U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.The DEAD-box protein UAP56 ( Nuclear pre-mRNA is extensively processed before it is exported to the cytosol for translation and splicing represents a central step in the mRNA maturation process. Assembly of the splicing machinery is mediated by a range of splicing factors including RNA helicases of the DEAD-box protein family, which belong to the superfamily 2 (SF2) of helicases U2AF65-associated protein prompting its name UAP56. U2AF65 recruits UAP56 to the mRNA, where it is required for stable interaction between U2 snRNP and the pre-mRNA branch point transcription-export) complex and mutations in its subunits cause nuclear mRNA accumulation In mammals, the 56-kDa DEAD-box RNA helicase UAP56 is an essential splicing factor that originally was identified as Arabidopsis genome, for instance, encodes a total of 113 helicases and 58 of them are putative members of the DEAD-box family of RNA helicases Arabidopsis and BrachypodiumArabidopsis AtDRH1 Arabidopsis UAP56, which is encoded by two genes. The Arabidopsis protein has RNA-stimulated ATPase activity and ATP-dependent RNA unwinding activity. Moreover, it binds both RNA and dsDNA, and it interacts with the mRNA export factors ALY2 and MOS11.In plants, RNA helicase gene families are larger and more diversified than in other systems. The Arabidopsis thaliana (Col-0) and the T-DNA insertion lines from the SALK collection http://arabidopsis.info/) were used. After sowing seeds were stratified (48 h at 4°C) and plants were grown in soil in a phytochamber at 22°C and 16 h photoperiods (LD) at ∼7,000 lux Agrobacterium-mediated transformation as previously described Taq DNA polymerase (Peqlab) and primers specific for DNA insertions and the target genes .The presence of T-DNA insertions was analysed by PCR-based genotyping Pfu DNA polymerase using an Arabidopsis thaliana cDNA library as template and the primers  listed in E. coli, the CDS was inserted into plasmid pQE9 (Qiagen) providing an N-terminal 6×His-tag and into plasmid pGEX-5X-1 (Amersham) providing an N-terminal glutathione S transferase (GST) fusion. For overexpression in plants, the CDS was inserted under control of the CaMV 35 S promoter into pGreenII0229myc providing an N-terminal myc-tag. The CDS of ALY2 was inserted into pBluescript (Stratagene) and the CDS of MOS11 into pQE9. All plasmid constructions were checked by DNA sequencing, and details of the plasmids generated in this work are summarized in The coding sequence (CDS) of UAP56 was amplified by PCR with E. coli M15 cells were transformed with the pQE9-UAP56 and pQE9-MOS11 expression vectors and the recombinant 6xHis-tagged UAP56 and MOS11 were purified by metal-chelate column chromatography using Ni-NTA agarose (Qiagen) from E. coli lysates essentially as described previously E. coli using the pGEX-5X-1and pGEX-5X-1-UAP56 vectors, respectively, and purified by glutathione-sepharose affinity chromatography as previously described E. coli, it was synthesized as a 35S-methione-labelled protein using plasmid pBluescript-ALY2 and a coupled in vitro transcription/translation system .Arabidopsis 6xHis-UAP56 was produced by commercial immunisation and tested as previously described An antiserum against purified recombinant To detect the localisation of total mRNA, we used an Alexa-488 end-labeled 48-mer oligo d(T) probe (MWG) as previously described Protoplasts of dark-grown tobacco BY-2 cells were transiently transformed using PEG-mediated transformation and analysed for the localisation of GFP fusion proteins by confocal laser scanning microscopy (CLSM) as previously described d corresponds to the protein concentration resulting in binding of 50% of the oligonucleotides. In addition, protein interactions with radio-labelled RNA and DNA oligonucleotides were examined by EMSAs as previously described 32P-labelled RNA or DNA probe  and different amounts of synthetic RNA oligonucleotides (MWG). The reaction in a final volume of 20 µl was allowed to proceed at 25°C for 30 min and was stopped by adding EDTA to a final concentration of 20 mM. ATP was separated from ADP by thin layer chromatography (TLC). Therefore 5 µl of the reaction mix was spotted onto polyethyleneimin cellulose TLC plates (Merck), which was developed in a buffer containing . The plate was dried and analysed by phosphorimaging.ATPase assays were essentially performed as previously described 32P]ATP  using T4 polynucleotide kinase (Fermentas). The reaction mix was boiled for 5 min to inactivate the kinase. The complementary unlabeled strand was added in a 1∶1.2 molar ratio (labelled to unlabelled). For annealing, the mixture was heated to 100°C for 5 min and then slowly cooled to 25°C. The labelled dsRNA was then purified by using G25 columns . The helicase assay was started by addition of 6xHis-UAP56 to the reaction mixture containing  and 3 mM ATP (unless stated otherwise) and 2.5 nM dsRNA. After incubation at 28 °C for 1 h, the reaction was stopped by adding 5 µl stop solution . After Proteinase K treatment, the unwound ssRNA was separated from the dsRNA substrate by electrophoresis on 16% polyacrylamide gels in 1xTBE. The dried gels were analysed by phosphorimaging.RNA helicase/unwinding assays were essentially performed as previously described Yeast two-hybrid assays were essentially performed according to the manufacturer (Clontech). In brief, yeast cells of the strain AH109 were cotransformed with pGBKT7 and pGADT7 plasmids and grown at 30 °C on SD/-Leu/-Trp. For interaction assays, cells were grown on SD/-Leu/-Trp/-His medium for 2 d. For β-galactosidase assays cells were grown on the same medium and enzyme activity was detected after incubating the filters of colony lifts in a solution containing X-gal. As positive and negative controls served the interactions between p53 and the SV40 large T-antigen, and between lamin and the SV40 large T-antigen, respectively, provided by the manufacturer. Pull-down assays with GST and GST-UAP56 were performed as previously described Arabidopsis database (http://www.arabidopsis.org/) with the BLASTP program using these two amino acid sequences as query. The search resulted in two prominent hits. Despite some variation of the adjacent loci  in the nucleotide sequences of the transcribed regions, the two closely related genes code for an identical UAP56 amino acid sequence of 427 residues . Aligning the Arabidopsis UAP56 sequence with the amino acid sequences of UAP56 proteins of other organisms revealed that the characteristic helicase motifs Arabidopsis UAP56 shares 89%, 70% and 61% amino acid sequence identity with rice UAP56, human UAP56 and yeast SUB2, respectively. Several plant genomes, for instance, of the monocot plant Oryza sativa and the dicot plant Populus trichocarpa have two genes encoding almost identical UAP56 proteins with ∼99% amino acid sequence identity. The relationship of (putative) UAP56 sequences of various plants and of other organisms is illustrated by a sequence similarity tree . The chromosomal HMGB protein encoded by the At2g34450 locus that localises to the cell nucleus h nuclei , confirmh nuclei  revealedh nuclei , and theh nuclei  indicateh nuclei .Arabidopsis UAP56 with nucleic acids in solution, we used microscale thermophoresis , while ssDNA was not bound. As shown before by MST 32P-labelled 25-nt long oligonucleotides, which differed in sequence from the 29-nt oligonucleotides used for the MST analyses. As seen with MST, UAP56 bound to the ssRNA, dsRNA and dsDNA probes, and not to the ssDNA, while Df31 interacted exclusively with ssRNA  the selected genes are expressed in two week-old plants used for the ChIP experiment, which was confirmed by rtPCR (not shown). In addition to these transcribed genes, the association with the non-transcribed retrotransposons Ta2 and Ta3 Arabidopsis UAP56 associates with sites of RNAPII transcription independent from mRNA splicing.We used chromatin immunoprecipitation (ChIP) to examine the association of UAP56 with RNAPII-transcribed loci in l sample . Using tl sample . Accordiin vitro for its catalytic activities. To study its ATP-hydrolysis activity, [α-32P]ATP (mixed with unlabelled ATP) was incubated with UAP56 or the chromosomal high mobility group HMGB2 protein, which served as a negative control. Afterwards ATP and ADP were separated by TLC and detected by phosphorimaging. In the absence of protein or in the presence of HMGB2 ATP hydrolysis was hardly detectable, whereas addition of UAP56 resulted in ATP hydrolysis, as evident from the clearly visible ADP spot  in the presence of ATP, no unwinding was observed  was found to interact with the nucleolar protein fibrillarin Arabidosis nucleoli In different organisms the RNA helicase UAP56 is a critical factor for pre-mRNA splicing and mRNA export from the cell nucleus. We have identified two Arabidopsis UAP56  were reported to interact with both RNA and DNA Drosophila Vasa helicase forms several specific interactions with 2′-OH groups of ssRNA The interaction of UAP56 with nucleic acids was studied using EMSA and MST analyses. Both approaches revealed that UAP56 can interact with ssRNA and dsRNA, but also with dsDNA, while ssDNA was not recognised. The interaction with dsDNA was unexpected, since DEAD-box helicases typically are specific for RNA (and RNA-DNA duplexes) Arabidopsis nuclei, in ChIP experiments the protein was found to associate with loci transcribed by RNAPII, but not with non-transcribed regions. UAP56 was detected at both intronless genes as well as intron-containing genes. This is in agreement with studies in other organisms, where UAP56 can be recruited in a splicing-dependent manner, but also independent from mRNA splicing In line with the immunofluorescence experiments, revealing that UAP56 primarily localises to the euchromatin of Arabidopsis enzyme. It displayed basal ATPase activity that is stimulated by the addition of RNA, and ssRNA was more efficient than dsRNA in promoting ATP hydrolysis, which was also observed with human UAP56 Arabidopsis UAP56 was analysed in unwinding assays, revealing that the unwinding of RNA duplexes is strictly ATP-dependent. Moreover, shorter RNA duplexes were unwound more efficiently than longer (more stable) RNA duplexes and RNA substrates with 5′or 3′overhangs were somewhat less efficiently unwound than a comparable blunt-ended substrate. With human UAP56 blunt-ended substrates and those with (longer) overhangs were unwound with similar efficiency, while the RNA length-dependence of the