RNA sociology investigates the behavioral motifs of RNA consortia from the social science perspective. Besides the self-folding of RNAs into single stem loop structures, group building of such stem loops results in a variety of essential agents that are highly active in regulatory processes in cellular and non-cellular life. RNA stem loop self-folding and group building do not depend solely on sequence syntax; more important are their contextual  needs. Also, evolutionary processes seem to occur through RNA stem loop consortia that may act as a complement. This means the whole entity functions only if all participating parts are coordinated, although the complementary building parts originally evolved for different functions. If complementary groups, such as rRNAs and tRNAs, are placed together in selective pressure contexts, new evolutionary features may emerge. Evolution initiated by competent agents in natural genome editing clearly contrasts with statistical error replication narratives. In contrast with the evolutionary narratives favoring selection of random replication errors of nucleic acids that constitute the genetic text, we do not know any real-life languages or codes that have emerged as randomly derived replication errors of sequences of characters ,2. EveryThe coordination of the social behavior of living agents follows rules that are additional to natural laws. This is the reason why natural sciences, such as physics and chemistry, cannot explain biological communication, because the rule-following of living agents that communicate is different to underlying unchangeable natural laws .If we look at behavioral motifs of RNAs in addition to their physical chemical reactions, we focus on an additional level of empirical investigations. Groups (consortia) and their single members can change their behavior according to adaptational and coordinative needs—this means there are certain categories of group behavior. One of the most basic group behaviors is the self/non-self differentiation competence, which implies activities that maintain group membership and in parallel with the rejection/defense of non-group members. Only if group identity can be reached, it is possible to generate and use commonly shared rules to correctly combine signs to sign-sequences according to contextual needs. Group behavior in this perspective is completely missing in abiotic nature, no syntactic, pragmatic and semantic rules of sign-use are present.If we look at the sequence structure of nucleic acid codes, some RNA stem loop groups are currently known to play vital roles in editing genetic text as a read and write medium ,14. This i.e., although different parts were derived originally for different functions, now all parts play an important role in coordinated interaction and the consortium must be established as a whole to function appropriately [Some ribonucleoproteins (RNPs) such as editosomes, spliceosomes and ribosomes are constituted of a variety of RNA stem loops that counter-regulate themselves during assembly in various steps and substeps. Also, small changes in the composition structure destroy their functionality. It must be assumed that the assembly occurs complementarily,priately .The RNA stem loop or “hairpin” loop is the basic motif with which RNA agents combine to form the more complex agents we know as tRNAs, rRNAs and self-splicing group II introns ,18,19. EA crucial competence for group building is RNA ligation ,23. WithRNAs that can form both simple base-pairing stems with non base-pairing loops by self folding, as RNA stem loops become subject to biological selection. RNA groups representing information and catalytic function provide themselves with a biotic feature such as self/non-self differentiation competence, acting together in variable contexts and with clear behavioral motifs, such as the natural genome editing of themselves, as well as of the DNA storage medium of their cellular host organisms. Now, we must ask how do RNA stem loop groups achieve the intriguing capability to colonize the DNA of host organisms? How do they organize themselves into being appropriate modules for reuse in DNA habitats by their cellular host or into being active (or silenced) as a regulatory element for gene expression?One of the most important behavioral motifs is termed ‘addiction module’, linking originally competing functions: the former competitive groups are now linked together in a counterbalancing manner, serving as the identity feature within a host genome. This implies that disturbances within this counterbalanced module can result in massive deregulation of genetic functions that we can identify in a variety of diseases.Generally, this behavioral motif of complementarity building without destroying one of the participating modules to create a unique structure that functions as an identity feature for the genotype of a host represents a key behavior in evolution: it transfers multiple genes within one infection event and contains multiple parts in parallel. In this respect I would like to predict that the more regulation features in living organisms we can look at, the more such addiction modules will be detected.Addiction modules can be defined as features that consist in general of a stable toxic component that is counterbalanced by an unstable component, which inhibits and suppresses the toxic component ,25,26. BSeveral kinds of addiction modules are known. First, and most prominent in bacterial life, is the restriction/modification addiction module, which is a common feature with immune functions. One part consists of an antitoxic modification enzyme, which is an unstable beneficial (protective) agent. The counterpart consists of a toxic restriction enzyme component, which is a stable but harmful (destructive) agent. Another kind of addiction module consists of two related features. There is an antitoxic antipore toxin that represents the unstable protective agent and a toxic component with a toxic pore, which represents the stable but destructive agent . A thirdAdditionally the basic motif of a counter-regulated module reminds us on structures with similar features, such as the nuclease/ligase regulation (see above), as basic competence for self/non-self differentiation within RNA stem loop groups ,33,34, tIf we look at the virosphere, we can find some basic motifs of group building and group cooperation between genetic parasites that act in a similar way and are cooperatively supported by subviral agents, defectives, or remaining viral parts that can be reused in other contexts. We can find them as genetic parasites, mixed parasites (epiparasites), hyperparasites (parasites of parasites), cooperating or competing according to changing circumstances in various ways .Key to generating the RNA structural motifs is the ability of a RNA to fold back to itself and build groups (consortia) of such stem loops via ligase procedure out of nuclease generated modules. The folding loop remains a single-stranded RNA sequence. Various motifs have been identified, yet all of them share a common function: they stabilize RNA tertiary formation. Such motifs include pseudoknots, kissing loops, A-minor motifs, A-platforms, kink-turns, S-turns, tetraloops and their receptors, and a variety of non-canonical base-pairs and base-triples . i.e., ribozymes, self-splicing introns, telomerase and ribosomal frameshifting in several viruses.Consortia of single RNA sequence strings build one of the most interesting structures: The pseudoknot is composed of two helical segments connected by single-stranded regions or loops that base-pair with complementary nucleotides . Bases iInterestingly, the base-pairing in pseudoknots is strictly context-sensitive and base-pairs overlap with one another in sequence positions. This imposes limits on algorithm-based prediction models, such as dynamic programming or stochastic context-free grammars .Sequence specificity is not required for the various functions, but forms the structure, that is, a tool for appropriate pseudoknot function. The building-up of ribosomal subunits, ribozymes, and reverse transcriptase, such as telomerase, depends on pseudoknots. Replication, as well as RNA processing and inactivation of toxins, gene expression control are additional functions. Last but not least, pseudoknots are involved in translation modification such as internal translation initiation, ribosome rescue and frameshifting . This inde novo generation of sequence space. Pseudoknotting seems to be a special viral competence, including the control of virus genome translation, replication, the switch between translation and replication, and genome packaging, later transferred to cellular domains via persistent infections.The most important role of pseudoknots seems to be genetic control and feedback via cooperative motifs of pseudoknots with riboswitches in destabilizing ligand-free or stabilizing ligand-bound conformations ,44. PseuA special RNA folding motif of the class of pseudoknotting is represented by stem-loop “kissing,” in that single-stranded regions of RNA stem loops bind according to molecular syntax rules to other single-stranded stem loop structures to unite and build more complex group identities . In kissFor several decades the mainstream perspective on non-coding RNAs resulted in the assumption that they are useless remnants of former evolutionary stages, which represent “junk”. This perspective is now falsified. Non-coding RNAs play vital roles and function in gene regulation and coordinate, as well as organize various actions, such as chromatin modification, epigenetic memory as a prerequisite of learning processes, transcriptional regulation, control of alternative splicing, RNA modification, RNA editing, control of mRNA turnover, control of translation and signal transduction .Most of these non-coding RNAs are divided into smaller RNAs that are integral parts of ribonucleoprotein (RNP) complexes. Small RNA species include micro RNAs, small interfering RNAs, small nuclear RNAs and small nucleolar and transfer RNAs . AlthougMicro RNAs and their associated proteins are ribonucleoproteins. Small non-coding RNAs also share a special competence regarding epigenetic regulation of gene expression and are derived from repetitive genomic sequences . The capInterestingly, small nuclear RNAs and small nucleolar RNAs counter-regulate activities which indicate similarity to an “addiction” module. Small nucleolar RNAs guide pseudouridylation and 2’O-ribose methylation of rRNAs in forming duplexes with their target. Small Cajal