-outrons. The first, designated SLi-RT (SL-intron Reverse Transcription), is a targetedreverse transcription from the 2′-arm (SL-intron) towards thevarying 5′-end of the outron . It is known thatM-MLV reverse transcriptase lacking RNase H activity, whenprimed from the 2′-arm, is able to quite frequently bypass thebranchpoint with the introduction of single-mismatch errorsand successfully reverse the 5′-segment . Among the expected drawbacks ofthe method are the reduced efficiency of such reverse transcription, the accumulation of products of mispriming, andthe underrepresentation of the 5′-regions of outrons due to afrequent accidental termination of the first strand cDNA synthesis. In accordance with the latter, the choice of enzyme andhigh temperature reaction conditions, including hot start, wereoptimized for maximum processivity and synthesis specificity.The second approach, SLi-BC (SL-intron Biotin Capture),is based on hybridization of the 2′-arm of outrons with a biotinylated single-stranded DNA probe homologous to theSL intron, followed by immobilization and purification ofthe corresponding fraction on magnetic particles coated withstreptavidin . This approach should be moresensitive to the proportion of the target within the total RNAsample, but it has a better chance of identifying full-lengthoutrons.Both approaches are aimed at the intact Y-structure of theprocessed outron and therefore would allow differentiatingthem from fragments of nascent transcripts that did not undergo trans-splicing. In addition, we also applied two methodsfor sequencing of the rRNA-depleted total RNA, that allowidentification of noncoding transcripts, including introns andoutrons. At the same time, since O. felineus is a non-modelspecies, available depletion kits based on hybridization withrRNA probes are not applicable. We therefore tested a protocol for treatment of total RNA with the Terminator exonuclease, which specifically hydrolyzes nucleic acids with aphosphorylated 5′-end (including rRNA), but does not affectcapped transcripts. Alternatively, we applied the commercialZymo-Seq RiboFree protocol, based on DSN-normalizationof RNA:DNA hybrids immediately after the synthesis of thefirst cDNA strand, as a result of which all overrepresentedtranscripts, including globins and rRNA, are hydrolyzed.Unlike the first two, these methods do not make it possible todistinguish the processed outrons from their precursors – unprocessed and nascent transcripts, and therefore cannot serveas direct evidence of the trans-splicing event.The sequences of the previously identified intact SL-RNA were used to design the correspondingprimers and oligonucleotide probes. Optimization of the enrichment conditions for Y-outrons in the SLi-BC and SLi-RTmethods was controlled by RT-PCR with primers to the outronand exons of the mmce gene, the trans-spliced product of whichis not polycistronic and is among the most highly expressedtranscripts . The enrichment was calculated as the difference of ΔCt  between thecontrol and the enriched cDNA samples. In the case of cDNAsamples used for the subsequent preparation of the SLi-BCand SLi-RT libraries, there was 4- and 100-fold enrichmentin the outron region relative to the mmce exons, respectively.RNA-seq libraries were prepared from the RNA or cDNAsamples obtained by various methods and then sequencedwith low coverage, which gives a reasonable indication ofthe overall performance for enrichment or depletion of targetsequences.Residual rRNA content in librariesTo compare the applied approaches against the standardmRNA-seq method, the obtained sequencing data from fivelibraries were mapped to a reference genome supplementedwith an rRNA repeat sequence. Since the latter repeat had asufficiently high coverage in all libraries, it turned out to beuseful not only for assessing the efficiency of rRNA depletion,but also for assessing the strand-specificity of the resultinglibraries . Of the libraries, only TerminatorExo had arelatively low degree of strand-specificity (68 %). Interestingly, each of the libraries was characterized by its ownspecific rRNA coverage profile. It is worth mentioning that, for a large number of protostomes, 28S rRNA is additionallyprocessed at the “hidden break” site with the formation ofapproximately equivalent in size 28Sα and 28Sβ fragments. This feature is also observed for O. felineus: a coverage drop in the 28S rRNA  anda single rRNA peak in the electropherogram The observed distribution of the few rRNA reads of theZymo-Seq library in comparison with mRNA-seq  reflects extremely effective subtractive depletion in the most overrepresented regions of thisrepeat.The TerminatorExo library profile indicates a fairly effectiveinitial enzymatic degradation of the 5′ regions of 18S, 28Sα,and 28Sβ rRNA , which,however, rapidly fades away at certain sites. This is probablydue to the sensitivity of the enzyme to the complex tertiarystructure of the substrate, or to the frequent hydrolysis ofrRNA at these sites with the formation of 5′-OH ends, whichprotect against further exonucleolytic cleavage by the enzyme.Consequently, the resulting TerminatorExo library containedabout 60 % of rRNA reads.Targeted enrichment methods SLi-BC and SLi-RT alsoshowed high levels of background rRNA (63.1 and 20.2 %).Namely, the SLi-RT library contained mainly the product ofoff-target reverse transcription from the 3′-region of 28SαrRNA. Since the 3′-region of 28Sα rRNA can potentiallyform a stable hairpin , it islikely that such a structure determines the priming of reversetranscription without any participation of the SLi_r1 primer.Representation of various genomic featuresin sequenced librariesFor the mapped data, we analyzed the distribution of readsacross various mutually exclusive groups of genomic elementsin the following priority order: rRNA repeats, mtDNA, exons,introns, promoter or outron regions (1 and 5 Kb upstream ofthe 5′-end of the predicted gene) and intergenic loci. The calculation was carried out separately for genes undergoing highlyefficient trans-splicing (SL) and for all other genes (TR). Ascan be seen from the results presented in Fig. 3, a, in the TerminatorExo, SLi-BC and SLi-RT libraries, the overwhelmingproportion of reads is represented by the uninformative rRNAfraction and the category of intergenic loci. In contrast, in theZymo-Seq and mRNA-seq libraries, more than 60 % of thereads are mapped to exons of the annotated genes. If we exclude the rRNA and mtRNA groups from consideration , the distribution of read countsacross categories of genomic elements, normalized to thetotal length of the latter, turns out to be quite similar for themRNA-seq, SLi-BC, TerminatorExo, and Zymo-Seq libraries.The SLi-RT library generally reproduces a random (uniform)distribution of reads across the genome. Obviously, SLi-RTcontains mainly noise, which was probably originated fromgenomic DNA, despite the identical DNase treatment of allRNA samples. It is worth noting that this library contained227 (0.12 %) read pairs containing the SLi_r1 primer at thebeginning of the fragment (next to the adapter sequence).These target sequences were expected to be overrepresentedin the SLi-RT library. Although they most likely do not belongto genuine Y-outrons, their presence confirms the efficiencyof the primer in the reverse transcription reaction.It was assumed that in the case of effective enrichment oflibraries by outrons we should observe a significant increasein the coverage of 5′-adjacent regions of trans-spliced genes(SL:up_1Kb and SL:up_5Kb) relative to the promoter regionsof ordinary genes (TR:up_1Kb and TR:up_5Kb). This enrichment was indeed observed for the SLi-BC, TerminatorExo andZymo-Seq libraries, but only in the case of the SL:up_5Kbcategory . In contrast, these libraries demonstrateddepletion in potential outrons in SL:up_1Kb. The depletionis most likely due to the fact that for many “TR” genes their5′-UTRs are not annotated correctly and therefore a largenumber of reads originating from mature mRNAs are falselyassigned to the TR:up_1Kb category. More revealing is thecomparison with mRNA-seq, in which all three mentionedmethods were more enriched in both categories of outronicregions. Since the coefficient of enrichment is approximatelythe same for the three libraries, it describes just the actualfraction of outrons in the initial total RNA. Consequently,the SLi-BC method did not result in preferential selection oftarget Y-outron sequencesThus, of all the tested methods, only Zymo-Seq protocolmade it possible to significantly get rid of the uninformative rRNA fraction, while preserving the original fraction ofnoncoding transcripts, including outrons. At the same time,the coverage of outrons compared to exons in the Zymo-Seqlibrary remains very low . Taken together, these results suggest that sequences corresponding to outrons including unprocessed or nascenttranscripts and intact or partially degraded Y-outrons, arerepresented by a very small fraction in the O. felineus RNApool. Apparently, intact Y-outrons make up the smallest partof them, and therefore the SLi-BC and SLi-RT libraries targeting the intact Y-outron structure contained mainly unspecificnoise. Although the mechanism and rate of degradation ofY-branched trans-splicing products remain unknown, it is assumed that they degrade rapidly .Thus, in an in vitro experiment on C. elegans, all trans-splicingintermediates were observed, except for the Y-branched outrons . The main source of noise in the SLi-BC method was thenonspecific sorption of RNA, while in the SLi-RT method,it was off-target reverse transcription of rRNA without the participation of a primer, as well as a minimal admixture ofgenomic DNA. 