reaction was also observed Arabidopsis UAP56 approximately threefold higher protein concentrations were required than with the human enzyme. Most likely this difference is due to the fact that our helicase assays were performed at 28°C, while the assays with human UAP56 were performed at 37°C. Both increased enzymatic activity and lower RNA duplex stability at higher temperature may contribute the different efficiencies. Despite the fact that DEAD-box proteins are bona fide helicases, they unwind RNA rather inefficiently when helix length rises above 10–15 bp, but helical elements within structured RNA substrates are rarely longer than 10 bp Arabidopsis UAP56 in addition to RNA can bind dsDNA, but not ssDNA. Therefore, we tested whether dsDNA stimulates the ATPase activity of UAP56, and we observed a similar efficiency as seen with ssRNA. However, in our helicase assay UAP56 was unable to unwind dsDNA. The two DEAD-box proteins, human DDX3 and yeast DBP9p , which also interact with RNA and DNA, were found to unwind both dsRNA and dsDNA Using recombinant UAP56 we have examined the catalytic properties of the Arabidopsis UAP56 can interact with the mRNA export adaptors ALY2 and MOS11, suggesting that also in plants the RNA helicase in combination with the corresponding export factors is involved in mRNA export from the nucleus. We have tested ALY2, because according to sequence alignments it is a close relative of human ALY Arabidopsis ortholog of human CIP29, and mutant mos11 cells display mRNA export defects Arabidopsis, according to database searches, there is no second helicase closely related to UAP56. Therefore, it is likely that Arabidopsis UAP56 is a functional interactor of both ALY2 and MOS11.Both yeast two-hybrid and GST pull-down experiments demonstrated that Drosophila, Caenorhabditis and yeast severely affected mRNA export and cell growth/survival SUB2 gene can be lethal, while overexpression of SUB2 impairs mRNA export in a dominant negative manner presumably by titrating mRNA export factors Arabidopsis however, the overexpression of UAP56 does not result in altered growth and development of the transgenic plants. In rice, down-regulation of the UAP56 (AIP1/2) transcript levels by amiRNA resulted in reduced fertility, presumably due to defects in anther/pollen development CP1 gene (encoding a Cys protease) by specifically binding to the CP1 promoter, thereby regulating tapetum degeneration in anthers. Due to the close proximity of the two Arabidopsis UAP56 genes on chromosome 5, it is unlikely to obtain double mutants  that do not express UAP56. We have attempted generating Arabidopsis plants with down-regulated expression of UAP56 using the RNAi strategy. So far we were unable obtaining plants with significantly reduced expression of UAP56 (data not shown), but this is clearly a goal of future experiments.Consistent with its essential role in splicing and mRNA export, depletion as well as overexpression of UAP56 in mammals, Figure S1Multiple sequence alignment of (putative) UAP56 sequences from different organisms.(PDF)Click here for additional data file.Figure S2Sequence similarity of UAP56 sequences.(PDF)Click here for additional data file.Figure S3Characterisation of Arabidopsis T-DNA insertion mutants.(PDF)Click here for additional data file.Figure S4Phenotypic analysis of T-DNA insertion lines uap56a-3 and uap56b-1.(PDF)Click here for additional data file.Figure S5Analysis of plants overexpressing UAP56.(PDF)Click here for additional data file.Table S1Oligonucleotide primers used in this study and construction of plasmids.(PDF)Click here for additional data file.Table S2RNA and DNA oligonucleotides used in different biochemical assays of this study.(PDF)Click here for additional data file.


It is still not clear how prebiotic replicators evolved towards the complexity found in present day organisms. Within the most realistic scenario for prebiotic evolution, known as the RNA world hypothesis, such complexity has arisen from replicators consisting solely of RNA. Within contemporary life, remarkably many RNAs are involved in modifying other RNAs. In hindsight, such RNA-RNA modification might have helped in alleviating the limits of complexity posed by the information threshold for RNA-only replicators. Here we study the possible role of such self-modification in early evolution, by modeling the evolution of protocells as evolving replicators, which have the opportunity to incorporate these mechanisms as a molecular tool. Evolution is studied towards a set of 25 arbitrary ‘functional’ structures, while avoiding all other (misfolded) structures, which are considered to be toxic and increase the death-rate of a protocell. The modeled protocells contain a genotype of different RNA-sequences while their phenotype is the ensemble of secondary structures they can potentially produce from these RNA-sequences. One of the secondary structures explicitly codes for a simple sequence-modification tool. This ‘RNA-adapter’ can block certain positions on other RNA-sequences through antisense base-pairing. The altered sequence can produce an alternative secondary structure, which may or may not be functional. We show that the modifying potential of interacting RNA-sequences enables these protocells to evolve high fitness under high mutation rates. Moreover, our model shows that because of toxicity of misfolded molecules, redundant coding impedes the evolution of self-modification machinery, in effect restraining the evolvability of coding structures. Hence, high mutation rates can actually promote the evolution of complex coding structures by reducing redundant coding. Protocells can successfully use RNA-adapters to modify their genotype-phenotype mapping in order to enhance the coding capacity of their genome and fit more information on smaller sized genomes. It has been generally accepted that replicators during the early stages of evolution, most likely formed an ‘RNA world’ Surprisingly, among the non-coding RNAs found in contemporary organisms, many are actually used to effectively influence the mapping between genetic content and the function it encodes (being other RNAs or proteins). That is, functional and structural inter-relationships of macro-molecules are widespread In contemporary life, these RNA-RNA modification machineries range in complexity from simple physical obstruction of translation by complementary base-pairing, to chemical modification, insertions and deletions of nucleotides by advanced RNA-editing processes, in which RNAs and proteins work together We hypothesize that such machinery could be a way for early replicators to cope with the major problem they have to face, known as the information threshold within replicating protocells. For this purpose we constructed a model of replicators (ie. protocells), evolving towards a fixed set of arbitrary RNA-structures functional by definition, concurrently avoiding any other (misfolded) structure. Note that, because the genetic content of reproducing protocells is copied at once, we do not study the coexistence of independent replicating RNAs within compartments, as for example in Major attempts to solve the problem of the information threshold have been made in the context of molecular organization With respect to the classical problem of the information threshold, it should however be mentioned that it becomes increasingly evident that the information content required for activity is much lower than indicated by the length of replicators Given the above considerations, the common reputation of the information threshold might have to be reconsidered. The dynamics of evolution itself imposes patterns on the coding structure of replicating units can replicators employ self-modification of genetic information to alter their genotype-phenotype mapping in order to evade the information threshold, and, how does the information threshold shape the structure of coding of evolved replicators?In a model-universe where only a small set of conformations of RNA-sequences is considered to be ‘functional’, while all misfoldings are considered to be ‘toxic’, we ask: The evolution of complexity is studied in protocells evolving towards a set of 25 pre-defined ‘functional’ secondary structures. Two distinct sets of 25 target-structures are used see  section.A minimal implementation for adapters is chosen, which could involve either chemical modification or binding induced alteration. Hence, RNA-sequences folding into a secondary structure consisting of a single hairpin-loop, are regarded as ‘RNA-adapters’. Note that early experiments (data not shown) showed that the exact predefined structure is not important for the results. RNA-adapters can bind reversibly, and only if binding free energy is less than 4 kcal/mol. When an adapter binds, it blocks certain nucleotides, which can possibly lead to a change in the conformational state of the bound RNA-sequence. How the new folding is achieved and the impact an adapter can cause on the structural conformation of the bound sequence, is illustrated in The total number of structures a protocell can possibly produce (codes for), defines the ‘phenotype’ of this protocell. With this multi-molecule genotype-phenotype mapping, the incorporation of a single RNA-adapter can thus provide a general self-modifying tool, potentially doubling the functional structures coded for by all other RNA-sequences in the cell. Note however, that such a one-to-many genotype phenotype mapping might potentially lead to a considerable amount of toxicity. Nonetheless, by having RNA-adapters, a protocell can code for more foldings than it has RNA-sequences. This is detailed in Population dynamics are given by competition and replication of protocells. Protocells transfer all genetic material  to their offspring at once, under consideration of mutations. In short, we defined a single level evolutionary system and only competition between protocells is considered, not between molecules within a cell. Focus is on the transfer of genetic material  by replicators. For further details, see the Now we will study the relationship between mutation rates, genome size and usage of coding, given the possibility of RNA-adapters. After identifying the general trends and characteristics of the system we will detail the evolved molecular interaction structures in the protocells and elaborate on the exact nature and implications of adapter usage by protocells. Finally we will scrutinize the evolved coding structures and communication