body-specific RNAs guide modifications of splicesomal RNAs. Small nucleolar RNAs have been identified as a family of mobile genetic elements  and otheSmall RNAs such as endogenous miRNAs and siRNAs share biogenesis and can perform interchangeable functions . They caSiRNAs are expressed by extended double-stranded regions of long inverted repeats, can inhibit the expression of nearly any target gene in response to double-stranded RNA and have a very efficient and ancient immune function against genetic parasites ,62. TheySmall interfering RNAs or microRNAs act in a coordinated manner in that they share the division of labor in hierarchical steps of suppression and amplification. This is indicated in transposable elements that encode both siRNAs and miRNAs . They caThe defense mechanism of host genomes against transposable element invaders through siRNA evolved into miRNAs with a new regulatory complexity and a new phenotype. First evolving as an immune function, they were later co-opted as a tool for complex regulatory pathways in host gene expression . This ex i.e., additionally to physical and chemical laws they coordinate group behavior in accordance with pragmatic, semantic and syntactic rules.If we look at the crucial difference between the physical chemical properties of RNAs and their social group behavior, we see that single RNA hairpins act exclusively according to physical and chemical laws, which do not underlie natural selection processes. In stark contrast, groups (consortia) of RNA stem loops generate behavior that underlies biological selection ,21,22. CSimple RNA structures can become biologically functional if they are used and integrated within higher order structures. This means single RNA nucleotides are not functional unless they become part of group identities. To encode content (information), social interactions (function) by duplex building and further ligations are necessary to form identity groups. This parallel set of levels—information and function—designates the fundamental split between inanimate and animate nature that can be found here, the absence or presence of social interactions based on natural codes. i.e., rule-governed interactions. The RNA sociology perspective therefore contrasts the narrative of the genetic code as a molecular random assembly, as being obviously the result of competent editing by social interacting RNA groups  . UnfortuA groups . i.e., the correct site of the nucleic acid sequence to edit nucleotides. We can speculate that they identify sequence syntax or specific markings on the sign sequences that are relevant to common interactions that are subject to change. Since the rise of epigenetics, the marking of sequences is an important designation of how to identify sequence syntax for modification. The evolutionary derivation of epigenetic markings is not answered so far, although agents of the virosphere are known to be most relevant in this competence.Let us take a look at some such code-editing RNA groups and the essential ribonucleoproteins that act on the nucleic acid language. They must share a group identity, which is able to reject/defense non-group members. Additionally they must identify content (information),We can start with an important context-dependent sequence operator, the editosome. RNA editing is a co- or post-transcriptional process that alters the RNA sequence derived complementarily to the DNA from which it was transcribed. RNA editing changes gene sequences at the RNA level. The edited mRNA specifies an amino acid sequence that is different from the protein that could be expected and is encoded by the genomic DNA of the primary transcript ,69. RNA cis-acting signals. In a second step, the editing site has to be processed by RNAs or proteins. In a further step, enzymatic agents have to be directed to the editing site to process the editing. All steps are catalyzed by a ribonucleoprotein complex known as the editosome. Additionally, the editosome is constituted of a variety of subcomponents, which counterbalance each other [RNA editing must “recognize” (identify) the site that has to be edited. This is not a random process and needs some ch other . RNA edich other . i.e., with more than one meaning (function). Additionally, the code modification can reach high levels of complex interactions, which multiply the possible outcomes. After the editosome another ribonucleoprotein complex acts on the result of the editosome: the spliceosome.The variable meanings of identical sign sequence syntax are an inherent feature of natural languages and codes. This optimizes energy costs in that informational content fixed in an information storage medium can be modified according to environmental needs in different ways,It is assembled in various steps and cuts out introns and splices exons together to form the final protein coding set . RNA ediInterestingly, the variety of steps in which the subunits of the final spliceosome are produced are counterbalanced within competing parts that regulate the stepwise processing of subcomplexes of the spliceosome. Besides the H complex, the E complex, the SR proteins within the E complex, the A complex, the B complex and the C complex are required steps before the final exon ligation processing occurs.cis-acting elements that precisely balance regulatory proteins. This means that the competition of exon ligation and exon rejection is instable from the beginning until the end of spliceosome assembly. As a consequence, alternative splicing is regulated until the latest stage of splicing actions.After this final splicing procedure of the mature spliceosome, the remaining RNA products are actively discharged from the spliceosome and recycled for further catalytic processes. Depending on the regulations, the end-product may vary concerning the context dependency of the regulation process, which is highly sensitive to various needs and circumstances. In consequence, spliceosomal regulation differentiates the inclusion or exclusion (enhancers or silencers) of exons in the final mRNA . SplicinThe crucial competence of the spliceosome is the appropriate and precise splice site “recognition” (identification) of consensus sequences that define exon-intron boundaries ,79. DepeAfter the RNA groups of editosome and spliceosome have acted to arrange a coherent nucleic acid sequence and are ready for translation into proteins, a third RNA group becomes active: The ribosome is the key player in translation after RNA editing and RNA splicing. Because the ribosome is at least the most important player in cellular life replication and is absent in viral genomes, it must have been a crucial evolutionary step from the capsid-encoding organisms of the pre-cellular RNA world and the ribosome-encoding organisms in the cellular life world .Transcription and assembly of the two ribosomal subunits occur in the nucleolus, which itself must be assembled by a highly regulated process during teleophase . The nucRibosomes are comprised of two-thirds RNA and one-third protein (by mass). As we understand it, ribosomal proteins are vital for structural stabilization. Around the catalytic site of the ribosome there are only RNAs and no ribosomal proteins . This meOne of the outstanding explorations in the last decades was the multiple ways how identical nucleotide sequences that serve as genetic storage medium undergo a variety of modifications during transcription as outlined above. The translational process from DNA via RNA and an abundance of regulatory processes of non-coding RNAs is object to a further process to dynamically modify genetic storage semantics. During the translational process, ribosomes may bypass and therefore ignore parts of the mRNA sequence. In this behavioral motif, translation is blocked at a certain codon, and a different stem loop RNA ensures the further translation process at a certain stage of the mRNA . In the Alan Weiner and Nancy Maizels showed convincingly that transfer RNAs represent a combination of two formerly different modules ,93. The Pre-tRNAs perform self-cleavage, which is clearly a ribozymatic reaction independent of translation ,99,100 aThe rise of epigenetics is more than an epiphenomenon within molecular biology. It marks an important perspective in which the given genetic information is marked according to different expression patterns depending on the tissue specificity and according to environmental conditions that make it necessary to change expression patterns ,103. OneChromatin marking represents a further behavioral motif that enables a kind of identity programming, which may or may not be heritable. This means a specific cell within an organism is able through epigenetics to obtain an identity or even to change its identity if environmental conditions make it necessary. Only RNAs are able to mark DNA sequences. RNA agents are mobile and can serve as signals throughout tissues, organs and whole organisms. In this respect the imprinting of new experiences that leads to variable meanings of genetic information depends on the ability of non-coding RNAs ,105,106.Both small RNAs and long non-coding RNAs are competent in terms of directing chromatin changes through histone modifications and DNA methylation. These non-coding RNAs are able to direct chromatin-modifying agents to specific targets. Interactions between non-coding RNAs and such agents are components of these regulatory networks . In smalAs mentioned before the evolutionary derivation of this special competence to identify specific sites for editing, splicing or epigenetic markings is not answered, although agents of the virosphere are known to be most relevant in this competence. Therefore epigenetic markings too, which are common in the virosphere for fixing identity of agents and enabling the differentiation between self and non-self, seem to be exapted to the world of DNA-based cellular life.Current knowledge about the virosphere and virolution indicate interactions of RNA viruses, DNA viruses, RNA-DNA viruses, viral swarms, viral and RNA-based sub-viral networks that cooperate and coordinate (regulate) within cellular genomes either as replication-relevant co-players or suppression-relevant silencers ,113,114.Persistent viral lifestyles are counterbalanced by viral properties found as addiction modules, that transfer complete genetic data sets into host genomes and alter the DNA genetic identity of the host and, additionally, the formerly competing viral agents, without damage to the host genome content . As we sgag, pol and env [Endogenous retroviral competences in