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CoV2), which should play a key role in a new drug development and in the interaction with RNA and other proteins are summarized. The structural feature of NCoV2 is first described. Next, simulation studies on the interaction between potential drug molecules and NCoV2 are summarized, which show the importance of molecular flexibility. Then, liquid-liquid phase separation phenomenon involving NCoV2, which has recently been reported, is described. This phenomenon also suggests the importance of molecular flexibility of NCoV2. Finally, a promising method using neutron scattering to characterize the structure and structural fluctuation of the droplets, which are formed through this phenomenon, is presented.Nucleocapsid protein is one of the essential proteins for viral replication including the coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) pneumonia leading to the ongoing pandemic. Whereas this protein has emerged as a potential drug target, its physicochemical properties and the molecular mechanism of how this protein is involved in viral replication remain unclear. In this review, structural and dynamical aspects of the SARS-CoV-2 nucleocapsid protein (NCoV2) is one of the abundant proteins and shows multiple functions including packaging the RNA genome during the infection cycle. It has also emerged as a potential drug target. In this review, the current status of the research of NCoV2 is described in terms of molecular structure and dynamics. NCoV2 consists of two domains, i.e., the N-terminal domain (NTD) and the C-terminal domain (CTD) with a disordered region between them. Recent simulation studies have identified several potential drugs that can bind to NTD or CTD with high affinity. Moreover, it was shown that the degree of flexibility in the disordered region has a large effect on drug binding rate, suggesting the importance of molecular flexibility for the NCoV2 function. Molecular flexibility has also been shown to be integral to the formation of droplets, where NCoV2, RNA and/or other viral proteins gather through liquid-liquid phase separation and considered important for viral replication. Finally, as one of the future research directions, a strategy for obtaining the structural and dynamical information on the proteins contained in droplets is presented.The latest coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) pneumonia leading to the pandemic, contains 29 proteins. Among them, nucleocapsid protein (N CoV2).A new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the cataclysmic pandemic through coronavirus disease 2019 (COVID-2019) pneumonia. SARS-CoV-2 is a member of β-coronavirus family that includes well-known SARS-CoV and MERS-CoV , and 4 mCoV2, which is composed of 419 residues in total, is shown in CoV2 consists of two domains, the N-terminal domain  and the C-terminal domain . The NTD is responsible for RNA-binding and the CTD for dimerization and oligomerization  and the C-tail (365–419), respectively. The atomic structures of the NTD have recently been solved by X-ray crystallography at 2.7 Å resolution /[polyU]) of 0.6 (wt/wt), heterotypic droplets (peptides-RNA droplets) were transformed into homotypic droplets (RNA-RNA droplets). Another study has shown that formation and dissolution of the peptide-polyU droplets are regulated by the phosphorylation state of the peptides [CoV2 droplets, indicating that interactions occurring at the amino acid level are prerequisites for droplet formation.In addition to proteins, small peptides are also known to show phase separation with RNA through electrostatic interactions ,74,75. INA ratio . In thisCoV2 droplets are dynamic objects: when NCoV2 or RNA molecules are added to the preformed NCoV2/RNA droplets, both types of molecules are incorporated into the droplets and simultaneously the NCoV2 and RNA molecules that constituted the preformed droplets diffuse out of the droplets, suggesting that both NCoV2 and RNA are exchangeable between the dense and dilute phases [CoV2 and RNA are crucial for dynamically maintaining the droplets. Moreover, when the complex of RdRp and RNA is added to the NCoV2/RNA droplets, the entire RdRp-RNA complex is also incorporated into the droplets [CoV2/RNA and G3BP1, there are three populations of the NCoV2 molecules that diffuse at different mobility [CoV2 molecules in the droplets might play an important role in the transcription and replication cycle. Since the diffusion of the whole protein molecules inside the droplets is largely affected by the strength of interaction between NCoV2 and other proteins/RNA, it would be of great interest to understand the relevant physical properties of NCoV2 such as side-chain fluctuations, which directly determine the rate and the manner of interaction with their binding partners.Fluorescence measurements using Alexa fluorophores have shown that the Ne phases . This aldroplets , suggestmobility . These rCoV2, RNA, other viral proteins and a stress granule protein are concentrated into a different phase from its surroundings suggest that the NCoV2 droplet formed through LLPS is the main arena where molecular events critical for the virus replication take place. It is thus indispensable to reveal the internal organization of NCoV2 droplets, especially the structure of NCoV2 in droplets formed with various binding partners to develop a new intervention technique targeting NCoV2 as well as to understand the molecular mechanism of biological functions of NCoV2 in detail. Furthermore, the molecules within the droplets are not frozen to the spot but fluctuate dynamically around their average conformation under the influence of thermal energy. The nature of these structural fluctuations is closely related to the interaction, the stability and the function of the proteins. Thus, unraveling the structure and structural fluctuation of NCoV2 in the droplets formed in various conditions is essential and the research in this direction would occupy an important position in this research field.The recent findings that NCoV2 in droplets would be small-angle neutron scattering (SANS). This technique has been used to characterize the structure of viral proteins in isolated state [CoV2 droplets. In neutron scattering, the strength of scattering (coherent scattering length) depends on the type of atoms bombarded by neutron as shown in 2O in the solvent. As the D2O content in the solvent increases, the labile hydrogen atoms in the solutes are exchanged for deuterium atoms, which modifies the SLD of the solutes. The SLD of perdeuterated proteins , meaning that the scattering contrast of the H-protein is zero. In this condition, the contribution of the H-protein to the scattering signal is effectively zero and hence it is “invisible” to neutron. Even though techniques to perdeuterate or partially deuterate RNA molecules by chemical synthesis, in vitro transcription and in vivo production have been established for NMR studies [A powerful method to characterize the molecular structure of Ned state ,77 and ied state ,79,80. Hed state ,82,83,84 studies ,89, prod studies . TherefoCoV2 structure in droplets. In the case where droplets are formed by NCoV2, RNA and another protein , (2) those formed by H-NCoV2, H-RNA and the perdeuterated another protein (D-AP) and (3) those formed by the perdeuterated NCoV2 (D-NCoV2), H-RNA and D-AP. The SANS measurements in these samples in the ~65% D2O solvent, the SLD of which matches that of H-RNA, provide three kinds of scattering data where each protein contribution is different. From these data, the scattering data arising from each protein can be extracted, leading to the elucidation of its conformation together with how each protein is arranged inside the droplets. protein c, three 2O buffer can minimize the solvent contribution to the iNS spectra. Since the hydrogen atoms are quasi-uniformly distributed throughout the molecules in the biological systems, the dynamical information averaged over the whole molecule is obtained. In the case of proteins, the observed motions reflect the fluctuations of amino acid side chains and backbones because hydrogen atoms are bound to these chemical groups. The schematic illustration of the dynamical analysis of the NCoV2 droplets is depicted in CoV2, RNA and another protein, two types of samples are required: (1) droplets formed by H-NCoV2, H-RNA and D-AP, (2) those formed by D-NCoV2, H-RNA and D-AP. By subtracting the iNS spectra of the latter from those of the former measured in 100% D2O, the remaining spectra arise from the structural fluctuation of the NCoV2 molecules, thereby providing dynamical information on NCoV2 inside the droplets. Instead, the dynamics of another protein in the droplets can be extracted in a similar manner.The structural fluctuation of the molecules in the droplets are obtained by incoherent neutron scattering (iNS). This technique measures the intensity of neutron scattered by the samples as a function of the momentum and energy change of the neutron, from which the amplitude, the frequency and their distribution