between sequences and RNA-adapters.Genome size  and fitness of evolved protocells is studied in simulations under increasing mutation rates. For the two target sets, four simulations are performed with the mutation rates The change in coding structures under high mutation rates is confirmed, on a general level, in The protocells in These observations show that mutation rates impose a restriction on the used coding length during evolution, similar to that described in other work The molecular composition in protocells under severe mutation rates in Concluding, protocells under high mutational pressure can compensate for a small genome size by adopting complex molecular coding structures, increasing the coding capacity of RNA-sequences by evolving general and/or specific code-modification machinery. The code used between such RNA-adapters and regular RNA-sequences play a key role in this and will be studied next.In our model, both sequence and adapter have to evolve a sequential ‘code’ to be able to correctly base-pair adapters and RNA-sequences in a protocell. The arbitrariness of such evolved codes is exemplified in  see . Each rogreen adapter 3). This shows that not only functional interactions did evolve, but also that all other interactions which produce toxic structures are successfully avoided.It is evident that, although evolved under exactly the same global circumstances, each simulation produces protocells with a distinct combination of primary and guided functions on their RNA-sequences. Not only the combinations of functions over sequences is evolved at liberty, but also the coding used for molecular communication is seemingly assigned at random. Notable is, that out of the 136 here reported function transitions only one leads to a toxic structure , and increased toxicity (by the introduction of a new RNA-adapter), cannot be crossed.Thus, redundant coding impedes the evolution of self-modification machinery on a genome, in effect restraining the evolvability of coding structures. However, when redundancy is restricted , coding capacity can be enhanced by adopting a complex coding structure, guided by adapters. Hence, an increase of mutation rates not only force protocells to employ complex coding structures, but also facilitates the evolution of adapters.But what happens when the protocells manage to decrease mutation rates? We observe that when subjecting a population initially evolved under high mutation rates, to low mutation rates, the adapter machinery is kept. However genome sizes can increase due to the reduced mutational pressure. This results in a coding structure which is not observed to evolve in simulations with constant low mutation rates: whereas redundancy prevents adapters to evolve, redundancy can evolve when adapters already have evolved. Hence, these results show that the limits posed by the information threshold are in fact responsible for the development of a compact and ‘complex’ structure of coding, observed in our model-universe.Not only in present day organisms RNA-modification has an important role. It has been claimed that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals However, we should mention that in the light of prebiotic evolution, ‘realistic’ protocells would not be able to transmit all the information of a parental protocell to its daughter protocells at once, as is assumed in our model. That is, here we study the information content of single level replicating systems, as “cells” which contain several RNA-sequences. This is a single level process, because the replication of the RNAs is directly coupled to the reproduction of the containing cells  determined genotype-phenotype mapping. The way information is accumulated and how coding structures are shaped, depends on limitations posed by the information threshold Finally, we showed that an arbitrary coding used for “communication” between adapters and other sequences is readily evolved. Such arbitrariness of biological coding is recently nicely demonstrated However, despite the power of the described self-modification machinery and its arbitrary code, there is the potential danger of interference between the functional structure of molecules and the coding used for adapters. Interference between different functions encoded on one RNA does occur in present day organisms. For example, the structure of RNA will constrain the variability and evolution at the protein level .The multi-molecule genotype-phenotype mapping consists of all RNA-sequences being folded . These structures were (hand)picked with ‘different’ as the most important criterion. Note that the major objective of this set is to show the independence of our results towards choice of target. The frequency of random sequences . Empty squares of the grid act as resource. Competition for an empty square is between all neighboring protocells. Between competing protocells, the probability that one is selected, is proportional to its We used this spatial setting as default. However, when the population is mixed at each time step, similar results are observed: the evolution of adapters can increase coding capacity at high mutation rates, however adapters evolve less frequently see . Thus, aFinally, all protocells decay with the probability of 0.4 plus an extra 0.02 for each non-functional folding it contains. Simulations are started with a population inhabiting Every 1000 time steps statistics on the population are recorded. The evolutionary trajectory of a simulation is best studied and visualized by determining the ancestor lineage. That is, all protocells during evolution carry a unique identifier and the identifier of its parent. After a simulation has finished, the protocells inhabiting the last population can be traced back and all ancestors (out of the recorded populations) leading to this last population can be identified. This gives us the ancestor lineage, which in hindsight contains all protocells which lead to the dominant genetic content of future generations.Figure S1Relation between mutation rates, genome size and fitness in a well-mixed population. We used spatial embedded populations as a default. However, in the current study spatial embedding is not necessary. When the population is mixed at each time step, similar results are observed: although adapters evolve less frequently, the incorporation of adapters still allows protocells to modify their genotype-phenotype mapping, enabling them to increase coding capacity at high mutation rates. Shown are mutation rates, genome size and fitness for the last common ancestors of four simulations for the different mutation rates with target set 2 in a well mixed population. Number of sequences is limited to 25. For each mutation rate four repetitions of a simulation (stars) with a different seed and the average, are shown. Upper squares point out those simulations which use adapters to modify their genotype-phenotype mapping. The evolution and incorporation of adapter-usage is observed less frequently (compare with results of (TIFF)Click here for additional data file.Figure S2Reproductive toxicity. To exclude dependency of our results on the implementation of toxicity as an increase in death rate, experiments are conducted where toxicity is implemented as a negative effect on reproductive fitness. Every misfolding leads to a decrease of 0.2 in fitness. Shown are mutation rates, genome size and fitness for the last common ancestors of four simulations for the different mutation rates with target set 2, number of sequences is limited to 25. For each mutation rate four repetitions of a simulation (stars) with a different seed and the average, are shown. The squares on top depict if adapters are used in that specific simulation. The same trends are observed as in the default (compare with results of (TIFF)Click here for additional data file.


Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3–9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR  but also with the newly characterized complex composed of two conserved Microrchidia (MORC) family proteins, MORC1 and MORC6. The effect of suvh2suvh9 on Pol IV-dependent siRNA accumulation and DNA methylation is comparable to that of the Pol V mutant nrpe1 and the DDR complex mutant dms3, suggesting that SUVH2 and SUVH9 are functionally associated with RdDM. Our CHIP assay demonstrated that SUVH2 and SUVH9 are required for the occupancy of Pol V at RdDM loci and facilitate the production of Pol V-dependent noncoding RNAs. Moreover, SUVH2 and SUVH9 are also involved in the occupancy of DMS3 at RdDM loci. The putative catalytic active site in the SET domain of SUVH2 is dispensable for the function of SUVH2 in RdDM and H3K9 dimethylation. We propose that SUVH2 and SUVH9 bind to methylated DNA and facilitate the recruitment of Pol V to RdDM loci by associating with the DDR complex and the MORC complex.RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in  Arabidopsis thaliana, an RNA-directed DNA methylation pathway is involved in transcriptional silencing. Noncoding RNAs produced by the plant-specific DNA-dependent RNA polymerase V are required for RNA-directed DNA methylation. A chromatin-remodeling complex was previously demonstrated to be required for the occupancy of DNA-dependent RNA polymerase V at RNA-directed DNA methylation loci. Our results suggest that two putative histone methyltransferases are inactive in their enzymatic activity and act as adaptor proteins to facilitate the recruitment of DNA-dependent RNA polymerase V to chromatin by associating with the chromatin-remodeling complex. In combination with previous studies, we propose that the inactive histone methyltransferases bind to methylated DNA, thereby linking DNA methylation to Pol V transcription at RNA-directed DNA methylation loci.Small RNA-induced transcriptional silencing at transposable elements and other DNA repeats is an evolutionarily conserved mechanism in plants, fungi, and animals. In  Arabidopsis thaliana, DNA methylation can be established through a well-characterized RNA-directed DNA methylation (RdDM) pathway DNA methylation is an important epigenetic modification that contributes to transposable element silencing, genome stability, and gene regulation in plants and animals DEFECTIVE IN MERISTEM SILENCING 3 (DMS3), DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), and RNA-DIRECTED DNA MEETHYLATION 1 (RDM1), is required for the recruitment of Pol V to RdDM loci and for the accumulation of Pol V-produced noncoding RNAs In