the persistent status are often characterized by features expressed only in the strict time window of a developmental process, such as axis formation, trophectoplast formation , or the S phase of the cell cycle. In these highly specialized contexts, they are replicated through signaling, which blocks the suppression of the replication process. After the function is fulfilled, a signal once again initiates suppressor function. Retroelements—with their (1) higher-order regulatory functions, (2) ability for genetic creativity and (3) capacity for innovation of new regulatory patterns and combinations—descended from retroviruses, which can easily be identified by their three essential parts  and env . Most en and env . This me and env .The lifestyle of endogenous retroviruses and other endogenous viruses that compete or cooperate was long assumed to be a rare and exceptional phenomenon. Today we know that this is common use and DNA generally serves as habitat for RNA inhabitants in abundant numbers . In mostWe saw a variety of behavioral motifs of RNA groups that, although constituted by molecules, behave like living agents. They infect host DNA, settle within nucleic acid sequences, differentiate between self and non-self group membership, reject foreign members, assemble to cooperative groups that build essential tools in DNA based cellular life and identify relevant syntax in certain sequence sites, store environmental experiences into DNA content and act according circumstantial needs, to faster adapt to changing environmental situations. This means, additional to underlying natural laws, they follow rules that are absent in inanimate nature.As in all known natural languages or codes, in the nucleic acid code we can also identify agents that generate and use code by an inherent competence to correctly combine signs to sign sequences according to contextual needs. These agents in most cases are infective RNAs, such as viruses, and sub-viral agents, such as viroids, and other related genetic parasites. Such competent agents interact, cooperate and integrate in host genomes where they are fully integrated or, in most cases, remain as defectives serving as module-like elements for co-opted adaptations in host gene regulation. As we now know, the RNA nucleic acid sequence is subject to a variety of alterations and modifications after or parallel with transcription out of DNA. Crucial for the function of such alterations is a precise site “recognition” (identification) where the change operators start or stop their competent action. This competence recalls phenomena such as self/non-self differentiation as a pre-requisite for memory and learning.de novo generation of behavioral patterns as well as de novo generation of new sequence content. Competent de novo generation of new sequence content is completely different from statistical error replication. This was the finding of a decade-long discussion on the essential features of natural languages or codes.Whereas interactions between molecules and atoms underlie natural laws strictly, social behavior of agents in animate nature follows additional motifs, such as rules that govern sign-mediated interactions. In contrast to natural laws, such rules may change according to contextual circumstances to adapt faster to appropriate behavior and additionally are completely absent in inanimate nature. Living agents in populations that interact according to rule-governed sign-mediated interactions are therefore both able to coordinate behavior and able to install de novo in terms of social sciences . Then, the empirical results of molecular biology would mean that RNA sociology could serve as empirical non-mechanistic explanation of social interactions: from single RNA stem loops to group (identity) building, self/non-self identification competence, the emergence of context-dependent interactions, and the cooperation between RNA groups and their host organisms, most of them counterbalanced by addiction modules. Additionally, RNA sociology could explain the de novo generation of nucleic acid sequences and their coherent integration into pre-existing ones, innovation by variations in RNA stem loops and, last but not least, innovative genetic identity by co-evolution.The challenge is to think about agents that are able to generate nucleic acid sequences


Ophioglossum californicum and Psilotum nudum. Using a relaxed automated approach to minimize false negatives combined with manual inspection to eliminate false positives, we identified 297 C-to-U and three U-to-C edit sites in the O. californicum plastid transcriptome but only 27 C-to-U and no U-to-C edit sites in the P. nudum plastid transcriptome. A broader comparison of editing content with the leptosporangiate fern Adiantum capillus-veneris and the hornwort Anthoceros formosae uncovered large variance in the abundance of plastid editing, indicating that the frequency and type of RNA editing is highly labile in ferns. Edit sites that increase protein conservation among species are more abundant and more efficiently edited than silent and non-conservative sites, suggesting that selection maintains functionally important editing. The absence of U-to-C editing from P. nudum plastid transcripts and other vascular plants demonstrates that U-to-C editing loss is a recurrent phenomenon in vascular plant evolution.The distinct distribution and abundance of C-to-U and U-to-C RNA editing among land plants suggest that these two processes originated and evolve independently, but the paucity of information from several key lineages limits our understanding of their evolution. To examine the evolutionary diversity of RNA editing among ferns, we sequenced the plastid transcriptomes from two early diverging species,  Numerous studies of RNA editing in seed plants (particularly angiosperms) have identified hundreds of C-to-U edit sites in the mitochondria and dozens of plastid C-to-U sites [Takakia and Haplomitrium appear to have many C-to-U sites [In land plants (Embryophyta), plastid and mitochondrial transcripts undergo a type of post-transcriptional processing called RNA editing, which converts specific cytidines to uridines (C-to-U) or uridines to cytidines (U-to-C) through undefined mechanisms (reviewed in –3). Surv-U sites –8. A few-U sites –12, alth-U sites –15. Howe-U sites –18, whil-U sites .Isoetes and Huperzia organelles, with pervasive editing in Isoetes mitochondria [Selaginella organelles, whereas no U-to-C editing was found, indicating a secondary loss of U-to-C editing in this genus [Adiantum capillis-veneris and Pteridium aquilinum [Equisetum and Psilotum [Equisetum and Psilotum [The pattern of RNA editing is more complex in hornworts, lycophytes, and ferns. Frequent C-to-U and U-to-C editing has been identified in plastid and mitochondrial transcripts from many hornworts –22. Amonchondria –26. C-tois genus , 28. Thequilinum , 30. In Psilotum , 31. AnaPsilotum –34.Selaginella and most  seed plants. Several ferns may also lack U-to-C editing, but complete organellar transcriptomes are needed to substantiate this possibility. To begin to understand the evolutionary diversity of RNA editing among ferns, we sequenced the plastid transcriptomes of two early diverging species, the adder’s-tongue fern  and the whisk fern (Psilotum nudum), using the Illumina platform.The above results illustrate the differential distribution of C-to-U and U-to-C RNA editing among land plants, which implies independent origins and subsequent evolutionary trajectories of the two editing processes. C-to-U editing has been observed in the mitochondria and plastids of all major land plant groups but in none of the closely allied green algal lineages nor in marchantiid liverworts, indicating an origin in the common ancestor of land plants followed by a single loss of editing from marchantiid liverworts. In contrast, U-to-C editing appears to be confined to ferns, lycophytes, and hornworts. Most current hypotheses about land plant relationships indicate that hornworts are the sister group to vascular plants –37, suggO. californicum and P. nudum plants, which were previously sequenced to obtain the plastid genome sequences [O. californicum and KC117179 for P. nudum), were obtained from the living collection at the Beadle Center Greenhouse (University of Nebraska–Lincoln). For each species, an organelle-enriched RNA sample was prepared. Mature, above-ground tissue (50–100 g) was homogenized in a Waring blender, filtered through four layers of cheesecloth, and then filtered through one layer of Miracloth. The filtrate was centrifuged at 2500 × g in a Sorvall RC 6+ centrifuge, and then the supernatant was centrifuged at 12000 × g for 20 min to pellet organelles. RNA was isolated from the pellet using the RNeasy Plant RNA Kit (QIAGEN) and treated with DNase I (Fermentas) according to manufacturer’s instructions. Ribosomal RNA content was reduced from the enriched organellar RNA using the RiboMinus Plant Kit for RNA-Seq (Invitrogen) according to the supplied protocol.equences  . Adapter and low-quality sequences were trimmed from the O. californicum and P. nudum raw reads using cutadapt version 1.4.1 (https://code.google.com/p/cutadapt/) with modified parameters (-a AGATCGGAAGAGC-q 20-m 35) before further analysis.The RiboMinus-treated organellar RNAs for both species were sent to the University of Nebraska Genomics Core Facility for 75 bp single-read sequencing on the Illumina GAII platform, generating 26.6 M raw reads for O. californicum and P. nudum plastid genomes using TopHat version 2.0.9 [O. californicum and P. nudum plastid transcriptomes by comparing the mapped transcript reads to the reference genome sequences. To do so, a pileup mapping summary file was generated with the mpileup command in SAMtools version 0.1.19 [All remaining data were mapped to the on 2.0.9  with relon 2.0.9 . Mismatcn 0.1.19 . All DNAThe read depth of the transcriptome mapping and the proportion of reads containing a mismatched nucleotide were calculated at each reference genome position by comparing the resulting pileup file with the reference plastid genome sequences. The pipeline required that all DNA:RNA mismatches have a minimum read depth of 3X and at least 5% of total reads or three individual reads supporting the mismatch, whichever is greater. Initial analysis on untrimmed data using these criteria  DNA heteroplasmy , 2) errors in the transcriptome reads due to imperfect binding of random hexamers during cDNA preparation, as described previously , 43, 3) To eliminate false positives from the automated