of atomic motions at pico- to nanosecond timescale at ångström length scale are estimated . The valD) of the identical NCoV2 molecule and the distance between domains of different NCoV2 molecules (RM) can be determined from structural analysis. This means that not only the conformation of NCoV2 in droplets but also the arrangements of the NCoV2 molecules in droplets are revealed. From the iNS measurements, efficient diffusion coefficients (Deff), which include translational and rotational diffusion, of the whole NCoV2 molecule and/or domains will be obtained. Furthermore, as shown in the lower panel of CoV2 in droplets with that in an isolated state (CoV2 in droplets. It is likely that the unique structure and arrangements of proteins inside the droplets form a field where RdRp molecules bind to RNA more easily than outside the droplets, promoting the viral genome transcription. The unique mode of protein motions inside the droplets would be the determinants of interactions between other molecules inside the droplets, affecting the efficiency of genome transcription. All the above structural and dynamical information will thus be useful not only for understanding the molecular mechanism of the virus replication/infection cycle but also developing medical intervention techniques based on the structure and dynamics of proteins. Moreover, since the time- and space-scales of iNS are comparable to those of MD simulation, dynamics parameters obtained from iNS would serve as a fundamental information in in silico drug development targeting the droplets in the future.That way, the structure and structural fluctuation of the proteins contained in the droplets, which play a crucial role in virus replication, are characterized. For example, following parameters characterizing the internal organization of the droplets are found: as shown in ed state b and in ed state c, structCoV2 plays a critical role in drug-binding, RNA-binding and formation of the droplets. Knowledge of the structure and dynamics of the NCoV2 molecules in isolated states in solution and in a variety of droplets would advance our understanding of the architecture and the physical properties of the droplets, thereby shedding light on the molecular mechanism of the viral infection and replication cycle. Furthermore, all this information would eventually be beneficial to the development of new drug molecules targeting NCoV2, of improved diagnostic techniques, of a new type of treatment for the COVID-19 pneumonia, and of a new combat strategy for a future pandemic.In conclusion, the molecular flexibility as well as conformations of N


CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of  CD45, also known as protein tyrosine phosphatase receptor type C (PTPRC), is mainly expressed on immune cells to initiate cross-membrane signal transduction ,2. The pRegarding the regulation of CD45 splicing, previous studies found that nucleotide polymorphisms at the splicing site of CD45 alternative exons influenced the expression of CD45 isoforms and the function of immune cells ,7,8, andPEBP1P3 (phosphatidylethanolamine binding protein 1 pseudogene 3) is a natural antisense gene of CD45 and annotated as a pseudogene in the human genome. Through epigenetic and gene structure analysis, we previously issued the viewpoint that the transcript of PEBP1P3 might be involved in the expressional regulation of CD45 isoforms in autoimmune diseases [diseases . To inveCD45 and its antisense gene PEBP1P3 were obtained from GenBank  and UCSC Genome Browser . Their coding potential was analyzed using the online software Coding Potential Calculator (CPC)  [https://www.gtexportal.org, accessed on 9 August 2018), which is a project creating and releasing RNA expression data from 8555 tissue samples obtained from 570 adult postmortem individuals [CD45 DNA was predicted through CTCFBSDB 2.0  [Genomic and cDNA sequences of ay 2021) . The levividuals . The motay 2021) .2.HuT 78 and Jurkat human T lymphocytes and RPMI 8226 human B lymphocytes were obtained from the Stem Cell Bank, Chinese Academy of Sciences. HuT 78 cells were cultured in IMDM  with 20% fetal bovine serum . Jurkat and RPMI 8226 cells were cultured in RPMI 1640 medium (Sigma-Aldrich) with 10% fetal bovine serum. The cells were incubated at 37 °C with 5% COThe siRNA oligonucleotides targeting PEBP1P3 (si-PEBP1P3 sense 5′-GUGAUCCUAAUACCAGAAUTT-3′ and si-PEBP1P3 antisense 5′-AUUCUGGUAUUAGGAUCACTT-3′) and negative control (si-NC sense5′-UUCUCCGAACGUGUCACGUTT-3′ and si-NC antisense 5′-ACGUGACACGUUCGGAGAATT-3′) were designed and synthesized by Genepharma . Synthetic RNAi was transfected into Jurkat, HuT 78 and RPMI 8226 cells, respectively, with Lipofectamine RNAiMAX Reagent (Invitrogen) for 48 h based on the manufacturer’s instructions.® reagent (Invitrogen) according to the manufacturer’s protocol. To detect the subcellular localization of the transcripts, the cytoplasmic and nuclear RNA fractions were prepared with a RNeasy Mini kit . RNA quality was examined by the ratio of OD260/OD280 and RNA electrophoresis, and then the RNAs were reverse-transcribed with a QuantiTect Reverse Transcription kit (QIAGEN) for genomic DNA removal and cDNA synthesis. The qPCR reactions were performed using SYBR Green qPCR SuperMix-UDG (Invitrogen) in an Applied Biosystems PRISM 7500 . All the qPCR primers used are listed in Total RNA of the cell lines was extracted with TRIzolThe genomic DNA of treated cells was prepared using a DNA extraction kit and subsequently treated with the DNA Bisulfite Conversion Kit (QIAGEN) according to the manufacturer’s instructions. For detecting DNA methylation of CD45 intragenic CpG sites, bisulfite sequencing PCR primers  were designed according to the bisulfite-treated DNA sequences. After amplification, PCR products were purified with a Universal DNA Purification Kit  and cloned into the pGM-T vector. The inserted PCR fragments from individual clones were sequenced to enable determination of the status of CpG sites.ChIP assays were performed with EZ-Magna ChIP A Kits  according to the manufacturer’s protocol. ChIP grade antibodies were used in the present study as follows: anti-histone H3 lysine 4 trimethyl , anti-histone H3 lysine 9 trimethyl , anti-histone H3 lysine 36 trimethyl , anti-H3acetyl , anti-histone H3 lysine 9 , anti-CTCF  and normal rabbit IgG. Immunoprecipitated DNA was analyzed by qPCR and normalized to the input DNA, and each experiment was repeated three times independently. The sequences of the primers in reference to the different regions of the CD45 gene are listed in Based on RNA-seq data from the GTEx from 51 specific tissues and two cell lines, the expression level of CD45 from high to low was whole blood > spleen > EBV-transformed lymphocytes > lung > small intestine > adipose > other types of tissues. Whole blood had the highest expression level, with a median expression of 969.96 CPM. For PEBP1P3, the expression level was testis > whole blood > spleen > lung > small intestine > EBV-transformed lymphocytes > other types of tissues A. The hiThe full-length CD45 RNA sequence recorded at GenBank was 5429 bp and composed of 33 exons, and PEBP1P3 RNA was 1452 bp and composed of two exons. Analysis of the RNA sequences with the online web server CPC predicted that the coding scores for CD45 and PEBP1P3 were 6.45 and −0.65, respectively, which indicated that the PEBP1P3 transcript was a noncoding RNA A. We appp < 0.05). Knockdown of PEBP1P3 also resulted in reduced RNA levels of the CD45RO isoform , increased CD45RA in Jurkat cells and increased CD45RB in RPMI 8226 cells  decreased in the RPMI 8226 cell line . For CTCF, the peaks at intron 2 and exon 5 increased after PEBP1P3 knockdown (p < 0.05)  D.CD45, we knocked down PEBP1P3 in cultured lymphocytes by RNAi. The RT-qPCR results showed that the knockdown of PEBP1P3 decreased the expression of CD45 in human cell lines of HuT 78 and RPMI 8226. The RNA levels of PEBP1P3 and CD45 in cultured lymphocytes HuT 78 and RPMI 8226 were consistent with their expression in human tissues based on the GTEx data. In addition to the effect on CD45 RNA, the RNAi results also indicated the regulatory effect of PEBP1P3 on the expression of CD45 alternative splicing isoforms, mainly to increase the expression of CD45 RB and decrease the expression of CD45 RO. Since CD45 isoforms indicate the development of lymphocytes and the activity of the immune system [Natural antisense transcripts are RNA sequences transcribed from opposite DNA strands at the same locus of the sense gene ,21, and CD45 DNA and contribute to the regulation of the transcriptional rate and the splicing of the alternative exons [The noncoding RNA PEBP1P3 had a relatively low expression level compared to CD45 and mainly existed in the nucleus, where the transcription and splicing of RNA occurs. A previous study indicated that DNA methylation and histone modification regulated the alternative splicing of weak exons in pre-mRNA at co-transcriptional level through controlling the RNA polymerase II transcription elongation rate. Methylated DNA sequences are correlated with fast transcriptional rate and skipping of the exons, and unmethylated DNA sequences are correlated with slow transcriptional rate and the enough time for spliceosomes to recognize each exon of the primary transcript ,14,25. Sve exons .CD45 after PEBP1P3 RNA was knocked down in the cell line of HuT 78, since HuT 78 cells manifested the best knockdown efficiency among the three PEBP1P3 RNAi cell lines as shown in CD45 DNA in the si-NC control or si-PEBP1P3 knockdown HuT 78 cells. H3K9me3 and H3K36me3 presented elevated levels at exon 4–5 of CD45. H3K36me3 is generally enriched at transcribed regions of genes, and it is believed to modulate exon definition by regulating the rate of RNA polymerase II elongation and alternative splicing [To explore the possible mechanism of PEBP1P3 in the splicing regulation of CD45, we detected the DNA methylation of splicing ,32,35. Hsplicing ,38. The CD45 DNA as in Among the weakly spliced exons 4, 5 and 6 of CD45, splicing out of exon 5 is the key step for the formation of CD45RO ,26,27, w


Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.  C. elegans and were observed to be pivotal for their development . Th. Th23]. Some of the shortcomings of the classical northern blotting technique have been resolved by modifications in the probe design, labeling, and detection aspects of the technique. For example, non-radioactive probes (such as those labeled with digoxigenin (DIG), biotin, or fluorescein) have been introduced as an alternative to radiolabeled probes used conventionally ,31,32. TUsing northern blots for small RNA molecules has further created challenges as well as opportunities for its improvement. One such improvement was introduced by Pall et al. , who modOther modifications have also been introduced for small RNA northerns. For example, Valoczi et al. have tested modified locked nucleic acid (LNA) probes to improve sensitivity and specificity towards mature miRNA detection by 10-folds. However, the usage of these modified probes is limited by their cost and commercial availability . Kim et With all the different variations introduced into the classical technique of northern blotting as well as liquid hybridization, in this study, we analyzed the different modifications and permutations introduced and used them to develop highly sensitive, much cheaper, and rapid liquid hybridization assays for not just one but three main categories of RNAs: the large class of mRNAs, the intermediate small RNAs, and the tiny miRNAs using easily available, non-radiolabeled, biotinylated probes. In parallel, we performed a systematic analysis of these liquid hybridization assays with northern blotting, to compare their relative levels of sensitivity, specificity, and utility for each category of RNAs. Throughout the manuscript, we highlight important experimental details which are critical to the success of both techniques as well as pinpoint their caveats, especially related to the use of exonucleases for excess probe digestion and detection of non-radiolabeled signals using commonly available biomolecular imagers. Finally, we compare our results with other noteworthy studies published in this area in the last few years to emphasize the enhanced sensitivity of our protocols and contributions made by our work.Overall, our results reveal that liquid hybridization is a much more sensitive, simpler, and faster technique than classical northern blotting for all three classes of RNAs and more amenable to larger sample size. Additionally, liquid hybridization was observed to be especially suited for the sensitive detection of miRNAs in the absence of specialized reagents compared to northern blotting. Evaluation of the sensitivity of our non-radiolabeled liquid hybridization assays with those published earlier revealed their enhanced sensitivity, making them comparable to radiolabeled assays. Thus, we present highly sensitive, rapid, and cost-effective liquid hybridization assays that do not need specialized probes like LNA or radioactivity for the detection of mRNAs, small RNAs, and miRNAs.Total cellular RNA was extracted from either the normal mouse mammary cell line, HC11 (RNA1 and RNA2) or 293T cells (RNA3 and RNA4) using 500 µL of TRIzol™ Reagent  according to the manufacturer’s instructions . The extAll primers used in this study were synthesized commercially  and their sequence is provided in The protocol of Wang et al. was used for liquid hybridization of mRNA, small RNA, and miRNA with a few critical modifications . BrieflyNon-denaturing, 1.2% agarose-MOPS (3-N-morpholinopropanesulfonic acid) gels with EtBr (0.05 µg/mL) were used for mRNA, while 12% TBE (Tris/Borate/EDTA)-acrylamide gels were used for small RNA and miRNA analyses. After polymerization, the gels were pre-run (running buffer: 1× MOPS for agarose and 1× TBE for acrylamide gel) for 30 min at 80 V and 150 V, respectively, followed by loading of RNA or hybridized samples mixed with 5× non-denatured RNA loading buffer. The agarose gels for mRNAs and acrylamide gels for small RNAs were run for 40–90 min at 80 V and 150 V, respectively, or until the bromophenol dye reached two-thirds of the gel.After the completion of gel electrophoresis, the acrylamide gels were stained with EtBr (0.05 µg/mL) for visualization using UVP Imager and/or Typhoon using EtBr filter. This was followed by pre-soaking the gels in either 1× MOPS (agarose gels) or 1× TBE buffer (acrylamide gels). RNA in the gels was transferred onto positively charged Hybond nylon membranes  pre-soaked in the same buffer used for equilibration. Electroblotting was performed at 80 mA constant current for 1 h for agarose gels and 18 mA for 1 h for acrylamide gels, respectively. After the completion of the transfer, membranes were placed on pre-soaked filter papers with either 1× MOPS or 1× TBE buffers and crosslinked using UV crosslinker at 700 µJ . This was followed by baking at 80 °C for two hours. After crosslinking and baking, membranes were wrapped and stored at −20 °C. This was followed by signal detection and imaging which will be discussed commonly for both northern blotting and liquid hybridization techniques in A 1.2% denaturing agarose-formaldehyde gel containing EtBr (0.05 µg/mL) was prepared for mRNAs, whereas 15% denaturing 7 M urea-acrylamide gels were prepared for small RNAs and miRNAs. Samples were prepared after a two-fold sequential dilution of both RNA samples, but starting from 25 μg RNA for northern blots, which is ten times the amount that was used in the liquid hybridization experiments. Thus, the RNA samples were diluted from 25 µg to 12.5 µg, 6.25 µg till 3.125 µg, respectively, and mixed with 2× denaturing RNA loading dye prior to loading. The running buffer used for agarose gels was 1× MOPS containing 0.1 M formaldehyde, while 1× TBE buffer was used for the 7M urea-acrylamide gels. Similar conditions for pre-run and run were followed for northern blotting, as described earlier, for liquid hybridization.After the completion of gel electrophoresis, the acrylamide gels were stained with EtBr (0.05 µg/mL) for visualization using UVP Imager and/or Typhoon using EtBr filter. This was followed by pre-soaking the gels in either 1× MOPS (agarose gels) or 1× TBE buffer (acrylamide gels). RNA in the gels was transferred onto positively charged Hybond nylon membranes  pre-soaked in the same buffer used for equilibration. Electroblotting was performed at 80 mA constant current for 1 h for agarose gels and 18 mA for 1 h for acrylamide gels, respectively. After the completion of the transfer, membranes were placed on pre-soaked filter papers with either 1× MOPS or 1× TBE buffers and first UV crosslinked at 700 µJ. This was followed by chemical crosslinking by EDC rather than baking, as described earlier . BrieflyAfter crosslinking, the membranes were prepared for hybridization with different probes. A modified Church Buffer was used as the hybridization buffer ,39. The The membranes from liquid hybridization samples were washed twice with high stringency buffer containing 0.1× SSC and 0.1% SDS for 5 min to remove residual buffers or contaminants prior to blocking, while the membranes from northern blotting were first washed twice in a low stringency buffer containing 2× SSC and 0.1% SDS for 15 min and then twice for 5 min with the high stringency buffer. After washing, membranes were blocked in a 25 mL blocking buffer containing 0.1% casein and 0.1% TBST for 60 min. Blocking was followed by incubation of the membranes with diluted horseradish peroxidase (HRP)-conjugated polystreptavidin protein  at room temperature in the blocking buffer. The HRP-conjugated streptavidin was used at a dilution of 1:300, 1:1000, or 1:3000 for miRNA, small RNA, and mRNA, respectively. After a 30-min incubation, the membranes were washed in the washing buffer (1× TBS/0.1% SDS) buffer for 30 min at room temperature and then either subjected to signal detection or stored at −20 °C.TM West Femto Maximum Sensitivity Substrate . The probe signal was detected by either exposing the membranes to an X-ray film in a darkroom or by using the biomolecular imager Typhoon FLA95000  by using the ECL plus filter or Sapphire . Exposure times varied between 5–30 s for the X-ray films and Typhoon biomolecular imager, depending upon the probe and kit used, and 2 min for the Sapphire biomolecular imager. The femto substrate was used only for experiments where the probe amounts were low due to purification after biotinylation on acrylamide gels.The HRP signals on the hybridized membranes were detected using the ECL chemiluminescent detection kit, as per manufacturer’s directions  or the SuperSignalAfter northern blotting, the resultant nylon membranes