the RdDM pathway, the multi-subunit DNA-dependent RNA polymerase IV (Pol IV) collaborates with RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) to produce double-stranded RNAs, which are subsequently cleaved into 24-nt siRNAs  by DICER-LIKE 3 (DCL3) Arabidopsis, H3K9 methylation is catalyzed by the conserved SU(VAR)3–9 homologs (SUVHs) and related proteins (SUVRs) In In this study, we found that SUVH2 and SUVH9 associate with the DDR complex and the newly characterized MORC1-MORC6 complex and act as adaptor proteins to mediate Pol V occupancy at RdDM loci. The conserved catalytic active site of SUVH2 is not required for the function of SUVH2 in RdDM and histone H3K9me2. The results revealed the functional mechanism of SUVH2 and SUVH9 in the RdDM pathway.SUVH2-3xMyc and SUVH9-3xFlag transgenic plants. The stably expressed SUVH2-3xMyc and SUVH9-3xFlag transgenic lines were used for affinity purification of the proteins that associate with SUVH2 and SUVH9. Mass spectrometric assay revealed that SUVH2 and SUVH9 were the most abundant proteins in affinity purification of SUVH2-3xMyc and SUVH9-3xFlag, respectively , in c plants . TherefoPAGE gel . The thrPAGE gel . These ransgenes . Furtheransgenes , which i. Pol IV-dependent 24-nt-siRNA regions were defined as the genomic regions in which the abundance of 24-nt siRNAs was significantly reduced in nrpd1 relative to the wild type. Consistent with previous reports suvh2 and suvh9 single mutants and the reduction was significantly enhanced in the suvh2suvh9 double mutant  of Pol IV-dependent and Pol V-independent siRNA regions are significantly decreased in 24-nt siRNAs in dms3  in ild type . In 63.8e  and 7.5% (434/5751) of the Pol IV-dependent siRNA regions show a significant decrease in 24-nt siRNAs, respectively  belong to Pol IV- and Pol V-dependent siRNA regions  show a decrease in 24-siRNAs, whereas only 5.2% (109/2081) of Pol IV-dependent and Pol V-independent siRNAs regions have decreased 24-nt siRNAs , AtGP1, AtSN1, and MEA-ISR loci. As expected, all these Pol IV-dependent siRNAs were blocked in the Pol IV mutant nrpd1, whereas AtSN1 siRNA and MEA-ISR siRNA but not 180 bp CEN siRNA and AtGP1 siRNA were reduced in the Pol V mutant nrpe1 and the DDR complex mutant dms3 >1; P<0.01) upregulated in nrpe1, dms3, and suvh2suvh9, respectively  are also upregulated in nrpe1  are upregulated in either nrpe1 or dms3  in the catalytic active site of the SET domain in SUVH2 and transformed the mutated SUVH2 sequences into suvh2suvh9 , suvh9 (SALK_048033), suvh2suvh9nrpd1-3 (SALK_128428), nrpe1-11 (SALK_029919C), dms3 , drd1-6morc1 , and morc6 (GK-599B06-023140). The seedlings were grown on MS medium plates under long-day conditions (16 h day time and 8 h night time) at 22°C. Two-week-old seedlings were directly used for the study or transplanted into soil when adult plants or flower tissues were needed.The Flag- and/or Myc-tagged SUVH2, SUVH9, MORC1, and MORC6 genomic sequences were constructed into the modified binary pCAMBIA1305 vector or pRI909 vector. The DNA primers used for constructing were listed in SUVH2-3xMyc sequence was transformed into suvh2suvh9. The wild-type SUVH2 genomic sequence was subjected to site-directed mutagenesis, introducing the SUVH2-H596K and SUVH2-P600A mutations in the SUVH2-3xMyc construct.The SUVH2-3xMyc, SUVH9-3xFlag, or MORC6-3xFlag transgenic plants was harvested for affinity purification by anti-Flag antibody. Flower tissue was ground in liquid nitrogen and suspended in Lysis buffer . The final supernatant was incubated with Anti-Flag M1 agarose  in Lysis buffer at 4°C for 2.5 h and then precipitated by centrifugation. The precipitant was washed four times, and the agarose-bound proteins were eluted with 3xFlag peptide .A 3-g quantity of flower tissue from Arabidopsis protein database on the Mascot server .The eluted proteins were run on a 10–12% SDS-PAGE gel and then subjected to silver staining with the ProteoSilver Silver Stain Kit . Total eluted proteins or specific target bands were cut from gels and extracted. Mass spectrometric analysis was performed as described previously SUVH2-3xMyc and SUVH9-3xFlag transgenic plants were used to determine whether SUVH2 and SUVH9 interact with DMS3 by co-immunoprecipitation. SUVH9-3xFlag transgenic plants were crossed to MORC6-3xMyc transgenic plants, and the offspring plants harboring both transgenes were used to determine whether SUVH9 interact with MORC6. For co-IP, protein extracts were incubated with the agarose-conjugated antibodies in Lysis buffer at 4°C for 2–3 h and precipitated by centrifugation. The precipitant was washed four times and then boiled for 5 min in 1×SDS sample buffer. After centrifugation, the supernatant was run on an SDS-PAGE gel, followed by Western blotting.For gel filtration, total protein extracts from wild-type or transgenic plants were loaded onto a Superose 6 10/300 GL column  and the eluted solution was harvested once per 500 µl. The eluted fractions were run on a 10–12% SDS-PAGE gel and were then subjected to Western blotting. Standard proteins were used to determine the sizes of the eluted fractions.GAL4-AD and GAL4-BD, respectively. The DNA primers for cloning were listed in The cDNA sequences were cloned into pGADT7 and pGBKT7 vectors in frame to the C-termini of Arabidopsis genome (TAIR10) using the Bowtie program Arabidopsis genome was split into 500-bp bins, and the appearances of 24-nt reads in each bin were counted. The read counts were then normalized by the library size. Reads per ten million (RPTM) were calculated for the adjusted small RNA abundance in each bin. The bins with low reads  were removed, and the regions were referred to as Pol IV-dependent siRNA regions if the RPTM value was five-time greater in the wild type than in the Pol IV mutant nrpd1. We also determine the RPTM value in Pol IV-dependent siRNA regions using 500-Kb windows to plot the profile of Pol IV-dependent siRNAs across chromosomes.Small RNA was extracted from two-week-old seedlings as described previously Arabidopsis genome (TAIR10) using TopHat The mRNA extracted from each genotype was used for Illumina library construction. The libraries were subjected to single-end Illumina sequencing. For data analysis, 45-bp sequences were mapped to the Total RNA was extracted by the routine method with Trizol as described previously DNA methylation was tested by chop-PCR. Genomic DNA was cleaved with the methylation-sensitive restriction enzyme HaeIII or AluI, followed by PCR. The abundance of PCR products indicates the DNA methylation levels of the amplified loci. The actin gene sequence, which lacks an HaeIII recognition site, was amplified as an internal control. The DNA methylation analyses were repeated at least two times.The levels of histone H3K9me2, NRPE1-Flag and DMS3 on chromatin were determined by CHIP assay as described previously Figure S1SUVH2-Myc transgenic plants were precipitated using anti-Myc antibody and the precipitates were subjected to Western blotting. The band labeled “*” is an unspecific band.  Detection of the interaction between SUVH9 and DMS3. The protein extracts from SUVH9-Flag transgenic plants were precipitated using either anti-Flag antibody (B) or anti-DMS3 antibody (C), and the precipitates were subjected to Western blotting.Detection of the interaction between the histone methyltransferases and the RdDM component DMS3 by co-IP. (A) Detection of the interaction between SUVH2 and DMS3. The protein extracts from (PDF)Click here for additional data file.Figure S2The interaction of MORC6 with MORC1 and MORC2 was determined by yeast two-hybrid assay. MORC1, MORC2, and MORC6 were separately cloned into pGADT7 and/or pGBKT7 vectors and transformed into the yeast strain PJ694a for yeast two-hybrid assay.(PDF)Click here for additional data file.Figure S3Detection of the interaction between truncated SUVH2 and SUVH9 sequences and RdDM components by yeast two-hybrid assay. Three individual yeast strains harboring the indicated GAL4-AD and GAL4-BD fusion constructs were streaked on the yeast synthetic dropout medium minus Trp, Leu, and His (SD-TLH) but supplemented with 3-AT and on the SD medium minus Trp and Leu (SD-TL). (A) Diagram of the truncated SUVH2 and SUVH9 sequences used in yeast two-hybrid assay. (B) The interaction between truncated SUVH2 sequences and the RdDM components DMS3 and MORC1. (C) The interaction between truncated SUVH9 sequences and the RdDM components DMS3, MORC6, and MORC1.(PDF)Click here for additional data file.Figure S4AtGP1, AtSN1, and MEA-ISR loci were counted based on small RNA deep sequencing data. The numbers of ta-siRNA255 reads in each corresponding library were used as controls to normalize the Pol IV-dependent siRNA reads. Shown are relative accumulation levels of indicated siRNAs in the wild type as well as in each mutant.Accumulation of Pol IV-dependent 24-nt siRNAs as determined by small RNA deep sequencing. Pol IV-dependent 24-nt siRNA reads from 180 bp CEN, (PDF)Click here for additional data file.Figure S5IGN5B, IGN23, and IGN25 were examined by quantitative RT-PCR. The mutants nrpe1, drd1, and dms3, which were previously demonstrated to be required for Pol V-dependent RNA transcripts, were included as controls. The nrpd1 mutant that has no effect on Pol V-dependent RNA transcripts was also used as a control.The Pol V-dependent RNA transcripts (PDF)Click here for additional data file.Figure S6NRPE1-3xFlag and DMS3 expression levels by Western blotting. (A) The NRPE1-3xFlag transgene was introduced into the wild type, dms3, and suvh2suvh9, respectively. The NRPE1-3xFlag expression was determined in transgenic plants as well as in the wild-type control by using anti-Flag antibody. The Ponceau S-stained rubisco protein is shown as a loading control. (B) The DMS3 expression level was determined in the wild type, dms3, and suvh2suvh9 by using anti-DMS3 antibody.Determination of the (PDF)Click here for additional data file.Table S1Summary of small RNA deep sequencing data.(XLS)Click here for additional data file.Table S2Arabidopsis genome in each ecotype.Numbers of Pol IV-dependent siRNA reads in 500-bp windows of the (XLS)Click here for additional data file.Table S3Summary of RNA deep sequencing data.(XLS)Click here for additional data file.Table S4nrpe1, dms3, and suvh2suvh9 detected by RNA deep sequencing.List of differentially expressed genes in (XLS)Click here for additional data file.Table S5List of DNA oligonucleotides used in this study.(XLS)Click here for additional data file.