approach, we used a defined set of criteria to identify DNA:RNA mismatches arising by one of the alternative explanations defined above. Heteroplasmic regions in the genomes were detected by mapping Illumina DNA sequence data from the plastid genome sequencing projects  onto theO. californicum, P. nudum, Anthoceros formosae (AB086179), and Adiantum capillus-veneris (AY178864) were aligned with clustalW [To compare editing content among species, genes from clustalW  and manuO. californicum and P. nudum plastid transcriptomes, we generated an automated bioinformatics pipeline that compared transcript reads to the plastid genome sequences that were previously sequenced from the same plants [To detect edit sites in the e plants . AlthougManual inspection of results identified several sources of error that generated false positive signals. First, heteroplasmy  occurring in transcribed segments of the genome produces polymorphism among transcripts, which generated a mismatch signal in our automated approach. To eliminate this issue, mismatches occurring at heteroplasmic sites (identified by mapping Illumina DNA reads onto the genome sequences) were removed from the results. Second, there was increased sequencing error at the ends of reads, mostly likely caused by imperfect binding of random hexamers during cDNA library preparation , 43. To O. californicum plastid transcriptome  and two stop codons (chIL and petD) by C-to-U editing and the removal of an internal stop codon (ycf1) by U-to-C editing  and the hornwort outgroup (Anthoceros formosae) shows a large amount of variation in the frequency and type of RNA editing among species , decreased conservation to homologous proteins (non-conservative sites), or did not alter the encoded amino acid (silent sites), and then we calculated editing efficiency as the proportion of reads that contained the edited nucleotide in the plastid transcriptome , while an edit site at position 74 in the psaI gene changes a conserved serine to a phenylalanine at 15% efficiency (41/267 transcripts). Both events substitute a small, hydrophilic amino acid with a large, hydrophobic amino acid, which may have negative effects on protein folding and function. In two more extreme cases, premature stop codons are introduced in the clpP gene at position 136 with 6.7% editing efficiency (5/75 transcripts) and in rpl20 at position 172 with 6.0% editing efficiency (16/267 transcripts). These events, which truncate >75% of the CLPP protein and half of the RPL20 protein, would almost certainly abolish their functional activity.The rarity and generally low editing efficiency of the non-conservative sites in the -editing . For exaP. nudum. Together, these findings show that the frequency and type of RNA editing is highly labile in ferns. Importantly, the discovered loss of U-to-C editing from the P. nudum plastid transcriptome, along with the independent losses of U-to-C editing from seed plant and Selaginella plastids and potentially from additional fern lineages, reveals a recurrent pattern of loss of this process from vascular plants ,–51,48–51ganelles , 52, 53.ganelles , 54, 55.O. californicum and inefficient editing (down to only 5% efficiency), although we acknowledge that sites edited at less than 5% efficiency or covered by fewer than three transcript reads would have been missed by our approach. Because these relaxed parameter values increased the number of false positives, we manually evaluated all detected mismatches to eliminate signals due to biological and technical issues. In fact, our manual examination of results eliminated all of the mismatches that could not be caused by C-to-U or U-to-C RNA editing, verifying that our manual approach is able to efficiently detect and eliminate false positives. In summary, our work demonstrates that automated detection approaches alone are not sufficient for accurate editing detection. It is imperative to manually curate the automated results to ensure that false edit sites are not reported and true edit sites are not missed due to the issues described above. Additional suggestions, such as sequencing from organelle-enriched RNA, using DNase I to ensure no DNA contamination, priming with random hexamers rather than oligo-dT primers, subtracting rRNA from the RNA preparation, and employing sequence aligners (such as TopHat) that can handle mismatches and splicing junctions, were recently proposed to ensure optimal analysis of plant mitochondrial transcriptomes . Many ofS1 FigThe reference genome sequence is shown on the first line, the consensus mapping sequence is shown on the second line, and the remaining sequences are individually mapped reads. Numbers in the ruler indicate positions in the reference genome sequence.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigShown at bottom is the unspliced reference genome sequence and the spliced RNA sequence.(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Here we discover an Xist antisense long non-coding RNA, XistAR (XistActivating RNA), which is encoded within exon 1 of the mouse Xist gene and is transcribed only from the inactive X chromosome. Selective truncation of XistAR, while sparing the overlapping Xist RNA, leads to a deficiency in Xist RNA expression in cis during the initiation of X inactivation. Thus, the Xist gene carries within its coding sequence an antisense RNA that drives Xist expression.The transcriptional imbalance due to the difference in the number of X chromosomes between male and female mammals is remedied through X-chromosome inactivation, the epigenetic transcriptional silencing of one of the two X chromosomes in females. The X-linked Xist long non-coding RNA functions as an X inactivation master regulator; Xist is selectively upregulated from the prospective inactive X chromosome and is required in  The X-chromosome linked long non-coding RNA, Xist, is a master regulator of the X inactivation. Here, the authors report that XistAR, an Xist anti-sense long non-coding RNA encoded within the mouse Xist gene and transcribed only from the inactive X chromosome, regulates Xist expression. In a shared nucleoplasm, one X chromosome of an identical pair undergoes transcriptional inactivation in mammalian females early during embryonic development. Once inactivated, replicated copies of the inactive X chromosome are transmitted as inactive through many rounds of cell division23789101112Xist in cis and is required for X inactivation.Here we describe the discovery of a novel Xist antisense transcript that is embedded within exon 1 of Xist. This transcript is co-expressed with Xist from the inactive X chromosome. On truncation of the antisense transcript, Xist induction is diminished by ∼90%, resulting in defective X-linked gene silencing on the inactive X. Thus, an Xist antisense RNA activates We previously derived F1 hybrid male and female mouse trophoblast stem (TS) and extra-embryonic endoderm (XEN) cell lines deficient in the expression of the Xist antisense transcript Tsix15161718XΔTsixY male TS and XEN samples did not display specific amplification of Tsix  on RNA purified from Tsix-mutant TS and XEN cell lines. We excluded amplification of genomic DNA in reactions lacking reverse transcriptase in all sets of RT–PCRs . As an a of Tsix . XΔTsixXamplicon . On SangXist/Tsix sequence. Whereas the X chromosome harbouring the intact Tsix locus is derived from the Mus molossinus JF1 strain (XJF1), the XΔTsix X chromosome is inherited from a Mus musculus laboratory strainExpression in mutant female but not male cells suggested that the transcript originated from the inactive paternal X chromosome and not the active maternal X chromosome, as would be expected of Tsix in these cellsMus molossinus-derived XJF1 X chromosome, the other X is derived from a M. musculus laboratory strain (XLab), as in i) and reciprocal (F1r) crosses displayed the specific amplification of antisense RNA(s) in males as well as females .We next assayed Xist antisense expression in wild-type (WT) F1 hybrid male and female TS and XEN cells. To distinguish parent-of-origin-specific from strain-specific bias in expression, we also tested F1 hybrid TS and XEN cells generated from the reciprocal parental cross females . SNP pro females . Since TWe also independently confirmed XistAR expression from the inactive X by strand-specific RNA fluorescence in situ hybridization (FISH). A single-stranded RNA FISH probe designed to hybridize specifically to Xist antisense transcripts detected a signal that coincided with or was adjacent to the Xist RNA-coated inactive X chromosome in a subset of nuclei in both TS and XEN cell lines 19212324 females . We conf females . Thus, sWe then wished to know if XistAR RNA is detected from the inactive X in cells of the developing embryo itself. We therefore assayed the expression of XistAR by strand-specific RNA FISH in embryonic day (E) 3.5 mouse blastocysts, which undergo imprinted inactivation of the paternal X in all cellsXistAR by 5′ RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) and by RT–PCR    in the antisense orientation at bp 933 of Xist (pA) ref.  