were stripped by washing two or more times in a pre-boiled 0.5% SDS solution for 10 min in a benchtop rocker. The membranes were then sealed in plastic pouches containing 0.5% SDS solution and incubated overnight at 60 °C in a shaking incubator. After overnight stripping, the membranes were washed once with 2× SSC solution and confirmed for lack of signal from the previous probe by exposing the membranes to an X-ray film for at least 45 min and re-stripped, if needed, or stored at −20 °C, or re-hybridized with a different probe. Membranes with no signal from the previous probe(s) were pre-hybridized with modified Church Buffer and the blotting procedure was repeated as stated above. The membranes prepared after liquid hybridization cannot be stripped. Hence, an individual hybridization process was conducted for every probe used.The sensitivity of the probes for liquid hybridized was tested using the slot blot technique. For slot blots, the Exo-I digested probes after liquid hybridization were mixed with pre-chilled 200 µL of denaturation solution (10 mM EDTA and 0.1 N NaOH). The membranes were placed into the slot blot (Bio-Rad) according to the manufacturer protocol and washed twice with chilled denaturation solution. Samples were transferred to the membranes by applying a vacuum followed by washing with denaturation solution. After washing, the membranes were removed from the slot blot and probes were crosslinked by UV crosslinker and 2-h baking at 80 °C. The crosslinked membranes were subjected to blocking, streptavidin-HRP incubation, and signal detection using the procedures described earlier.The intensity of signals produced after mRNA, small RNA, or miRNA probing was measured/quantified using GelQuant.Net 1.8.2 software. GraphPad Prism 7 was used to create the graphs.An ideal northern blot result primarily depends on the quality and integrity of the extracted RNA. Although multiple protocols have been described for RNA extraction, RNA extracted through TRIzol is still preferable for molecular biology studies, especially when it comes to the isolation of all classes of RNAs, from the larger ribosomal and mRNAs in the kilobase range, to the small RNAs in the 100 to 200 nucleotide (nt) range, to the miRNAs in the 20–25 nt range. Thus, in this study, RNA from the mouse mammary epithelial cell line HC11 was extracted through TRIzol that revealed the three distinct expected bands for 28S, 18S, and 5S RNAs . Our resNext, we studied whole cell RNA upon electrophoresis on 10% 7 M urea-acrylamide gels to determine the banding pattern obtained, with a focus on the smaller RNA species upon ethidium bromide staining which have been less well studied. As can be seen in Next, we compared the sensitivity of the liquid hybridization technique with northern blotting for the detection of mRNAs. For northern blotting, RNA was loaded as two-fold serial dilutions on 1.2% formaldehyde-agarose (FA) denaturing gels starting from 25 µg per well, along with ethidium bromide. In case of liquid hybridization, we started with 10-fold less RNA, starting from 2.5 µg, and diluted it two-folds to 1.25, 0.625, and 0.3125 µg that were subjected to in-solution hybridization. Less RNA was used for the liquid hybridization since it is thought to be more sensitive than northern blotting ,36,43,44As can be seen, upon liquid hybridization and Exo-I treatment, the RNA now appeared as a smear as opposed to the distinct ribosomal bands that were apparent earlier. After transfer and signal development, clear single bands of decreasing intensity could be observed for both the β-Actin and c-Myc d. Quanti6 copies of 5S RNA per cell compared to only 4 × 105 copies of U6 RNA [Next, we compared northern blotting with the liquid hybridization technique in detecting small RNAs using the same sequentially diluted RNA samples. The RNA samples were electrophoresed on 15% denaturing 7M urea-acrylamide gels to allow detection of smaller sizes of RNAs and tested by transfer to nylon membranes for classical northern analysis. The membranes were hybridized with a biotinylated oligonucleotide probe against the small RNA, 5S RNA , and thef U6 RNA .For liquid hybridization, the RNA samples were hybridized with either the 5S or U6 DNA oligonucleotide probes end-labeled with biotin at the 5′ end, digested with Exo-I, and the hybridized samples were electrophoresed on non-denaturing 12% TBE acrylamide gels, followed by detection. As observed with the ethidium bromide-stained gels for liquid hybridized samples, the RNA samples for the small RNAs also showed a diffused pattern of staining, starting from the wells up to about halfway down the gel b. Upon tLiquid hybridization for U6 small RNA after signal detection revealed three bands similar to the 5S blot. However, all three of these bands were quite strong: one band was specific for the U6 RNA (~100 nts long), one again ~50 nts long that we suspect represents primer/dimers, and the third one banding at ~20 nts, revealing the presence of unhybridized/undigested probe e. Both tNext, we asked if we could get rid of the primer/dimers that were being observed in the liquid hybridization technique to improve visualization and ensure the results were not compromised. Increasing the temperature of hybridization or decreasing primer concentrations did not help remove the primer/dimer smears in the liquid hybridization reactions. To determine whether the primers being used could have been the problem, the biotinylated single-stranded oligonucleotides were analyzed on 12% acrylamide non-denaturing gels followed by their transfer onto nylon membranes, incubation with HRP-linked streptavidin, and test of the HRP activity using luminol substrate . The cheBased on these results, we extracted the correct size oligos from the acrylamide gels and used only those as probes in our liquid hybridization analysis for 5S RNA described earlier. As can be seen in Next, we compared the sensitivity of liquid hybridization with northern blotting for miRNAs for which it was originally developed ,36. For The liquid hybridization for miR-18-5p was performed both in the presence or absence of Exo-I so that one could distinguish between the signal coming from the probe bound to the target miRNA versus signal from unhybridized probe alone that would have remained single due to the excess concentration used. As can be observed, the dilution effect of the RNA was apparent in the samples with Exo-I, but not in samples without Exo-I, an effect which could be quantitated c. TogethThese results raise the question that if liquid hybridization is so sensitive, why was it unable to detect the ~60 nt pre-miRNA band that could be effectively detected upon northern blotting a. This bIn order to confirm this point, the northern blot analysis was repeated for miR-18-5p after probe purification to determine whether the ~60 nt form could be detected using this technique. As suspected, we were unable to detect any band on the northern blot for miR-18-5p after probe purification (data not shown). This observation suggests that: (1) the 60 nt band being observed earlier in miR-18-5p northern was an artefact of the probe primer/dimer prior to its purification, and (2) that northern blot is unable to detect miRNA expression under the conditions used in this study for either the pri-, pre-, or mature forms of miRNAs, while liquid hybridization can detect the mature form.To confirm these observations, liquid hybridization was performed for the detection of three additional miRNAs, miR-381-3p, miR-10b-5p, and miR-10b-3p, where gel-purified biotinylated oligonucleotides were used as probes to determine if they could be successfully detected. As can be seen from Our assay was unable to detect the precursor forms of the tested miRNAs (the pri- and pre-forms) despite the high sensitivity of the liquid hybridization assay. This probably is due to the location of the probes used that encompasses the “seed” region of the miRNAs a. This rFinally, we asked whether liquid hybridization could be coupled with slot blotting for better quantitation purposes since only one species of mRNAs, small RNAs or miRNAs could be detected using this technique rather than their pre-forms. In this approach, the products of liquid hybridization were immobilized onto membrane and UV cross-linked rather than size fractionated, saving time and allowing better quantitation . Thus, iThe biotin-labeled product is visualized using the chemiluminescent detection kit. For detection, the membrane containing the biotinylated products are incubated with streptavidin protein conjugated with horseradish peroxidase (HRP). HRP provides enzymatic activity for detection using the substrate system like luminol (ECL). Upon enzymatic reaction, luminol results in chemiluminescence that can be captured either by the classical X-ray imaging or by the more modern imaging systems. In this study, we compared the signal detection sensitivity of both X-ray and the widely used imaging system, Typhoon (FLA9500). The Typhoon machine has prescribed wavelengths and filters for specific dyes that can selected. To detect HRP-luminol, the ECL wavelength was selected whereas, for ethidium brome, the EtBr wavelength was employed. In northern blotting, a stringent quality check is routinely performed before transfer to positively charged membranes. To check for RNA quality and integrity before transfer, agarose or