Small secreted membrane vesicles called exosomes have recently attracted a great interest after the discovery that they transfer mRNA that can be translated into protein in recipient cells. Surprisingly, we found that for the majority of exosomal mRNAs only a fraction of their corresponding probes is detectable on the expression microarrays. Exosomal mRNA fragmentation is characterized with a specific structural pattern. The closer to the 3′-end of the transcript the fragments are localized, the larger fraction among the secreted RNAs they constitute. Since the 3′-ends of transcripts contain elements conferring subcellular localization of mRNA and are rich in miRNA-binding sites, exosomal RNA may act as competing RNA to regulate stability, localization and translation activity of mRNAs in recipient cells.This article was reviewed by Neil Smalheiser and Sandor Pongor. Exosomes are small nano-sized 50–150 nm) membrane vesicles secreted by most cell types including hematopoietic, neuronal, fibroblastic and various tumor cells 50 nm mem, they weOne possible explanation for this observation could be that exosomes are enriched in mRNAs encoding very short proteins. However, Frith with colleagues  analyzedNext generation sequencing-based method RNA-Seq allows accurate determination of transcript boundaries and thus could be used to verify the hypothesis that exosomes carry RNA fragments. However, in case of exosome microvesicles, this approach was applied only for the analysis of small RNAs ,17. InteTo detect possible presence of mRNA fragments in exosomes we utilized a microarray dataset from the published study  that anaClasses ii-iv, representing putative transcripts secreted in fragments, constituted 68.5% of all secreted transcripts. Moreover, we observed that with increase of the enrichment in exosomes the fraction of partially secreted transcripts of both classes increased Figure B. The frP = 0.0013) and in proteins participating in extracellular transport (P = 0.028). On the other hand, the proteins encoded by the full-length secreted mRNAs are specialized at cell surface receptor linked signal transduction (P = 2.04∙10-4), cell communication (P = 8.06∙10-4) and system development (P = 0.029). Interestingly, the products of 17 of these transcripts are localized in the extracellular matrix (P = 0.0046). Thus it can be concluded that secreted transcript fragments might have specific functions.We performed the analysis of gene ontologies (GOs) associated with each class of secreted transcripts   , with the probe localization in the UTRs correlating with secretion positively   , the stronger was the tendency of the probes to be localized in the 3′-end of the transcripts Figure C. The prTo validate the accuracy of the results obtained with the microarrays, we examined the presence of various transcript parts in exosomes using quantitative real-time PCR (qPCR). RNA was isolated from exosomes secreted by human glioblastoma cells SF295. Three exemplary mRNA targets were selected for qPCR analysis, for which we observed unequal distribution of probe intensities on the microarray between cellular and exosomal RNA - CNDP2, RHO, PPFIBP1 mRNAs  to microRNA response elements on target transcripts resulting in their degradation or translational reppression. A single 3′-UTR contains many miRNA binding sites. We can imagine that the 3′-UTR derived mRNA fragments carried by exosomes could directly compete for binding of miRNA or specific RNA-binding proteins to the recipient cell mRNA and lead to deregulation in protein production. Lee et al. reported that expression of versican 3′-UTR induces organ adhesion in transgenic mice through binding miR-199a* and freeing mRNAs of versican from being repressed by miR-199a* . The 3′-In addition to controlling translation efficiency of mRNAs, the 3′-UTRs are also critical for the subcellular localization of mRNAs . The 3′-In summary, our results provide evidence that exosomes secreted by human cells transport largely mRNA fragments derived from the 3′-ends of mRNA. This finding suggests the need to reassess the assumption that RNA messages delivered by exosomes are mainly translated into proteins by the recipient cells. Instead, we propose that RNA delivered by exosomes play largely regulatory roles. The secreted mRNA may act as competing RNAs to regulate stability, localization and translational activity of mRNAs in target cells, because 3′-UTRs contain elements that confer subcellular localization of mRNAs and are rich in miRNA-binding sites.Expression data, measured with Agilent human gene expression G4112F microarray, were obtained from .For every selected gene the number of probes enriched in the exosomal and cellular fractions were determined at a given ECER cutoff level. We confirmed absence of correlation between probe expression and its localization for weakly secreted and intracellular transcripts (ECER < 3) . The methods for collecting and isolating exosomes do not employ RNAse inhibitors, and there are no internal controls to monitor the extent of RNA degradation. I have seen (in other studies) great variation in the ribosomal RNA profiles of isolated exosomes, ranging from intact 28S and 18S RNA to total absence, and I strongly suspect that is due to RNA degradation since (in our own unpublished data) we find abundant partial rRNA reads in exosomal fractions.Authors’ response: We thank the reviewer for critical reading of the manuscript and raising several very helpful comments. Indeed, alternative explanation for the observed preferential stability of 3′ fragments would be the partial degradation of exosomal mRNA with RNA binding proteins protecting 3′fragments. However, RNases present in cell culture conditioned medium are unlikely to contribute to mRNA degradation. Several studies (including our unpublished data) have demonstrated that RNA in exosomes is well protected from the attack by exogenous RNases. In the present study, we utilized a microarray dataset from the study analyzing mRNA in exosomes released by cultured glioblastoma primary cells . RNase treatment of the glioblastoma exosomes in that study led to a very insignificant (less than 7%) decrease in RNA suggesting that exosomal RNA is inaccessible for the RNase from outside the vesicles. We cannot exclude though a possibility that RNases originating from donor cells could be incorporated inside exosome vesicles. So far, the presence of RNases inside exosome vesicles has not been investigated and this poses a question that remains to be addressed. Regarding the levels of intact 28S and 18S RNA found in various exosome preparations, indeed it is very variable. Interestingly, in line with the reviewer’s observation, analysis of several exosome preparations in our lab revealed that even when 18S and 28S intact rRNA peaks were barely detected , rRNA sequences represented the majority of the reads in the RNA-Seq data suggesting that rRNA is extensively fragmented (unpublished data). It is, however, unclear whether the fragmentation is non-specific and occurs randomly or is purposeful and occurs at specific sites in rRNA. Another example is tRNA. It is represented in exosomes mainly by its fragments and the most abundant tRNA hits in exosomal RNA are all located at the 5′end of mature tRNAs . Regarding the site of mRNA fragments generation, we believe it is likely to be intracellular. We noted, for example, that all three transcripts selected for RT-PCR validation experiment, CNDP2, RHO, PPFIBP1 are present in various cDNA libraries not only in their full length forms but also as smaller transcript isoforms truncated at 3′-UTR, as well as, fragments derived entirely from 3′-UTR .” Regardless the site and the cause of mRNA fragmentation, the preferentially produced 3′-end fragments, we believe, have the potential to act as competing RNA to regulate stability, translation activity and localization of mRNAs in recipient cells.roduction. RegardiReviewer’s comment: The second problem with their data is that they have not defined the size distribution or specific lengths of specific 3′-UTR fragments, which would be suggestive of specific processing . They cite Mercer et al. as providing a precedent for the existence of discrete 3′-UTR RNA fragments, but Mercer et al. did define specific lengths, and this should be done here too.Authors’ response: Indeed, Mercer et al. study provided defined specific lengths of mRNA fragments because they are based on the data obtained by sequencing of cDNA libraries. Our analysis has been based on previously published microarray data which have a limitation of interrogating levels of mRNA based on hybridization of small probes (60 oligomer in case of Agilent) to different parts of mRNA. Since the probes were designed to match few specific regions of the target mRNAs, their coverage of the transcripts is essentially incomplete and too sparse to use them as positional markers for direct estimation of the transcripts’ sizes.Nevertheless, considering the importance of the raised question, we performed an additional analysis using the available data. We attempted to estimate the upper bound of fragment lengths by treating the locations of Agilent probes and gene borders as limits. We observed a large variation in the fragment lengths originating from two sources: the natural variation of UTR lengths (ranging from a few hundred to a few thousand nucleotides) and the design of the probes (the probes are distributed highly unevenly within transcripts and there is no common pattern in probe location among different transcripts). The combination of these two factors resulted in over-representation of transcripts with atypically longer UTRs (more than 1000 nt long) among the ones for which fragment length could be reliably