Fig. 6aVS) refs , 35  by RT–qPCR, we found that the XXpA blastocysts did not exhibit a significant increase in XistAR abundance compared with XXIVS and XX embryos  to induce Xist. eRNAs are broadly transcribed from enhancers and may contribute to their function .TsixpA and XistIVS strains has been described previouslyTsixpA and XistIVS females with C57Bl/6 males. The X-linked GFP transgenic (X-GFP) and JF1 strains have also been described previously153655The generation and characterization of −1 BSA . Zona pellucida surrounding E3.5 embryos were removed through incubation in cold acidic Tyrode's solution , followed by neutralization through several transfers of cold M2 medium . Green fluorescent protein (GFP) fluorescence conferred by the paternal transmission of the X-GFP transgene was used to distinguish female from male embryos, since only females inherit the paternal X chromosome. Embryos were either lysed for RNA isolation or plated onto gelatin-coated glass coverslips in 1 × PBS with 6 mg ml−1 BSA for RNA FISH staining. Excess solution was aspirated, and the plated embryos were air dried for 15 min. After drying, embryos were permeabilized and fixed in 50 μl solution of 0.05% Tergitol  and 1% paraformaldehyde  in 1 × PBS for 10 min. Excess solution was tapped off, and coverslips were rinsed thrice with 70% ethanol and stored in 70% ethanol at −20 °C before RNA FISH.E3.5 embryos were isolated essentially as described−1 BSA. Embryos were dissected from the decidua, and the Reichert's membranes surrounding post-implantation embryos were removed using fine forceps. For separation of extra-embryonic and epiblast portions of E6.5 embryos, fine forceps were used to physically bisect the embryos at the junction of the epiblast and extra-embryonic ectoderm. The epiblast was further distinguished by GFP fluorescence conferred by the paternally transmitted X-GFP transgene; the transgene is mosaically expressed in the epiblast due to random X inactivation but is silenced in the extra-embryonic tissues because of imprinted X inactivation of the paternal X153655−1 BSA onto gelatinized coverslips. The samples were permeabilized, fixed and stored in 70% ethanol as described above for E3.5 embryos, before RNA FISH.For isolation of E6.5 embryos, individual implantation sites were cut from the uterine limbs and decidua were removed with forceps in 1 × PBS/6 mg mlL-glutamine , 37.5 ng ml−1 FGF4  and 1.5 μg ml−1 heparin . Following 5 days of growth at 37 °C with 5% CO2, blastocyst outgrowths were dissociated with 0.05% trypsin . Dissociated cells were plated on MEFs and cultured at 37 °C with 5% CO2. Once TS lines were established, TS cells were collected for total RNA preparation using Trizol . For RNA FISH, TS cells were split onto gelatin-coated glass coverslips and allowed to grow for 2–3 days. The cells were then permeabilized through sequential treatment with ice-cold cytoskeletal extraction (CSK) buffer  for 30 s, ice-cold CSK buffer containing 0.4% Triton X-100  for 30 s, followed twice with ice-cold CSK buffer for 30 s each. After permeabilization, cells were fixed in 3% paraformaldehyde for 10 min. Cells were then rinsed thrice in 70% ethanol and stored in 70% ethanol at −20 °C before RNA FISH.TS cell derivation, culture and characterization was carried out essentially as described previously1415−1 penicillin/streptomycin , 20% FBS, 1 mM sodium pyruvate, 100 μM β-mercaptoethanol, 2 mM L-glutamine, 100 μM nonessential amino acids , 1,000 units ml−1 leukemia inhibitory factor (Millipore # ESG1107). Following 6–8 days of growth at 37 °C with 5% CO2, blastocyst outgrowths were dissociated with 0.05% trypsin. Dissociated cells were plated into individual wells of a 96-well dish containing MEFs and cultured at 37 °C with 5% CO2. The cells were then split into a single well of a four-well dish containing MEFs. After confluency, the cells were trypsinized and plated into a gelatinized well of a six-well dish in XEN growth media . Total RNA was collected from XEN cells using Trizol for RT–PCR. For RNA FISH, XEN cells were split onto gelatin-coated glass coverslips and allowed to grow for 2–3 days. The cells were then permeabilized, fixed and stored in 70% ethanol as described above for TS cells.XEN cells were derived, cultured and characterized as described previously15L-glutamine, 1 × nonessential amino acids and 100 μM β-mercaptoethanol. After 5–6 days, outgrowths were partially dissociated with 0.05% trypsin. The partial dissociates were plated individually on MEFs in a well of a four-well dish and cultured for an additional 4–6 days in K15F5 medium. The culture was then passaged by a brief exposure (2–3 min) to 0.05% trypsin/EDTA  with gentle pipetting to prevent complete single-cell dissociation of pluripotent clusters, and plated on MEFs in a single well of a six-well dish in K15F5 medium. Morphologically distinct EpiSC colonies became evident over the next 4–8 days. EpiSC colonies were manually dissociated into small clusters using a glass needle and plated on MEFs in a single well of a four-well dish in EpiSC medium consisting of knockout DMEM supplemented with 20% knockout serum replacement, 2 mM Glutamax , 1 × nonessential amino acids, 100 μM β-mercaptoethanol and 10 ng ml−1 FGF2 .EpiSC lines were derived, cultured and characterized as described212223Total RNA was purified from cultured TS, XEN and EpiSC cell lines using Trizol reagent . Total RNA was treated with DNase  to remove any contaminant genomic DNA. mRNA was isolated from DNase-treated total RNA using Dynabeads mRNA DIRECT Micro Kit .Total RNA from E3.5 embryos was purified by lysis in 10 μl extraction buffer of the PicoPure RNA Isolation Kit, followed by the manufacturer's instructions (Life Technologies #KIT0204). Purified total RNA was resuspended in 30 μl of elution buffer. mRNA from E3.5 embryos was purified by lysis in 100 μl lysis/binding buffer of the Dynabeads mRNA DIRECT Kit, followed by the manufacturer's instructions. Purified mRNA was resuspended in 50 μl of elution buffer.To exclude the RT and amplification of the sense Xist RNA by cell intrinsic primersTsixpA and XistIVS blastocysts in TsixpA and XistIVS mutations. Similarly, in the distal amplicon the reverse primer in the initial round of PCR resides in the inserted cassette. The RT primers were AUAP coupled to provide further specificity during PCR amplification; AUAP was then employed in the initial and nested PCR steps.For the proximal and distal RT–PCRs from XJF1XLab, XJF1XIVS and XJF1XpA blastocysts was assessed by RT–PCR using Invitrogen SuperscriptIII one-step RT–PCR system  using the primer pair TF5 and TR732  with some modifications. Briefly, 5′ RLM-RACE exploits the A unit of 10 μg of total RNA was treated with calf intestine alkaline phosphatase (CIP) to remove free 5′ phosphates from molecules such as ribosomal RNA, fragmented mRNA, tRNA and contaminating genomic DNA. The cap structure found on intact 5′ ends of transcript is not affected by calf intestine alkaline phosphatase. The RNA was then treated with tobacco acid pyrophosphatase to remove the cap structure from full-length transcript, leaving a 5′ monophosphate. A 45 base RNA adapter oligonucleotide (provided with the kit) was then ligated to the RNA population using T4 RNA ligase and primed with oligo XF1851  for cDNAAllele-specific expression was quantified using Qiagen PyroMark sequencing platform. Amplicons containing SNPs were designed using the PyroMark Assay Design software. cDNAs were synthesized using Invitrogen SuperScript III One-Step RT–PCR System . Following the PCR reaction, 5 μl of a total of 25 μl was run on a 3% agarose gel to assess the efficacy of the RT and amplification. The samples were then prepared for pyrosequencing according to the standard recommendations for use with the PyroMark Q96 ID sequencer.Mus domesticus-derived Bl/6 female to M. molossinus-derived XJF1 male (F1i), as well as from the reciprocal cross (F1r)), F1rXistIVS ), F1rTsixpA ) embryos. Sex of the embryos was then assayed by amplifying Xist RNA by RT–PCR and profiling a SNP in the amplicon to determine if the cDNA maps to the paternal X; and, by examining if the X-linked Pdha1 gene is biallelically detected by RT–PCR and Sanger sequencing. mRNAs from each of five WT F1i fully grown blastocysts, in which Xist RNA is derived solely from the XJF1 paternal X, was individually mixed with an equivalent amount of mRNA from each of six fully grown blastocysts from each of three different F1r genotypes, WT, XistIVS or TsixpA, all of which express Xist only from the M. domesticus-derived paternal X. RT–PCR was then performed with 2 μl of the mixed mRNAs using the Invitrogen SuperScript III One-Step RT–PCR System. In WT F1i blastocyst embryos, Xist is only expressed from the paternal XJF1 allele due to imprinted X inactivation. In the XistIVS and TsixpA mutant F1r blastocysts, on the other hand, the only transcriptionally competent Xist allele is M. domesticus derived. In the cDNAs generated from an equal mixture of F1i WT with F1rXistIVS or TsixpA mutant female mRNAs, a SNP in Xist was exploited to distinguish and quantify the relative expression of Xist RNAs in XistIVS or TsixpA compared with WT embryos by pyrosequencing. In the mRNA mixtures, the F1i WT Xist RNA originates from the M. molossinus-derived XJF1; in the F1rXistIVS or TsixpA mutants, the expressed Xist allele is M. domesticus derived. Expression of M. molossinus and the M. domesticus Xist alleles was normalized by comparing Xist expression in WT F1i and F1r blastocysts by pyrosequencing. In WT F1i and F1r blastocysts, Xist expression was undetectable from the maternal X chromosome by pyrosequencing.To quantify the relative expression of Xist RNA between embryos of different genotypes, mRNA was isolated from individual E3.5 F1 hybrid WT , following the manufacturer's instructions. Control reactions lacking reverse transcriptase for each sample were also performed to rule out genomic DNA contamination. For amplification of Xist, XistAR and Tsix in P values for all RT–qPCR results were calculated using Welch's two-sample t-tests.RT–qPCR was performed using SYBR Green-based (Kapa Biosystem #KK4650) relative quantification method on an Eppendorf Realplex Mastercycler. g for 20 min at 4 °C. The resulting pellet was washed in 70% ethanol, then washed in 100% ethanol, dried and resuspended in deionized formamide . The resuspended probe was denatured via incubation at 90 °C for 10 min followed by immediate 5 min incubation on ice. A 2 × hybridization solution consisting of 4 × SSC, 20% dextran sulfate  and 2.5 mg ml−1 purified BSA  was added to the denatured solution. The probe was then pre-annealed by incubation at 37 °C for 1 h to minimize probe hybridization to repetitive sequences. Probes were stored at −20 °C until use.RNA FISH probes from double-stranded DNA templates were created by randomly priming DNA using BioPrime DNA Labeling System . FISH probe templates and procedures have been described previously1536Strand-specific RNA FISH (ssRNA FISH) probes were labelled with Fluorescein-12-UTP  or Cy3-CTP  using the Invitrogen MAXIscript Kit . To detect XistAR, a DNA template spanning Xist bp 953–1,440 was transcribed to generate single-stranded RNA probe. A single-stranded probe detecting Xist RNA was generated using PCR amplified DNA from Xist exon 7. Labelled probes were column purified  and precipitated in a 0.25-M ammonium acetate solution essentially as described above for double-stranded RNA FISH probes, but without the addition of COT-1 DNA. Probes were resuspended also as described for double-stranded RNA FISH probes and stored at −20 without pre-annealing. Embryos and cells were hybridized to the probe overnight in a humid chamber at 37 °C. The samples were then washed thrice for 7 min each while shaking at 39 °C with 2 × SSC/50% formamide, twice with 2 × SSC and twice with 1 × SSC. A 1:250,000 dilution of 4,6-diamidino-2-phenylindole  was added to the third 2 × SSC wash. The embryos were then mounted in Vectashield .XistAR promoter sequence . The plasmids were transfected into female XEN cells plated in a 96-well format. Each well was transfected with 100 ng of pNL-NanoLuc reporter construct and 6 ng of transfection control plasmid encoding the firefly luciferase gene , with Lipofectamine 2000 (Invitrogen) transfection reagent, following the manufacturer's instructions. Each construct was tested in triplicate in each of three separate transfections. Seventy-two hours after transfection, the activities of firefly and NanoLuc luciferase were measured using the Nano-Glo Dual-Luciferase reporter assay system . The NanoLuc luciferase activity was normalized with firefly luciferase activity for each transfection.The selected promoters and enhancer sequences analysed are listed in Stained samples were imaged using a Nikon Eclipse TiE inverted microscope with a Photometrics charge-coupled device camera. The images were deconvolved and uniformly processed using NIS-Elements software.How to cite this article: Sarkar, M. K. et al. An Xist-activating antisense RNA required for X-chromosome inactivation. Nat. Commun. 6:8564 doi: 10.1038/ncomms9564 (2015).Supplementary Figure 1 and Supplementary Table 1