acrylamide gels are stained with EtBr and viewed either by UVP gel imaging system or by Typhoon imager using the EtBr filter.Northern blot is considered a gold standard to determine RNA transcript form and expression semi-quantitatively. Despite this, several caveats make northern blotting unpopular amongst modern molecular biologists. Unlike other techniques like western or Southern blotting, northern analysis requires huge amounts of RNA that is labile and highly sensitive to degradation when compared to DNA or proteins. Northern blotting is also a time-consuming protocol and detection of small RNAs could take from three days up to a week or more, depending upon how many transcripts need to be analyzed. Moreover, northern shows the least sensitivity when compared to other techniques like microarrays, RT-PCR and ribonuclease protection assays ,24,25. OWith the discovery of miRNAs, liquid hybridization has been introduced as a possible alternative to classical northern blotting since miRNAs are difficult to detect due to their small size and low levels ,36,43. TIn this study, we developed non-radiolabeled, biotin-based-northern blots as well as highly sensitive liquid hybridization assays and systematically compared them for all three sizes of RNAs: mRNAs, small RNAs, and miRNAs. The liquid hybridization technique was taken several steps forward by not only using it to detect miRNAs via the biotin-streptavidin detection system, but extending it to the study of small RNAs and mRNAs, an aspect that has not been reported before to the best of our knowledge, making our study more versatile and broader than reported previously. Our work further highlights several technical aspects of the technique that have not been addressed before, including: (1) introduction of a pan-hybridization step that allows probes with different melting temperatures to be hybridized using one program, (2) use of poly-HRP to enhance signal, (3) demonstration of artefacts of probe biotinylation that necessitate their gel purification, (4) test of RNA samples in the presence and absence of Exo-I to confirm target specificity and allow study of RNA biogenesis, (5) use of EDC to enhance cross-linking for northern blotting, and (6) uncovering caveats of modern image analyzers for the detection of luminol signals in the presence of ethidium bromide, etc. Thus, the incorporation of these technical aspects helped us develop more sensitive, efficient, cheaper, and faster liquid hybridization assays (as well as northern blots) for RNA detection.For example, incorporation of improvised hybridization conditions for liquid hybridization assays based on published studies ,43,44,50Compared to northern blotting, we found liquid hybridization technique to have a much shorter procedure that could be completed in essentially a day versus 3–5 days or more for northern blotting, especially in cases of re-use of blots for multiple transcripts. In this study, we used overnight Exo-I digestions to ensure complete digestion, a step that can easily be reduced to 30 min by increasing enzyme concentrations (data not shown). We opted for low exonuclease concentrations for the overnight time periods to reduce confounding factors (discussed below) as well as ease of work since the experiment could be set up in the evening and easily completed the next day. We also observed liquid hybridization to be 10—100 folds more sensitive than the classical northern blotting when using biotinylated probes for the detection of mRNAs, small RNAs, and miRNAs . FinallyThe As mentioned above, for mRNA detection, liquid hybridization showed excellent sensitivity and was able to detect signals in 10 times less RNA when compared to the classical northern . Unlike Among the three classes of RNAs tested, liquid hybridization was most efficient at sensitively detecting miRNA expression. In fact, liquid hybridization was able to detect mature form of miRNAs undetectable even using 25 times higher amounts of total RNA in the classical northern blotting technique . This loWe were also able to detect the same sensitivity of liquid hybridized signals after transferring the liquid hybridized-Exo-I-digested small RNA or miRNA samples directly on to the positively charged nylon membranes using slot-blot . This meOne important observation in our study was that primer probes that are biotinylated must be gel-purified prior to their use in liquid hybridization assays if multiple bands are observed other than the expected one upon their gel electrophoresis . This obAnother important observation in our study was the detection of false-positive signals after using machine-based detection of the HRP-catalyzed chemiluminescent signal using an ECL detection kit. After hybridization, mRNA, small RNA, or miRNA expression can be detected using the biotin-streptavidin-HRP conjugation method. X-ray has been traditionally used for the detection of HRP-luminol . HRP oxiIn summary, in this study, we developed and compared non-radiolabeled, sensitive, rapid, and cost-effective northern blots and liquid hybridization assays for mRNAs, small RNAs, and miRNAs that require minimum specialized reagents. Overall, we observed the sensitivity of our biotin-based northerns comparable to traditional northerns for mRNAs and small RNAs, while our liquid hybridization assays were even better and demonstrated sensitivity (>10–100 folds) for the detection of all three classes of RNAs. Unlike northern blotting, liquid hybridization was able to efficiently detect mature miRNAs using biotin-labeled probes and total RNA. Moreover, the traditional X-ray film method to detect HRP-luminol showed superiority over advanced laser machine-based technology. Overall, our study provides an effective experimental framework for investigators researching different RNA molecules and their functions at the molecular level in an economical and efficient manner.


Alternative polyadenylation (APA) is a widespread and conserved regulatory mechanism that generates diverse 3′ ends on mRNA. APA patterns are often tissue specific and play an important role in cellular processes such as cell proliferation, differentiation, and response to stress. Many APA sites are found in 3′ UTRs, generating mRNA isoforms with different 3′ UTR contents. These alternate 3′ UTR isoforms can change how the transcript is regulated, affecting its stability and translation. Since the subcellular localization of a transcript is often regulated by 3′ UTR sequences, this implies that APA can also change transcript location. However, this connection between APA and RNA localization has only recently been explored. In this review, we discuss the role of APA in mRNA localization across distinct subcellular compartments. We also discuss current challenges and future advancements that will aid our understanding of how APA affects RNA localization and molecular mechanisms that drive these processes. For many transcripts, there are alternative cleavage and polyadenylation sites (PAS) that can generate multiple mRNA isoforms with different 3′ UTRs. This phenomenon, termed alternative polyadenylation (APA), generates distinct 3′ termini on mRNAs and thus allows for a single gene to create multiple transcripts, each with different 3′ UTR contents. These alternative 3′ UTR isoforms allow for inclusion or exclusion of APA isoforms can generally be classified into two different forms. The first form, which we will define as “tandem UTRs”, occurs when multiple APA sites are present within the same terminal exon . In thisThe core 3′-end processing machinery is comprised of four main complexes: cleavage and polyadenylation specificity factor (CPSF), cleavage factor I (CFIm), cleavage factor II (CFIIm), and cleavage stimulation factor (CstF) . The polDrosophila neurons by suppressing the use of proximal PAS  that bind with RNA to form ribonucleoprotein complexes (mRNPs) and mediate specific transport  and neuropil (axon- and dendrite-enriched) layers from the rat hippocampus showed that localized transcripts contained strong preferences in APA site usage compared to non-localized transcripts. For example, dendrite-localized lization .Cdc42, a small GTPase that controls the actin cytoskeleton and can influence cellular morphology. In the brain, Cdc42 is responsible for regulating axon and dendrite outgrowth, dendrite arborization, and spinal development. APA of Cdc42 RNA yields two isoforms with different last exons whose encoded proteins have distinct functions and localization in neurons. The proximal ALE isoform encodes CDC42E6, which localizes to the dendritic spines and is involved in their formation. On the other hand, the distal ALE isoform encodes CDC42E7, which has a role in axonogenesis , and membrane-associated, where components of the endoplasmic reticulum (ER), mitochondria and plasma membrane are found. RNA is isolated from each fraction, allowing for identification of localized transcripts . FurtherGeneral trends of RNA localization to membranes and 3′ end choice have been revealed by LABRAT, an APA quantification software . LABRAT Because the ER comprises a large fraction of cellular membrane, it is assumed that most RNAs in the membrane-associated fraction originate from the ER. Surprisingly, RNAs localized to the membrane fraction by more distal APA usage are depleted for known peptide-encoded ER localization signal sequences . This suIn order to