estimated . At ECER cutoff 2 (moderately secreted fragments), within UTRs up to 1000 nt long the median fragment length 122 nt was observed with IQR = 168.25. At ECER cutoff 3 (strongly secreted fragments), the median and the IQR were 60.5 and 32.5 respectively. Understanding the limitations on the biological interpretation of this result, we decided not to include them in the text of the manuscript.Reviewer’s comment: A minor issue is that having 9 supplemental files are too many and not very crucial to their story.Authors’ response: We combined the former Figures S1, S2, S3 and S4 figures into a single file, Figure S1, and renamed them S1A, S1B, S1C and S1D, respectively. We also combined the former Figures S5 and S6 into a single file, Figure S2, and renamed them S2A and S2B, respectively. As the result, the number of supplemental files is 5 in the new version.Reviewer’s comment: My concerns have not been addressed; the revised ms. is essentially the same as the original version.Authors’ response: We appreciate the reviewer’s time to revise our manuscript. His previous comments were most helpful to improve the manuscript and strengthen the biological insights gained from this study. We understood that Reviewer 1 raised no concerns regarding the main results and conclusions. His concerns touched upon the possible mechanisms of mRNA fragmentation and specific lengths of the fragments. Our study did not focus on the causes of mRNA fragmentation but rather on establishing the fact that the majority of exosomal mRNA is fragmented and possible regulatory roles of 3′UTR-derived mRNA fragments in the recipient cells. Regarding the second concern of determining the specific lengths of the fragments, in our reply we described intrinsic limitations of the used experimental platform (microarrays) for such sort of analysis. Nevertheless, we estimated the upper bound of fragment lengths by treating the locations of Agilent probes and gene borders as limits. The numbers were provided in our reply to this comment, but the data were not included in the main text, again, because the platform is not suitable.We have accepted nearly all the reviewer’s suggestions and have revised the paper accordingly. Specifically, we provided an evidence that exosomal RNA is inaccessible for the RNases from outside the vesicles. Moreover, we provided several potential scenarios of mRNA fragmentation that were incorporated into the revised version of the manuscript. We also modified Figure Reviewer 2: Prof. Sandor PongorReviewer’s comment: Exosomes and other microvesicles released by prokaryotic and eukaryotic cells have been known or suspected already early in the 20th century. Their functional properties have only recently reached the focus of rigorous investigation as it was discovered that they can also transfer RNA and one seminal paper showed by microarray studies that this RNA can be translated into proteins by the recipient cell. In this analysis, the authors show that microarrays detect only a small fraction of microvesicle sequences detected by NGS, and the analysis of this greater sample reveals a definite structural pattern, namely the sequences tend to be at the 3′ end of the RNA transcripts. Since the 3′-ends of transcripts are rich in miRNA-binding sites and also contain elements conferring subcellular localization the authors suggest that exosomal RNA may act as competing RNA to regulate stability, localization and translation activity of mRNAs in recipient cells.The paper is concisely written, the conclusions are underpinned by the data. Recently, the authors of the present paper suggested that a combination of several linear motifs can mediate targeting of secreted RNA. The present contribution represents another important step towards the structural characterization of RNA transmitted by microvesicles. The author’s suggestion that RNA delivered by exosomes plays largely regulatory roles may provoke discussions, but in my view this is what science is about.Authors’ response: We appreciate the overall positive reaction of the reviewer to this work.Reviewer’s comment: I find the authors have adequately addressed the issues raised previously.The authors declare that they have no competing interests.AOB and IVK conceived the idea and wrote the paper. AOB performed the computational work. IVK carried out the experiments. Both authors read and approved the final manuscript.Gene ontologies associated with exosomal transcripts.Click here for file(A) Composition of the fragmented transcripts for probes located in the untranslated regions at ECER cutoff varying from 1 to 32. (B) Composition of the fragmented transcripts for probes located in the translated regions at ECER cutoff varying from 1 to 32. (C) Composition of the fragmented transcripts for probes located in the untranslated regions at ECER cutoff varying from 10 to 32. (D) Composition of the fragmented transcripts for probes located in the translated regions at ECER cutoff varying from 10 to 32.Click here for fileComparison of the expression ratios obtained from Agilent and qPCR for selected transcripts.Click here for file(A) Distribution of individual probe expression by their relative location within the transcript/gene for weakly secreted and intracellular transcripts, ECER < 3. (B) Distribution of individual probe expression by their relative location within the transcript/gene for strongly secreted and intracellular transcripts, ECER ≥ 3.Click here for fileExperimental part.Click here for file


Several mechanisms have been defined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially important tool in drug discovery.Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. Using recently developed reconstitution strategies, transporters can be inserted in artificial bilayers with the same orientation as in the cell membranes and in the absence of other interfering molecular systems. These methodologies are very suitable for studying kinetic parameters and molecular mechanisms. After the first applications on mitochondrial transporters, in the last decade, proteoliposomes obtained with optimized methodologies have been used for studying plasma membrane transporters and defining their functional and kinetic properties and structure/function relationships. A lot of information has been obtained which has clarified and completed the knowledge on several transporters among which the OCTN sub-family members, transporters for neutral amino acid, B0AT1 and ASCT2, and others. Transporters can mediate absorption of substrate-like derivatives or drugs, improving their bioavailability or can interact with these compounds or other xenobiotics, leading to side/toxic effects. Therefore, proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H Membrane transport systems play pivotal roles in cell homeostasis. This is indirectly but definitely demonstrated by the occurrence of many human pathologies of various severity caused by defects of genes coding for transporters ,2,3,4,5.The functions of transport systems are at the basis of metabolite exchanges among cells and extracellular environment and, within a cell, among different sub-cellular compartments, allowing completion of compartmentalized biochemical pathways. It was previously believed that a large number of molecules could diffuse through membranes, but it is now established that transmembrane proteins are necessary for translocating virtually all the molecules with the exception of very few small compounds such as oxygen ,7. Trans−1. The driving force for transport derives from ion concentration gradients. The high transport rate of ions generates measurable currents which can be detected by electrophysiological methods. Important exceptions are aquaporins which do not generate currents . This compound displays a chemical structure very similar to carnitine. It strongly inhibited the transport activity of CACT when externally added to the proteoliposomes together with the physiological substrate. Also in this case, according to the work plan of ldronate .2O2 has been revealed [2O2 is formed in cells, mostly in mitochondria, playing signaling roles; its concentration can increase reaching local millimolar range under pathological conditions [2O2 at physiological concentrations, i.e., less than 50 μM, is about 30%. The inhibition became much more effective at higher concentration of the reactant  pOHMB was observed [50 and Ki values for each metal cation has been performed (Effects of agents ranging from pharmaceutical and toxic compounds to physiological effectors have been tested on the mitochondrial ORCT which is highly expressed in liver where it is essential for the functioning of the urea cycle ,125,126.observed . The traobserved  and it herformed  allowingProteoliposomes have been used since long time, for studying membrane transporters in isolated environment. In the last decade, improved methodologies allowed to study kinetics and transport mechanisms of several plasma membrane transporters. More interestingly, the summarized novel results, highlight the power of proteoliposomes in revealing the molecular mechanisms of the interactions of transporters with different types of xenobiotics, from toxic molecules to commonly used drugs or newly synthesized compounds. In particular, aspects of “off-site” interactions have been revealed, which may be important in predicting side effects and, hence, in improving drug design. Bioinformatic analyses correlated well with the experimental data thus confirming the suitability of proteoliposomes for obtaining information on molecular mechanisms. Therefore, proteoliposomes can be considered a new tool, among those already available, for studying the role of transporters in drug development and clinical pharmacology .