Bunyaviridae, genus Orthobunyavirus. These viruses are associated with fever and encephalitis, although INKV infections are not usually reported and the incidence is largely unknown. The aim of the study was to determine the prevalence of anti-INKV antibodies and associated risk factors in humans living in northern Sweden. Seroprevalence was investigated using the World Health Organization Monitoring of Trends and Determinants in Cardiovascular Disease study, where a randomly selected population aged between 25 and 74 years  was invited to participate. The presence of anti-INKV IgG antibodies was determined by immunofluorescence assay. Seropositivity for anti-INKV was significantly higher in men (46.9%) than in women . In women, but not in men, the prevalence increased somewhat with age (P = 0.06). The peak in seropositivity was 45–54 years for men and 55–64 years for women. Living in rural areas was associated with a higher seroprevalence. In conclusion, the prevalence of anti-INKV antibodies was high in northern Sweden and was associated with male sex, older age, and rural living. The age distribution indicates exposure to INKV at a relatively early age. These findings will be important for future epidemiological and clinical investigations of this relatively unknown mosquito-borne virus.The mosquito-borne Inkoo virus (INKV) is a member of the California serogroup in the family  Togaviridae, Flaviviridae, and Bunyaviridae. The genus Orthobunyavirus in the Bunyaviridae family has a growing number of strains. Among them, the viruses in the California serogroup are commonly found in the northern hemisphere, particularly in North America, although some―such as Inkoo virus (INKV), Tahyna virus (TAHV), and Snowshoe hare virus (SSHV) or the closely related Chatanga virus―are also present in Europe.Mosquito-borne viruses cause a variety of clinical syndromes in humans, and they include members of the Ochlerotatus communis mosquitoes in Finland, Sweden, and Russia, and from Ochlerotatus hexodontus and Ochlerotatus punctor in Russia.17INKV is known to circulate in northern Europe and Russia.The degree of exposure of the human population to INKV infection in Sweden is not known, so we wanted to investigate the prevalence of anti-INKV IgG antibodies. In this large randomized, stratified, and population-based survey in northern Sweden, we also investigated possible associated risk factors.In this study, we used data from the northern Sweden component of the World Health Organization Monitoring of Trends and Determinants in Cardiovascular Disease (WHO MONICA) study.18Of the 2,500 subjects invited, a total of 1,729 participated (69.2%) in the MONICA study in northern Sweden, which was used in the screening for anti-INKV IgG antibodies. Data on individuals who declined participation have been published.18Serological samples from 7.4% of subjects were missing. Thus, valid data were available for 1,607 subjects: 50.7% men and 49.3% women.The measurement procedures included blood pressure measured twice in a sitting position with an interval of 5 minutes. Subjects were weighed on an electronic scale. The highest level of education attained was classified as 1) primary school or secondary school (0–12 years of school) or 2) university studies. Regular smokers smoked at least one cigarette a day; all other subjects were considered to be nonsmokers. Subjects who answered yes to the question, “Do you have diabetes mellitus?” or the question, “Have you been treated for high blood pressure in the past two weeks” in the questionnaire were reported as having “known diabetes” and “hypertension.” Subjects who owned a farm were reported as being “farmers.”The study was approved by the Regional Ethical Review Board, Umeå, and all subjects gave informed consent.Serum samples obtained from the participants were stored at −80°C before analysis. Anti-INKV IgG antibodies were detected using a previously validated anti-INKV IgG immunofluorescence assay (IFA).N = 90) with negative, weakly positive, or strongly positive INKV IgG results from the IFA were verified with a neutralization assay to determine whether the IgG antibodies were specific to INKV, TAHV, or SSHV.3A subset of sera N = 0 with net test or χ2 test, and means and proportions were reported. When accounting for differences in sex and age between groups, general linear models were used. All P values < 0.05 were considered significant.Differences between groups were tested with P < 0.001). The proportion of men who were seropositive varied between the different age groups  but there was no linear relationship with increasing age . In women, the prevalence increased somewhat with age   than in women  (P < 0.03). No other background factors or prevalent disease showed any relationship with previous INKV infection. In an analysis taking gender into account, we found no differences in seropositivity for variables such as level of education, living on a farm, diabetes, and myalgia or arthralgia in either sex with the exception of men living on a small farm . However, the 95% confidence interval for this 18.2% difference in prevalence was 1.2–32%.Living in smaller communities or rural areas was associated with a higher prevalence of seropositivity (< 0.001)  . This pSeropositive subjects generally had higher body mass index, waist circumference, systolic and diastolic blood pressure, and serum creatinine (data not shown). After adjusting for the differences in sex and age, no differences between groups remained significant.Supplemental Table 1). As expected, there was some cross-reactivity with TAHV and SSHV. However, anti-INKV titers were approximately 4-fold higher than the antibody titers to the other viruses tested , with geometrical mean titers of 77 for anti-INKV, 28 for anti-SSHV, and 24 for anti-TAHV). One IFA-positive sample that did not neutralize any of the three viruses was observed, and interestingly, one of the weak IFA-positive samples had a high neutralization titer to TAHV only . Of the anti-INKV positive samples that neutralized INKV, 11 were further verified by PRNT, and they were all confirmed to be positive (data not shown).Because of the large number of anti-INKV antibody positives, a subset of serum samples (64 positive and 26 negative) was selected for verification of the anti-INKV IgG IFA results by a neutralization assay. The assay involved neutralization of three viruses from the California serogroup: INKV, TAHV, and SSHV. Altogether, 62 of 64 (97%) of the anti-INKV IgG IFA–positive samples, either strongly or weakly positive, specifically neutralized INKV and all anti-INKV IFA–negative serum samples were confirmed to be negative (There are numerous kinds of mosquito-borne viruses that may be a threat to public health. Members of the California serogroup are less well known, and there have been very few studies of the epidemiological and clinical properties of the European orthobunyavirus INKV. In this study, we found a high prevalence of anti-INKV antibodies in a population-based cohort from northern Sweden, and more so in men than in women. A similarly high seroprevalence has been shown in a previous study in Finland.9The strength of this study was the use of a large randomized and stratified population-based sample. The only background variable that was significantly associated with seropositivity was living in small communities or rural areas, which suggests more exposure to mosquitoes. All other associations noted could be explained by age and sex differences.We used a previously validated9We cannot completely rule out cross-reactivity to other members of the California serogroup, but positive samples had higher neutralization titers to INKV than to TAHV and SSHV, indicating the presence of anti-INKV antibodies. It is possible that some individuals had undergone infections by other orthobunyaviruses of the California serogroup, either through travel or locally. Interestingly, a SSHV-like Chatanga virus has been identified in the neighboring country Finland.3Intriguingly, the high prevalence of anti-INKV antibodies is not reflected by recognized cases of human infections. The high proportion of seropositive individuals already in the younger age group, 25–35 years, suggests that there is relatively early exposure to this mosquito-borne virus. Interestingly, for the mosquito-borne Sindbis virus, which is also present in northern Sweden, we found that virus antibodies were uncommon in young people, with no age-dependent increase, suggesting a recent introduction of Sindbis virus and increase in this region.21Ochlerotatus communis and Ochlerotatus punctor are very commonOur results indicate that INKV is widespread and highly endemic in northern Sweden, which may be explained in part by the fact that two of the suggested mosquito vectors, In conclusion, the high prevalence of anti-INKV antibodies indicates that this mosquito-borne viral pathogen has been very common in northern Sweden. The seroprevalence was found to be higher in men and in rural areas. These findings draw attention to the possible impact of this infection, which is still relatively unknown to the medical community. However, the clinical importance of INKV requires further studies of febrile cases during the mosquito season in endemic areas. More arbovirus studies are also needed to determine the factors that are important for its circulation and human infection.Supplemental Table.


Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. Replication of retroviruses including the Human immunodeficiency virus type 1 (HIV-1) occurs in the nucleus of infected cells. The incoming viral RNA genomes are reverse transcribed in the cytoplasm and the resulting DNA transported to the nucleus where it is integrated into chromatin . The chrThree features distinguish the HIV-1 provirus from the host genes: (i) cis-acting DNA/RNA elements present in the provirus/viral RNA; (ii) the activity of the viral trans-activator of transcription Tat and (iii) the activity of the viral regulatory protein Rev. All other factors required for efficient viral transcription and RNA processing are of cellular origin, including the RNA polymerase (RNAPII). The generation of infectious retroviral progeny requires the synthesis and export to the cytoplasm of spliced subgenomic mRNAs for protein translation, of partially spliced RNAs that function as the mRNA for the viral proteins Gag-Pol and Env and of the viral genomic RNA ,14. HencHIV-1 RNA starts its journey at the provirus integration site, where RNAPII transcribes the viral genome, and reaches the nuclear pore to exit the nucleus. HIV-1 integrates with high frequency within active genes, possibly because of a better accessibility of open chromatin . Investi1–5) and nine 3’ splice acceptors (SA1–9) [cis-acting repressor (CRS) sequences [nrb complex, were shown to bind specifically to INS elements [After RNA synthesis, the HIV-1 transcript is processed through alternative splicing. The transcripts are grouped into three size classes: the unspliced, 9 kb RNA that encodes Gag and Gag/Pol but also forms the genomic RNA, the 4 kb, singly spliced RNAs that encode Vif, Vpr, Vpu and Env, and the 2 kb, fully spliced RNAs that express Tat, Rev and Nef. Generation of the various viral RNA species is achieved through the use of five 5’ splice donors (SD (SA1–9) ,36,37. H (SA1–9) ,44,45,46equences ,50,51,52equences . Severalelements ,54,55,56Rev is an essential HIV-1 protein encoded from two exons that are joined by splicing to produce a monocistronic transcript, early in the viral replication cycle ,58. In tRev exports the RRE-containing HIV-1 mRNAs from the nucleus through interaction with Crm-1 (chromosome maintenance region 1) . Crm-1 iThe Rev-RRE-Crm-1 complex also engages the activity of the cellular RNA helicase DDX3 that functions to enhance the Rev-dependent pathway and it is believed to facilitate the passage of large unspliced HIV-1 RNAs through the nuclear pore . RNA helThe nucleolar protein B23 associates tightly with Rev through the basic domain . Rev andA number of other interactions have been investigated . Some ofHIV RRE-dependent RNAs are modified by the peroxisome proliferator-activated receptor-interacting protein with methyltransferase domain (PIMT) that induces a modification of the 7-methylguanosine (m7G) cap to create a trimethylguanosine (TMG)-capped RNA . Like ceA recent novel approach to identify HIV-1 RNA binding factors exploited the MS2-tagging of RNA method to affinity purify the ribonucleprotein complex in the cell nucleus . Proteinin vitro with nuclear or cytosolic extracts in conditions emulating intracellular environments of Rev, where the association of functionally relevant binding factors could be predicted to be different [Rev appears to be a versatile adaptor of cellular factors on RRE-containing HIV-1 RNAs and recent literature has provided attempts to identify the interactome of Rev. Of note the work of Gerace and collaborators who carefully designed a purification scheme to dissect Rev complexes assembled ifferent . Reassurifferent . Of noteAlthough it is well established that the primary effect of Rev is to promote the nuclear export of RRE- containing HIV RNAs, many aspects of this process are still poorly understood. Rev integrates several cellular posttranscriptional mechanisms, such as mRNA splicing, RNA stability and nucleocytoplasmic transport. However, Rev does not possess any enzymatic activity and functions as an adaptor between RRE-containing RNAs and cellular factors. Hence, the activity of Rev is to engage in a promiscuous menage-a-trois with RRE and cellular proteins to shift the equilibrium from spliced/RRE-containing viral RNAs towards export of the latter.The journey of the viral RNA starts at the transcription site. As soon as RNA is synthesized by RNAPII, its post-transcriptional processing begins through binding of proteins to the nascent RNA. These include the splicing factors described previously. For example, the splicing factor SF2/ASF appears enriched at the transcription site on nascent RNA C. In addStudies of Cochrane and co-workers found that Rev could only export newly synthesized HIV RNA indicating that Rev acts on nascent transcripts rather than on downstream pathways engaged in splicing or RNA degradation . AnotherRev recognizes RRE as a monomer  and was The journey of viral RNAs bound to Rev in the nucleus finally ends at the nuclear pore where the Crm-1/RRE/Rev complex is translocated to the cytoplasm as described previously. Little is known about this process. Certain nucleoporins were shown to be associated to the Crm-1/RRE/Rev complex and may be involved in the initial steps of pore recognition, while helicases such as DDX3 may assist RNA translocation. The kinetics of the process is also unknown, the application of single cell live imaging could eventually address this issue ,133.The mechanism of Rev function in the nucleus is likely to be governed by kinetic competition. The rate of viral RNA biogenesis determines the amount of unspliced and partially spliced RNAs that are formed at the transcription site. In fact the high transcription rate observed for the HIV-1 provirus also resulted in the presence of a large fraction of unspliced RNAs at the transcription site possibly indicating an effect of RNAPII velocity on splicing efficiency as was recently proposed ,34. ThisSeveral questions remain unanswered. What are the cellular factors that determine the fate of unspliced HIV-1 RNA in the absence of Rev? Does Rev localize on nascent RNA? Where does Rev engage Crm-1? Future studies on HIV-1 Rev will need to address these issues with a combination of standard molecular biology techniques and the new tools that are available for high resolution live cell analysis ,134.


We analyzed the complete genome sequence of the A/Shanghai/01/2014 (H7N9) strain, which will provide a better understanding of the evolution of influenza A(H7N9) virus. Influenzavirus, family Orthomyxoviridae, contains 8 single-stranded negative-sense RNA (-ssRNA) segments that encode 12 proteins. Recently, a novel avian-origin influenza A(H7N9) virus has caused >410 cases of infection in China, including 60 deaths from March 2013 to 8 April 2014 (Influenza A virus (IAV), which is a member of the genus All gene segments of the A/Shanghai/01/2014 (H7N9) virus were compared with those of the vaccine component strain A/Anhui1/1/2013 and those of the currently circulating strains. The complete coding region of A/Shanghai/01/2014 (H7N9) is 13,090 nucleotides long. Segments 1 to 8 are 2,280, 2,274, 2,151, 1,683, 1,497, 1,398, 969, and 838 nucleotides (nt), respectively. The strain has an amantadine resistance mutation (S31N substitution) in the viral M2 protein and no oseltamivir resistance mutation (R292K substitution) in the viral neuraminidase (NA) protein (n = 34) were all >99.9%, while the similarity of the PB2 gene was only 96.8%. Phylogenetic analysis indicated that the PB2 gene of the A/Shanghai/01/2014 (H7N9) virus was not clustered with those of the human H7N9 strains in 2013 but clustered with those of A/Chicken/Wenzhou/598/2013 (H9N2) and A/Chicken/Wenzhou/642/2013 (H9N2). The similarities of the PB2 nucleic acid sequences between the A/Shanghai/01/2014 (H7N9) and A/Chicken/Wenzhou/598/2013 (H9N2) strains were as high as 99.6%, which suggests that the origin of the PB2 gene fragment in the A/Shanghai/01/2014 (H7N9) virus strain was different from those of human strains in 2013. The sequence information reported here will facilitate further investigations of the evolution of human infected avian-origin H7N9 influenza virus.The similarities of the PB1, polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), NA, matrix (M), and nonstructural (NS) genes between the sequence of A/Shanghai/01/2014 (H7N9) and the consensus sequence of 2013 strains (KJ411975 to KJ411982.The complete genome sequence of the A/Shanghai/01/2014 (H7N9) strain has been deposited and updated in GenBank under the accession no.


RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functions in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. This review summarizes evidence for mammalian RNAi factors in cell nuclei and mechanisms that might contribute to the control of gene expression. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes. The nucleus is a hub of RNA synthesis, processing and regulation –3. TransRNA interference (RNAi) provides a mechanism for selective recognition of RNA sequences. Unlike protein transcription factors that are characterized by relatively limited selectivity for target sites and relative difficulty evolving new selectivities, recognition of RNA by small RNAs would exploit the high specificity of Watson–Crick base-pairing and the potential for developing new selectivities by nucleotide mutation. The potential evolutionary advantages of RNA recognition in cell nuclei appear compelling, but how might it be achieved?RNAi is well known as a regulatory mechanism for controlling gene expression in cell cytoplasm. If the protein machinery and small RNAs responsible for cytoplasmic RNAi were also found in mammalian cell nuclei, it is possible to imagine these factors promoting recognition of nascent transcripts, splice junctions, and other regulatory control points. Such recognition would constitute a previously unappreciated level of regulatory control over gene expression.Our goal is to describe the current state of research into mammalian nuclear RNAi. We begin by briefly addressing nuclear RNAi in non-mammalian organisms and by addressing the origins of controversy and skepticism towards the existence of RNAi in mammalian somatic cells. We describe reports of nuclear RNA affecting gene repression, gene activation, miRNA function, and gene splicing.RNAi is a powerful mechanism for using small RNAs to control protein translation in the cytoplasm of mammalian cells ,5. NaturThe small RNAs are typically duplex RNAs that include a guide strand complementary to the cellular RNA target sequence. During RNAi the guide strand RNA is loaded into an RNA-induced silencing complex (RISC) consisting of protein factors including Argonaute 2 (AGO2) –10 and TDrosophila protein GW182  . AGO2 isin GW182 ,20. TNRCin GW182  and within GW182 .The guide strand RNA directs the complex to target sequences through Watson–Crick base-pairing . RecogniThe protein domains of RISC serve multiple functions. Single-stranded RNA is subject to digestion by nucleases and its interaction with proteins helps ensure its survival. The guide RNA binds directly to AGO2 –25. The The potential for RNA to induce sequence-specific changes in the nucleus of non-mammalian organisms has been recognized for over two decades and has been extensively reviewed –31.Nuclear RNAi was first discovered in plants and shown to induce DNA methylation of sequences at gene promoters  Figure . In thisS. pombe, RNAi factors modulate gene expression by inducing histone modifications  . Since tMultiple independent methods have recently been used to examine localization of endogenous AGO2 and other RNAi factors by microscopy, cell fractionation, size exclusion chromatography, and activity assays . Wide-fiC. elegans  promoter to inhibit expression of chromosomally encoded PR expression  complementary to the progesterone receptor  expression in human mammary ductal carcinoma T47D cells, a cell line with high basal expression of PR. To further test the hypothesis that RNA might activate expression in one context while inhibiting in another we evaluated promoter-targeted RNAs in MCF7 cells that have a low basal level of PR expression. We identified several duplex RNAs capable of activating gene transcription    and LDL  (COX-2) .Whenever nucleic acids are introduced into cells, there will be a potential for off-target effects . ActivatThe advent of CRISPR/CAS9-mediated technology has facilitated sequence-specific modification of genomes and has the potential to provide further rigor for analyses. For example, mutation of a seed sequence would be expected to block transcriptional activation or silencing. While CRISPR/CAS9 is an inviting technology, the use of mismatched RNA duplexes remains a quicker and less expensive alternative. In addition, some cell lines where effects have been observed contain multiple chromosomes, making them poor targets for efficient CRISPR-mediated mutation. For targets near transcription start sites, mutations might affect basal expression and obscure interpretation. Regardless of the potential obstacles, well-designed use of CRISPR-CAS9 to validate RNA-mediated transcriptional control would be an important step forward.The RNAs used to achieve transcriptional silencing or activation are duplex RNAs that are identical in structure to those used for standard post-transcriptional RNA interference . BecauseSeveral laboratories have observed that silencing expression of AGO2 reversed transcriptional silencing and transcriptional activation ,129–131.COX-2  or they may act at a distant site (in trans). Whether an RNA acts in cis or trans relative to its target gene is one determinant of its mechanism of action and careful characterization is necessary to achieve persuasive results  is caused by a truncation in dystrophin protein due to a premature stop codon . AntisenAntisense oligonucleotides act by binding pre-mRNA and blocking association of splicing factors . Our labRNAi-regulated splicing of endogenous genes has not been reported to date, but RNA sequencing of AGO2-bound species in human cell nuclei has revealed many potential binding sites near exon/intron boundaries , suggestThe mammalian nucleus contains many different species of RNA, each of which must be regulated and many of which have the potential to be regulators. Experimental data have built a strong case for the existence of RNAi factors in mammalian somatic cell nuclei, consistent with their presence in yeast, worms, and plants. These nuclear RNA factors can act in conjunction with small RNAs to regulate transcription and splicing. Data suggest that multiple mechanisms are involved and that these mechanisms share considerable similarity to mechanisms of protein transcription or splicing factors. The distinguishing feature relative to protein factors is that the small RNA provides a programmable specificity module that can be more readily adapted for recognition of many different RNA species.This programmable specificity of nuclear RNA:RNAi factor complexes has the potential to confer a significant evolutionary advantage and may represent a new area for miRNA function. Experiments using model endogenous genes have demonstrated that miRNAs can regulate gene transcription, but the broad importance of miRNAs in cell nuclei is not yet clear. Gaining a better appreciation for the impact of nuclear RNAi on mammalian physiology and a more detailed mechanistic understanding will be primary goals for future research.