increase the resolution and specificity to isolate ER-associated transcripts, polysome profiling was performed on the membrane associated fraction isolated from myoblasts. These translating RNAs are enriched in the ribosome coated rough ER where substantial protein synthesis occurs. ER-localized RNAs identified through this method were also found to be longer and have more distal APA usage  Cheng e. FurtherCo-translationally ER localized RNAs are enriched for known ER localization signal sequences that create a nascent peptide signal recognized and localized by the signal recognition particle complex. While these translation-dependent ER localized RNAs tend to have longer 3′ ends and use more distal APA, this effect is accentuated when translation is inhibited. These results bolster the notion that two distinct mechanisms of RNA localization to the ER exist: co-translational localization, and translation-independent, 3′ end mediated localization. However, further experimentation with more precise techniques such as proximity labeling assays, like APEX-seq  or HaLo-RBP binding to alternative 3′ UTRs is one of many mechanisms that can regulate RNA localization at the 3′ end isoform level . When thCD47 have the same RNA localization and encode identical proteins, the resulting protein products are differentially localized to the ER and plasma membrane respectively. This localization mechanism, termed 3′ UTR-dependent protein localization, is mediated by a transfer of proteins bound to the 3′ UTR during translation to the nascent peptide determining both the protein’s distinct localization and cellular function.In some cases, the localization of a protein depends on the APA isoform from which it was produced in an RNA localization-independent manner. The protein localization of CD47 was found to be dependent on the 3′ end choice of its RNA transcript . It was CD47 contains multiple AU-rich elements and binds to TIS11B to form membraneless organelles called TIS granules. These granules are intertwined with the ER creating a subcellular compartment called the TIGER domain that enables formation of specific protein-protein interactions that are dependent on 3′ UTRs of mRNAs . These structures are particularly abundant in the introns and 3′ UTRs of transcripts in the human genome  to the outer mitochondrial membrane (OMM) . Loss ofthaliana . Additiocis-elements called zipcodes, most often present in its 3′ UTR. Given this crucial role of 3′ UTR in mRNA transport, the formation of alternate 3′ UTR isoforms can serve as an important mechanism to create diverse subcellular RNA localization patterns and ultimately, contribute to cellular physiology. However, we still lack a detailed understanding of how the majority of the RNAs are transported to their destination. For instance, while thousands of localized transcript have been identified in projections of neuronal cells, we only know handful of examples where the corresponding cis-elements and trans-factors that govern its transport are known. This lack of known zipcodes hinders our ability to unambiguously define many cases where one 3′ UTR isoform contains a zipcode while another doesn’t.RNA localization as a post-transcriptional regulatory process is highly prevalent across species. mRNA localization is mediated by While we have an extensive knowledge of core processing factors that regulate APA, the transcript-specific RBPs that regulate differential localization of APA isoforms remain to be identified. The advent of transcriptome-wide RBP/RNA interaction identification methods such as RIP-seq, CLIP-seq, HITS-CLIP, and eCLIP has enabled high-resolution mapping of RBP-RNA binding interactions . Howeverin-situ hybridization (FISH)  , single-n (FISH)  and genen (FISH) . HoweverOne approach toward bridging these knowledge gaps is the use of large scale screens. For example, one such RNAi screen surveyed 489 RBPs to identify regulators of PAS usage . SimilarIn the future, implementing long-read sequencing methods to directly sequence poly(A) RNAs without fragmentation offer higher accuracy and sensitivity to detect and quantify APA isoforms . Additio


Comparative RNA interactome capture identifies potential regulators of RNA metabolism in fission yeast and reveals RNA exosome–dependent buffering of stress-responsive gene expression networks. The nuclear RNA exosome plays a key role in controlling the levels of multiple protein-coding and non-coding RNAs. Recruitment of the exosome to specific RNA substrates is mediated by RNA-binding co-factors. The transient interaction between co-factors and the exosome as well as the rapid decay of RNA substrates make identification of exosome co-factors challenging. Here, we use comparative poly(A)+ RNA interactome capture in fission yeast expressing three different mutants of the exosome to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identify multiple RNA-binding proteins whose association with RNA is altered in exosome mutants, including the zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc-finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight the importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress. Regulation of RNA maturation and degradation is crucial to accurate gene expression . The nucThe nuclear RNA exosome is a 3′–5′ exonuclease complex that consists of a nine-protein core (EXO-9) and two catalytic subunits, Rrp6 (EXOSC10) and Dis3/Rrp44 (hDIS3). EXO-9 forms a double-layered barrel-like structure that comprises six ribonuclease (RNase) PH-like proteins  and three S1/K homology (KH) “cap” proteins  . The twoSchizosaccharomyces pombe (S. pombe), Mtr4 shares its function with the highly homologous Mtr4-like helicase Mtl1 . Our data suggest that the nuclear exosome plays a more prominent role in controlling the fission yeast transcriptome than previously anticipated. In addition, analysis of the impact of mutations in different exosome subunits on the poly(A)+-bound proteome has identified proteins with increased RNA binding that could function as potential regulators of the exosome in fission yeast. We selected 10 RBPs with significantly altered RNA association and demonstrate that deletion of each of these proteins phenocopies at least one of various phenotypes typical of inactivated nuclear exosome, namely, suppression of transposon RNAs, telomeric silencing, and nuclear RNA retention. We focus on the uncharacterised zf-MYND -type zinc-finger protein Mub1, which is highly enriched on poly(A)+ RNA in the exosome mutants. Mub1 physically interacts with the exosome and its deletion leads to increased levels of a specific subset of exosome substrates, supporting its role in exosome regulation.In addition to the RNAs recognised by Mmi1, levels of multiple other transcripts increase in nuclear exosome mutants, suggesting that Mmi1-independent mechanisms contribute to their recognition . We therrrp6Δ, lacking exonuclease Rrp6; dis3-54, a Dis3 mutant containing an amino acid substitution (Pro509 to Leu509) within the RNB domain, which reduces its catalytic activity + RNA . Specifiactivity ; and mtlh domain  Fig 1A)rrp6Δ, lah domain . To gainh domain . The thrWhole cell extract normalisation data of comparative RNA interactome capture MS data.Table S1 List of proteins with > 2-fold changes (up/down) in enrichment in the mutant poly(A)+ RNA interactomes relative to the WT.Table S2 rrp6Δ and mtl1-1, fewer RNAs in total increased in the dis3-54 mutant , possibly reflecting that Dis3 function is only partially inhibited under the condition tested. Approximately half of the transcripts with increased levels in the dis3 mutant (54%) were also dependent on Mtl1, suggesting that these correspond to nuclear targets of the exosome. In agreement with previous reports, transcripts with increased levels in the mutants included many mRNAs , confirming that the nuclear exosome regulates mRNA levels in addition to its well-described role in degradation of ncRNAs , in agreement with the suggested functional connection between Mtl1 and Rrp6 , levels of multiple nc transcripts were increased in all three exosome mutants  .RNAs increased in the oligo-dT pull-downs of exosome mutants (RNA interactome capture samples) , resulting in a quantitative data set for a total of 1,146 RBPs. 152, 180, and 83 RBPs were ≥2-fold enriched in mtl1-1, rrp6Δ, and dis3-54 over WT, respectively . Consistent with the RNA-seq data, the overlap between RBPs with increased RNA association was substantially larger for rrp6Δ and mtl1-1 than for dis3-54 and mtl1-1, further supporting a functional link between Mtl1 and Rrp6  and 31 of 86 RBPs that are underrepresented in dis3-54  are annotated with “cytoplasmic translation.” Altogether, RIC analyses of exosome mutants demonstrate significant changes in protein–RNA interactions in exosome-deficient cells, highlighting the important role of the exosome in nuclear RNP metabolism.To assess how inactivation of the different subunits of the exosome impacts the RNA-protein interaction profile, we next performed comparative analysis of RBPs differentially enriched in the interactomes of and Rrp6 . ConversGene ontology analysis of proteins enriched/depleted more than 2-fold in RNA interactome capture.Table S4 rrp6 mutants (The strong increase in the association of poly(A)+ RNA with RBPs in the nucleus combined with the lower association with proteins involved in cytoplasmic