The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as  in vitro and in vivo. Our experiments clearly show that the antiviral mechanism of IFIT1 is based on sequestration of viral RNA lacking cap 2′O methylation, thereby selectively preventing translation of viral RNA. Our data establish IFIT1 as a general sensor for RNA 5′ end structures and provide an important missing link in our understanding of the antiviral activity of IFIT proteins.Cellular messenger RNAs of higher eukaryotes are capped with a methylated guanine and, in addition, methylated at the 2′O position of the first ribose. Viruses unable to methylate their RNA at the 2′O position of the cap and viruses generating uncapped RNA with 5′ triphosphate groups are inhibited by an antiviral complex of different IFIT proteins. How IFIT proteins restrict viruses lacking 2′O methylation at the RNA cap remained unclear. We used a mass spectrometry-based approach to identify proteins binding to capped RNA with different methylation states. We found that IFIT1 directly binds to capped RNA and that this binding was dependent on the methylation state of the cap. Having identified IFIT1 as being central for recognition of 2′O-unmethylated viral RNA we further examined the mode of action of IFITs  Remarkably, only human and murine IFIT1 were detected when CAP-RNA was used as bait  and mutant, Ifit1-deficient (Ifit1−/−) MEFs. The overall precipitation efficiency was comparable in both cell types, as evidenced by equal enrichment of the RNA-binding protein Syncrip and the cap-binding protein Ncbp1  mice were infected with a wild-type murine coronavirus  and a mutant strain carrying the equivalent amino acid substitution (D130A) in its 2′O methyltransferase in vivo. MHV-WT grew to high titres in the spleens of infected Ifit1+/+ mice, whereas no viral replication could be detected upon infection with MHV-DA  and lysine (Lys8). Newly synthesized proteins incorporate the heavy label and pre-existing proteins remain in the light form, which allows to measure relative changes in the translation of individual proteins, regardless of the absolute amount of RNA present. We pulsed Ifit1+/+ and Ifit1−/− MΦs infected for 5½ h with either MHV-WT or MHV-DA for 2 h with SILAC medium  as compared to all other conditions tested  C medium  and analnfection , boxes, nfection . Howeverh MHV-DA . Variatis tested , and amos tested . Taken t−/− MEFs than from lysates of Ifit1+/+ MEFs , license nr. SG 11/03.Renilla luciferase were expressed from constructs obtained by Gateway cloning into pCDNA-REN-NT-GW (a kind gift from Albrecht v. Brunn). For expression in bacteria, human EIF4E cDNA was cloned into pETG10A-GW E. coli and purified using HisPur Ni-NTA resin (Thermo Scientific). Streptavidin-agarose beads were obtained from Novagen. Polyclonal antibodies directed against human and mouse IFIT1 were described previously +/+ and Ifit1−/− mouse embryonic fibroblasts) were described previously 13C615N4 L-arginine and 146 mg/L 13C615N2 lysine (Cambridge Isotope Laboratories) was used for SILAC experiments. Murine bone marrow-derived macrophages were generated in vitro by cultivating bone marrow from mouse femur and tibia in DMEM supplemented with 10% (v/v) fetal calf serum, 5% (v/v) horse serum, 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, 10 mM L-glutamine and 20% (v/v) L929 cell-conditioned medium (containing macrophage colony-stimulating factor) for 6 days. Reagents for transfection with plasmid DNA (Nanofectin) or siRNA duplexes (siRNA Prime) were obtained from PAA Laboratories. Wild-type and 2′-O-methyltransferase-deficient recombinant coronaviruses  and the green fluorescent protein  were synthesized by the Core Facility at the MPI of Biochemistry.Interferon-α (IFN-α A/D) was a kind gift from Peter Stäheli. Expression constructs for human and murine IFIT proteins Renilla luciferase , and purified by ammonium-acetate isopropanol precipitation. Aliquots of PPP-RNA were then mock-treated, dephosphorylated with alkaline phosphatase , or modified with different 5′ cap structures using the ScriptCap 2′-O-Methyltransferase and m7G Capping System (CellScript) according to the manufacturer's instructions. Briefly, 20-µg samples of RNA were heat-denatured at 65°C for 5 min, cooled on ice, then incubated with ScriptCap Buffer in the presence of 500 µM GTP, 100 µM SAM, 100 U 2′-O-methyltransferase (VP39), 10 U Vaccinia Capping Enzyme (VCE) and 40 U RNase inhibitor for 1 h at 37°C. Capped RNAs were further treated with FastAP to dephosphorylate any residual PPP-RNA, and then column-purified using the NucleoSpin RNA II kit (Macherey-Nagel). To add radioactively labelled methyl groups to in vitro transcribed RNA, 500 ng of each RNA was incubated with 100 U 2′-O-methyltransferase or 10 U of VCE in 0.5 µM S-adenosylmethionine and 1.4 µM S-[3H-methyl]-adenosylmethionine  for 1 h at 37°C. Reactions were purified on SigmaSpin Post-Reaction Clean-Up columns (Sigma) and eluates were mixed with 2 ml Ultima Gold scintillation fluid for measurement of 3H incorporation with a Packard Tri-Carb liquid scintillation counter (Perkin Elmer).Triphosphorylated PPP-RNA was synthesized by in vitro transcription with SP6 or T7 polymerase , in the presence or absence of biotin-16-UTP (Enzo), from plasmids encoding antisense 7SK RNA (7SK-as) AUAUUAGGUUUUUACCUACCC)-NH2) and a corresponding 2′O-ribose methylated RNA-oligomer [PPP-r(AmUAUUAGGUUUUUACCUACCC)-NH2] were ordered from ChemGenes Corporation  and capped as described above using the m7G Capping System (CellScript). Capped RNA oligomers were then HPLC-purified, biotinylated with biotin-N-hydroxysuccinimide ester (Epicentre) according to the manufacturer's instructions and again HPLC-purified. As control we used a corresponding 3′-terminal biotinylated and HPLC-purified oligoribonucleotide harbouring a 5′ hydroxyl group [OH-r(AUAUUAGGUUUUUACCUACCCU)-biotin].Capped m7Gppp-oligoribonucleotides matching the first 22 nucleotides of the 5′ untranslated region of Severe Acute Respiratory Syndrome Coronavirus HKU-39849 were prepared by adding N7-methylated cap structures to chemically synthesized RNA oligomers with a 3′-terminal C6 amino linker. A triphosphorylated RNA oligomer [PPP-r] in the presence of 40 U RNase inhibitor (Fermentas) for 60 min at 4°C on a rotary wheel. Control or RNA-coated beads were then incubated with 2-mg samples of HeLa cell lysate for 60 min, washed three times with TAP buffer, and twice with TAP buffer lacking Nonidet-P40 to remove residual detergent. Three independent affinity purifications were performed for each RNA. Bound proteins were dentatured by incubation in 6 M urea-2 M thiourea with 1 mM DTT (Sigma) for 30 min and alkylated with 5.5 mM iodoacetamide (Sigma) for 20 min. After digestion with 1 µg LysC  at room temperature for 3 h, the suspension was diluted in 50 mM ammonium bicarbonate buffer (pH 8). The beads were removed by filtration through 96-well multiscreen filter plates , and the protein solution was digested with trypsin (Promega) overnight at room temperature. Peptides were purified on stage tips with three C18 Empore filter discs (3M) and analyzed by mass spectrometry as described previously For quantitative purification of RNA-binding proteins, streptavidin affinity resin was first incubated with 1-µg aliquots of biotin-labelled OH-RNA, PPP-RNA, CAP-RNA, CAP0-RNA or CAP1-RNA  in TAP buffer [50 mM Tris pH 7.5, 100 mM NaCl, 5% (v/v) glycerol, 0.2% (v/v) Nonidet-P40, 1.5 mM MgClRenilla luciferase-tagged proteins were transiently expressed in 293T cells. Three micrograms of each construct were transfected into 6×106 cells using 9.6 µl nanofectin (PAA Laboratories) in 10-cm dishes according to the manufacturer's instructions. After 24 h, the medium was removed, and cells were lysed in ice-cold TAP lysis buffer. An aliquot (10%) of the lysate was removed to determine input luciferase activity. The rest was added to streptavidin-agarose beads coated with 250 ng of RNA as described above, and incubated on a rotary wheel at 4°C for 60 min. Beads were washed three times and resuspended in 50 µl TAP buffer. Luciferase activities present in the suspension and in the input lysate were assayed in Renilla reaction buffer  containing 10 µM coelenterazine as substrate. The reactions were performed in triplicate and results were quantified using an Infinite 200 PRO series microplate reader (Tecan). For affinity purification of recombinant proteins with different RNAs, 50 to 250 ng of biotinylated RNA were coupled to streptavidin-agarose beads for 60 min at 4°C. Beads were washed three times with TAP buffer and incubated with recombinant His-tagged proteins for 60 min at 4°C. After three washes beads were boiled in Laemmli buffer for 10 min at 95°C and subjected to SDS-PAGE and Western Blot analysis.N-terminally Renilla luciferase (5′-CGAAAGTTTATGATCCAGAAC-3′ and 5′-AATCATAATAATTAATAAATG-3′), hCycB (5′-CAGCAAGTTCCATCGTGTCATCAAGG-3′ and 5′-GGAAGCGCTCACCATAGATGCTC-3′), mTBP (5′-CCTTCACCAATGACTCCTATGAC-3′ and 5′- CAAGTTTACAGCCAAGATTCA-3′), mIFN-β (5′-ATGGTGGTCCGAGCAGAGAT-3′ and 5′-CCACCACTCATTCTGAGGCA-3′), MHV-N (5′-GCCTCGCCAAAAGAGGACT-3′ and 5′- GGGCCTCTCTTTCCAAAACAC-3′), 229E-N (5′-CAGTCAAATGGGCTGATGCA-3′ and 5′- AAAGGGCTATAAAGAGAATAAGGTATTCT-3′), mIfit1 (5′- CCATAGCGGAGGTGAATATC-3′ and 5′- GGCAGGACAATGTGCAAGAA-3′), mIfit1c (5′-AATCAGAAGAGGCAGCCATC-3′ and 5′-CATGGCTTCACTTGTGTTCC-3′), mIfit2 (5′-TCAGCACCTGCTTCATCCAA-3′ and 5′-CACCTTCGGTATGGCAACTT-3′), and mIfit3 (5′-GCTGCGAGGTCTTCAGACTT-3′ and 5′-TGGTCATGTGCCGTTACAGG-3′).Total RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel), including on-column DNase digestion, and 200 to 500 ng of RNA was reverse transcribed with the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). RNA levels were then quantified by real-time RT-PCR using the QuantiTect SYBR Green RT-PCR kit (Qiagen) and a CFX96 Touch Real-Time PCR Detection System (BioRad). Each cycle consisted of 15 sec at 95°C, 30 sec at 50°C and 30 sec at 72°C, followed by melting curve analysis. Primer sequences were as follows: −/−Ifit1 mice have been described −/−Ifit1 mice (kindly provided by Michael Diamond) were injected intraperitoneally with 5,000 plaque-forming units of MHV. Virus titers in samples of spleens removed and frozen 48 h post infection were assessed by standard plaque assay on L929 cells. Bone marrow-derived macrophages or mouse embryo fibroblasts (1 to 5×105 cells) were treated or not with IFN-α and infected with the indicated viruses at a multiplicity of infection (MOI) of 5. For synchronised infection, cells were infected with virus on ice and pre-warmed DMEM growth medium was added 1 h later. To quantify the effects of siRNA-mediated knockdown of IFIT1, aliquots of 105 HeLa cells that had been transfected for 48 h with 15 pmol siRNA and 500 ng expression plasmid for human APN using the siRNA Prime reagent (PAA Laboratories) according to the manufacturer's instructions, were pretreated with IFN-α as indicated and infected with HCoV-229E at an MOI of 1 for 24 h.C57BL/6 mice were obtained from Charles River Laboratories , and g at room temperature. Protein concentration was determined by Lowry assay , and 50-µg aliquots were reduced with 10 mM DTT for 30 min, alkylated with 55 mM IAA for 20 min at room temperature, and precipitated with 80% acetone for 3 h at −20°C. After centrifugation for 15 min at 16,000× g at 4°C, pellets were washed with 80% acetone, dried for 30 min at room temperature and dissolved in 6 M urea-2 M thiourea. Proteins were digested with LysC and trypsin at room temperature and peptides were purified on stage tips and analysed by LC-MS/MS using a Easy nano LC system coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptide separation was achieved on a C18-reversed phase column  using a 95-min linear gradient of 2 to 30% acetonitrile in 0.1% formic acid. The mass spectrometer was set up to run a Top10 method, with a full scan followed by isolation, HCD fragmentation and detection of the ten most abundant ions per scan in the Orbitrap cell.For pulsed SILAC, mouse macrophages labelled with heavy isotopes (see above) were lysed in SDS lysis buffer . The lysate was then heated for 5 min at 95°C, sonicated for 15 min with a Bioruptor (Diagenode) and centrifuged for 5 min at 16,000× Raw mass-spectrometry data were processed with MaxQuant software versions 1.2.7.4 and version 1.3.0.5 S0 was empirically set between 0.2 and 1, to separate background from specifically enriched interactors.In analyzing mass spectrometry data from RNA affinity purifications, only proteins identified on the basis of at least two peptides and a minimum of three quantitation events in at least one experimental group were considered. Label-free quantitation (LFQ) protein intensity values were log-transformed and missing values filled by imputation with random numbers drawn from a normal distribution, whose mean and standard deviation were chosen to best simulate low abundance values. Significant interactors of RNAs with different 5′ end structures were determined by multiple equal variance t-tests with permutation-based false discovery rate statistics For data analysis from pulsed SILAC experiments, we used log-transformed heavy to light protein ratios. Only proteins with valid values were considered for analysis, and normalized by dividing by the row median. Profile plots were generated using LFQ intensities of log-transformed heavy-labelled protein intensities. We excluded proteins containing less than 10 valid values in all 12 measurements, and missing values were filled by imputation. LFQ intensities were then normalized by dividing by the row median.www.R-project.org) and GraphPad Prism version 5.02. Multiple sequence alignments were generated with ClustalW .Results were plotted using R  and further refined with MODELLERs align2d. Human IFIT1 and IFIT5 share approximately 75.6% sequence similarity, with 57.2% of all residues being identical. Cavity volumes in both structures were calculated in a two-step process with the rolling probe method using 3V A homology model of human IFIT1 was obtained with MODELLER Figure S1Generation of 5′end modified in-vitro transcribed RNA. (a) Schematic overview of synthesis of the biotinylated RNA used in this study. 5′ triphosphorylated (PPP-) 7SK-antisense RNA obtained by in vitro transcription with SP6 polymerase was modified enzymatically at the 5′ end by incubating with alkaline phosphatase (AP) to remove 5′ phosphates (OH-RNA), with recombinant Vaccinia virus capping enzyme (VCE) to produce unmethylated capped RNA (CAP-RNA), with VCE in the presence of S-adenosyl methionine (SAM) to generate N7-methylated capped RNA (CAP0-RNA), or with VCE and recombinant Vaccinia virus 2′O methyltransferase (VP39) in the presence of SAM to generate N7-methylated capped RNA methylated at the 2′O position of the first ribose (CAP1-RNA) b) Agarose gel image showing 200 ng of in vitro transcribed, biotinylated RNA following the enzymatic treatments depicted in (a). (c) Evaluation of the N7- and 2′O-methylation efficiency of recombinant Vaccinia virus enzymes. Capped RNAs produced as in (a) were incubated either with VCE or VP39 in the presence of 3H-labeled SAM, and the incorporation efficiency was measured by scintillation counting. 3H-labeled methyl groups were transferred from SAM only if the RNA had not previously been methylated , showing that methylation of RNA by both VCE and VP39 was maximally efficient.(TIF)Click here for additional data file.Figure S2RNA affinity purifications from HeLa cell lysates. (a) Heatmap of all proteins identified in RNA affinity purifications from HeLa cell lysates. Hierarchical clustering of proteins was performed on logarithmic LFQ protein intensities using Euclidean distances. The colour code represents LFQ intensities in rainbow colours . (b) Heatmap showing hierarchical clustering (Euclidean distances) of interactors that were significantly enriched (see c) Volcano plots showing enrichment  and p-values  of CAP1-RNA to CAP-RNA. Data are from three independent affinity purifications. Significantly enriched interactors (see ched see  in fracttors see  are sepa(TIF)Click here for additional data file.Figure S3RNA affinity purifications from lysates of mouse embryo fibroblasts. (a–b) As in b) the saturation threshold is set at −1. 5 and +1. 5. The asterisk indicates the Ifit complex.(TIF)Click here for additional data file.Figure S4Characterisation of the murine IFIT complex. (a) Expression of Ifit genes in wild-type (Ifit1+/+) and Ifit1-deficient (Ifit1−/−) mouse embryonic fibroblasts (MEFs). MEFs were left untreated, treated with 1000 U/ml IFN-α, or infected with Rift Valley fever virus Clone13 or a mutant version of vesicular stomatitis virus (VSV-M2) at a multiplicity of infection of 1 or 0.01, respectively. Sixteen hours later RNA was analysed by quantitative RT-PCR for mIfit1, mIfit1c, mIfit2 and mIfit3. In each case, one representative experiment of three is shown, with means ±SD after normalization to the TATA-binding protein (TBP) mRNA. (b) Heatmap of selected proteins identified in RNA affinity purifications from cell lysates of Ifit1+/+ and Ifit1−/− MEFs. The plot shows the means of log-transformed label-free quantitation protein intensities in rainbow colours . (c) Alignment of murine and human IFIT proteins using ClustalW. (d) Matrix showing amino acid similarity  of all murine and human IFIT proteins. Percent similarity is indicated as color coded from white to red, and the exact similarity is shown within each element of the matrix.(TIF)Click here for additional data file.Figure S5Comparison of the RNA binding cavities of IFIT5 and IFIT1. Sections of surface representations of the solvent-accessible surfaces of IFIT5 (top) and IFIT1 (bottom) are shown, with PPP-RNA bound as in IFIT5 , and the corresponding cavity volumes V calculated as described in 3. The calculated volume of the corresponding cavity of the modelled IFIT1, at 12627 Å3, is about 700 Å3 larger.(TIF)Click here for additional data file.Figure S6Induction of interferon-β in wild-type and Ifit1-deficient mouse cells. Interferon-stimulated bone marrow-derived macrophages (MΦs) from C57/BL6 (Ifit1+/+) or Ifit1-deficient (Ifit1−/−) mice were left untreated, or infected with wild-type MHV (WT), 2′O-methyltransferase-deficient MHV (DA), or Sendai virus (SeV). Twelve hours later total RNA was harvested and analysed by quantitative RT-PCR for interferon β (IFN-β) mRNA. Data from three independent experiments showing fold change relative to untreated cells (mean ±SD) after normalization to the TATA-binding protein (TBP) mRNA.(TIF)Click here for additional data file.Figure S7Translation profiles of individual proteins in MHV-infected macrophages. Translation profiles based on pulsed SILAC of macrophages from C75/BL6 (Ifit1+/+) and Ifit1-deficient (Ifit1−/−) mice infected with wild-type MHV (WT) or 2′O methyltransferase-deficient MHV (DA) as shown in (TIF)Click here for additional data file.Table S1Quantitative MS data from RNA affinity purifications with HeLa cell lysates. Proteins identified by LC-MS/MS from lysates of HeLa cells upon affinity purification with different RNA baits. Table contains log-transformed and imputed label-free quantification (LFQ) intensities of all identified proteins. Significantly enriched proteins, p values and mean differences from t-test based analyses are indicated.(XLSX)Click here for additional data file.Table S2Quantitative MS data from RNA affinity purifications with MEF lysates. Proteins identified by LC-MS/MS from lysates of mouse embryo fibroblasts (MEF) upon affinity purification with different RNA baits. Table contains log-transformed and imputed label-free quantification (LFQ) intensities of all identified proteins. Significantly enriched proteins, p values and mean differences from t-test based analyses are indicated.(XLSX)Click here for additional data file.