translation may reflect a change in subcellular localisation of poly(A)+ RNA in these strains. Indeed, it was previously reported that poly(A)+ RNA is retained in the nucleus in  mutants .rrp6Δ + RNA foci are detected inside the nuclei of mtl1-1 cells. Last, rrp6Δ cells show a mixed phenotype with foci present in addition to a strong diffuse nuclear poly(A)+ FISH signal. We had previously reported that RNA association with the exosome complex is increased in dis3-54 (reflecting its inability to efficiently degrade RNA substrates), whereas RNA association with the exosome complex is strongly reduced in mtl11, suggesting a disruption of substrate recruitment as well as decay + RNA in the nucleus, we performed FISH with oligo(dT) probes. Poly(A)+ RNA accumulated inside the nucleus in all three mutants . Quantifrrp6Δ : In dis3as decay . This blas decay .FISH quantification data relative to Fig 3B.Table S5 mtl1-1 and rrp6Δ RIC, respectively, compared with WT cells ; in dis3-54, the change was not significant  . Similarly, RNA association of the nuclear cap-binding complex Cbc1, Cbc2 and Pir2 increased (>4-fold) in all three mutants (Table S1). Together, this illustrates the capacity of comparative RIC to discover RBPs with bona fide roles in exosome targeting to substrate RNAs.To validate our hypothesis that exosome co-factors are indeed differentially enriched in the comparative interactome capture experiment, we first analysed how RNA association of Mmi1 is affected in the exosome mutants. As expected, the association of Mmi1 with poly(A)+ RNA increased two and fourfold in = 0.768) . The inc dis3-54 . MoreoveSPCC126.11cΔ (ratio of poly(A)+ signal ∼1.5). In all cases, the pattern of RNA accumulation in the nucleus resembled the diffuse staining of rrp6Δ and dis3-54 rather than the pronounced punctate staining characteristic of the mtl1-1 mutant , a compound that is toxic when a functional version of the ura4 gene is expressed. The deletion of either SPCC126.11c, srp40, or SPAC222.18 led to moderate growth on −URA plates and attenuated growth on 5-FOA compared with WT . Tf2-1 is up-regulated in rrp6Δ but independent of Mmi1 . To assess the contribution of Mub1 to regulation of RNA levels, we compared the transcriptomes of WT and mub1Δ cells by RNA-seq normalised to S. cerevisiae spike-in . The levels of 248 transcripts (162 mRNAs and 86 ncRNAs) increased more than 1.5-fold  in mub1Δ cells . 144 tr [mtl1-1 , in suppeLi tool ), potentanalyses . No addi pathway .mub1Δ.List of up-regulated genes (>1.5-fold) in Table S7 mtl1-1List of up-regulated genes (>1.5-fold) in Table S8 mub1 deletion + RNA interactome suggested that Mub1 is an RBP. We hypothesised that the zf-MYND domain (471-528) could mediate an interaction with RNA. Multiple other proteins that associate with the exosome contain zinc-finger domains, for example Red1, Red5, and Air1, a component of the TRAMP complex . For Reddeletion  . To test whether the dysregulation of stress-responsive transcripts extended to stress conditions, we performed Northern blot for hsp16 mRNA, which encodes a heat shock protein . We observed no significant change in Pol II occupancy in mub1Δ and mub1-ΔZ-3xFLAG compared with WT cells  with inhibitors  using a bead beater (Cryoprep). After addition of 275 μl ice-cold lysis buffer with protease inhibitors, the extracts were clarified by two successive rounds of centrifugation. Samples were incubated for 1 h at 4°C with anti-MYC magnetic beads . Beads were washed five times with lysis buffer without inhibitors. Proteins were eluted with hot Laemmli buffer at 95°C for 5 min. For reverse co-immunoprecipitation, lysis was performed in ice-cold buffer  with inhibitors . Samples for each strain were distributed to two tubes. One was treated with 2.5 μg of RNase A  for 20 min on ice. Samples were immunoprecipitated for 1 h at 4°C with anti-FLAG antibody  coupled to μMACS protein G .To prepare WCEs, cells were collected, rinsed in ice-cold PBS with1 mM PMSF, and frozen on dry ice. Lysis was performed in 125 μl ice-cold lysis buffer  using PCR-generated DNA templates produced from genomic DNA isolated from a wild type S. pombe strain (YP71) with the oligonucleotides listed in Table S11. Probes were added to the membrane and hybridised at 42°C overnight. After repeated washes in 2× SSC, 0.1% SDS, blots were exposed to Amersham Hyperfilm MP .Northern blot experiments were essentially performed as described . RNA wasS. pombe poly(A)+ RIC was performed as described (mtl1-1 = First eXperiment (FX); WT2 + rrp6Δ + dis3-54 = Second eXperiment (SX)) were performed. S. pombe cells were grown at 30°C in Edinburgh minimal media supplemented with glutamic acid (EMMG) with limited amounts of uracil (10 mg/l) and labelled with 4-thiouracil (1 mg/l) for 4 h 30 min. Cells were harvested by filtration, snap-frozen in liquid nitrogen after UV-crosslinking at 3 J/cm2 in 50 ml PBS and lysed by grinding in liquid nitrogen. The grindate was resuspended in oligo-d(T) lysis buffer  1:10,000; and 5 mM DTT) and cleared by centrifugation. 1/48th of the total volume of WCE was used for proteomics analysis, the rest was subjected to pull-down with oligo-d(T)x25 magnetics beads (NEB-S1419S), 1 ml of slurry per 1 liter of cell culture during 1 h at 4°C. Immobilised oligo-d(T)x25 magnetics beads were washed two times with oligo-d(T) wash buffer 1  at 4°C, two times with oligo-d(T) wash buffer 2  at room temperature and two times with oligo-d(T) low salt buffer  at room temperature. RNA–protein complexes were eluted from beads with oligo-d(T) elution buffer , 330 μl/l of culture for 10 min at 55°C. 1/33 of the oligo-d(T) pull-down total volume was used for RNA sequencing analysis. The rest was subjected to RNase-A and RNase T1 treatment and subjected to mass spectrometry analysis as described in escribed , 2020b. 2 transformed, normalised to the same median, and analysis was followed by the imputation of missing values using a minimal value approach (MinDet–where each sample is considered independently). Data manipulations, principal component analysis, and Pearson correlation plots were performed with the DEP package implemented in R . To test the changes between whole-cell extract-normalised  proteomes of mutants and WT cells, we used modified scripts from the DEP package. Briefly, this software takes advantage of the Limma package that calculates moderated t-statistics on a linear model fit to the expression data  > 1 were considered to be specifically enriched in exosomes mutants. All other analyses were performed with custom scripts or ones modified from the DEP package. S. pombe GO term annotations and information on individual proteins were retrieved from PomBase , immediately resuspended in 1 ml of spheroplast buffer  with 1% 100T zymolyase  and incubated for 60 min to digest the cell wall. The reaction was stopped by washing with 1 ml of cold buffer B. Cells were incubated for 20 min in 0.01% Triton X-100/1X PBS and washed with 10% formamide/2× SSC at room temperature. Before hybridization, 50 ng of the oligo-d(T) probe was mixed with 2 μl of a 1:1 mixture between yeast transfer RNA  and salmon-sperm DNA  and the mixture was dried in a vacuum concentrator. Hybridization buffer F  was added, and the probe/buffer F solution incubated for 3 min at 95°C. Buffer H  was added in a 1:1 ratio to the probe/buffer F solution. Cells were resuspended in the mixture and incubated overnight at 37°C. After three washes , cells were resuspended in 1× PBS/DAPI and mounted on glass slides for imaging. Z-planes spaced by 0.2 μm were acquired on an Ultraview spinning-disc confocal with an Olympus UPlanSAPO 100× objective. Acquisition was carried out with DAPI (405 nm) and FITC  filters.Poly(A)+ RNA FISH was carried out as described , using at test) was carried out with GraphPad Software. Raw data for each sample and the data used to generate the graph in Images were analysed using ImageJ software . BrieflyS. cerevisiae cells were added to S. pombe at a 1:10 ratio before RNA isolation. Total RNA was extracted from cultures in mid-log phase using a standard hot phenol method and treated with RNase-free DNase RQ1  to remove DNA. For total RNA sequencing, experiments were performed in duplicates. Ribodepletion was carried out with the ribo-minus transcriptome isolation kit . Poly(A)+ RNA sequencing was performed by using 1/33 of the oligo-d(T) pull-down total volume, subjected to proteinase K treatment for 1 h at 50°C. Poly(A)+ RNA was recovered by a standard hot phenol method. Experiments were performed in triplicate. cDNA libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina  for 50 ng of total RNA and using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina  for 100 ng of WT1, mtl1-1, rrp6Δ, and dis3-54-purified oligo-d(T) RNA. Paired-end sequencing was carried out on the Illumina HiSeq 500 platform.For spike-in normalisation, S. pombe genome (ASM294v2.19) using Bowtie 2 (TopHat) , a web-based tool, was used . Reads were aligned to the (TopHat) . For spiSAMTools  and readSAMTools  in R andSAMTools  in R waswas used .fbp1).Chromatin immunoprecipitation (ChIP) assays were performed as described . ImmunopGSE148799 and GSE149187. Mass spectrometry data are available via ProteomeXchange with the identifier PXD016741.Raw (fastq) and processed (bedgraph) sequencing data can be downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus with the GEO numbers