In parallel, many computational methods have been developed to analyze and interpret these data. The interplay between RNA secondary structure and RBP binding has also been a growing theme. Integrating RNA–protein interaction data with observations of post-transcriptional regulation will enhance our understanding of the roles of these important proteins.RNA-binding proteins (RBPs) are important regulators of eukaryotic gene expression. Genomes typically encode dozens to hundreds of proteins containing RNA-binding domains, which collectively recognize diverse RNA sequences and structures. Recent advances in high-throughput methods for assaying the targets of RBPs  RNA-binding proteins (RBPs) have diverse roles in post-transcriptional gene expression, including regulation of alternative splicing, RNA export and localization, RNA stability and translation . Many RBin vitro and in vivo, as well as methods to determine the proteins bound to an RNA molecule. Finally, we discuss computational methods for analyzing high-throughput data and predicting RBP binding.Here we introduce the major classes of sequence- and structure-specific RNPs, and discuss aspects of RNA secondary structure that impact RBP binding; knowledge of how proteins interact with RNA is important for the interpretation of high-throughput data. We then review current methods for high-throughput experimental determination of the RNA targets of RBPs Different classes of RNA-binding domains (RBDs) use different strategies for binding to RNA. Nonetheless, there are some general features of RBP–RNA interactions: RNA recognition is commonly a combination of recognition of the RNA by the overall protein fold (involving hydrogen bonds with backbone atoms) as well as specific amino acid side chain–nucleotide interactions. Target specificity is often accomplished by way of hydrogen bonding and electrostatic interactions; the latter also contribute to the protein’s affinity for RNA along with stacking interactions . IndividThe RNA recognition motif (RRM) is found in all domains of life; in metazoans, it is the most common RNA-binding domain. A single RRM spans ∼90 amino acids and contains two conserved motifs, RNP-1 and RNP-2, consisting respectively of 8 and 6 mostly positively charged or aromatic amino acids , 11.Structurally, the RRM consists of a four-strand antiparallel β-sheet backed by two α-helices. RRMs are highly versatile in their mode of RNA recognition: in canonical RRM–RNA interactions, the bound RNA lies across the β-sheet and contacts one or more key residues in the conserved RNP-1 and RNP-2 motifs; however, the amount of the β-sheet surface directly contacting the RNA varies considerably . NoncanoThe β-sheet surface of a single RRM can contact up to four nucleotides, while engaging the loop regions external to the β-sheet can allow binding of up to six nucleotides . ApproxiK homology (KH) domains, named after the founding member of the family, hnRNP K , are alsDouble-stranded RNA binding domains (dsRBDs) are involved in diverse aspects of post-transcriptional regulation, including RNA editing , miRNA bBecause of the nature of the A-form RNA double helix, in which the major groove is narrow and deep, it is gIn contrast to the other major RNA binding domains, for which the elucidation of a recognition code has proven elusive, RNA recognition by Pumilio Homology Domains (PUM-HD) is understood to the extent that custom proteins can be designed to bind new sequences. The PUM-HD typically consists of 8 PUF (Pumilio and FBF) repeats of a 36 amino acid motif, and the entire domain forms a curved structure that binds RNA in the concave side of the domain, while the convex side mediates protein–protein interactions . Each PUZinc fingers are a large and diverse class of domains with the common property of coordinating zinc. The different types of zinc fingers have varying three-dimensional structure and likely have independent evolutionary origins. Nonetheless, several types act as DNA-, RNA-, and protein-binding domains. The extent to which individual zinc finger proteins bind and recognize each class of biopolymers is unknown, however there are some trends: C2H2 zinc fingers are usually DNA binding, while CCCH zinc fingers are primarily single-stranded RNA binding. CCHC zinc knuckles in viral and metazoan proteins also bind RNA; however, RNA binding by metazoan CCHC zinc knuckles is understood only in the context of proteins that also contain another RBD , 34. In The human genome encodes ∼60 proteins with CCCH zinc fingers (a larger number than contain KH domains), of which 11 have evidence of single-stranded RNA (ssRNA) binding . RNA recin vivo RNA structure is impacted both by the presence of ATP-dependent RNA helicases, as well as a number of proteins that could bind co-transcriptionally. Despite the development of high-throughput methods for measurement of RNA structure, there is ongoing controversy regarding the degree of RNA structure present in cells and the accuracy of both computational algorithms and these experimental techniques to predict RNA folding.RNA structure is a critical aspect of describing, measuring and predicting RBP binding: many RBPs recognize specific structures, while those that bind single-stranded RNA presumably compete with RNA structures. In addition, RBP binding likely has some impact on RNA structure, and in vivo in Arabidopsis [The secondary structure of an RNA molecule can be determined by footprinting techniques: cutting the RNA using RNases specific to ssRNA or dsRNA, or small molecule reagents that cleave or modify RNA at positions in a manner proportional to their accessibility . The clebidopsis  and yeasbidopsis .Arabidopsis using DMS [Arabidopsis using DMS [The overall trends identified by these studies vary. RNA accessibility around the start codon was associated with translational efficiency (as measured by ribosome profiling) in yeast using PARS  and in Asing DMS ; howeversing DMS . Using Psing DMS . This cosing DMS  as well sing DMS  all of wsing DMS  support Computational prediction of mRNA secondary structure generally conforms to one of the two approaches. The first relies on the assumption that thermodynamically stable structures are more likely to exist than unstable structures, exemplified by the Zuker MFOLD algorithm  and exteAs an alternative to the free-energy based algorithms, covariation-based approaches take advantage of the fact that functional RNA secondary structures are more likely to be conserved through evolution. Covariation algorithms use a number of simplifying heuristics (reviewed in ), as simGiven the inconsistencies among the experimental methods for assessing mRNA secondary structure and uncertainty about the accuracy of computational predictions, it is important to evaluate the accuracy of both these types of estimates. However, doing so has been troublesome because of the lack of gold standards for mRNA secondary structures. Classic RNA secondary structure benchmarks are likely inappropriate because they are composed of highly structured ncRNAs like ribosomal RNAs and ribozymes. In addition, mRNAs are longer than most well-characterized ncRNAs, such that windowed approaches  are often preferred both for their speed and potentially increased accuracy .Lange and colleagues performed an analysis to determine the accuracy of predicted secondary structures using yeast PARS data  and a cuin vitro approaches , also known as r an RBP , 63. In ratories . In addiratories . While Sratories , 63, andCaenorhabditis elegans PUM domain RBP FBF-2 to a 20 nt randomized pool alone and in the presence of a non–RNA-binding peptide fragment of the CPEB protein CPB-1, and found that FBF-2’s binding specificity was altered in the presence of the CPB-1 fragment. As well, they reported an alternate binding mode for FBF-2 with a slightly different motif.SEQRS is a method that applies high-throughput sequencing to SELEX to monitor the enrichment of optimal and alternate binding motifs by sequencing after each round of selection . SimilarRNAcompete  is a metNeither SEQRS nor RNAcompete, in their current versions, are optimal for determining the structural specificity of RBPs. SEQRS uses a 20 nt pool, which is not long enough to encompass all but the simplest of secondary structures, while RNAcompete, owing to its microarray-based strategy, is able to robustly represent only primary RNA sequence in the ∼244 k RNA sequences in the pool, and so the current version of the RNAcompete pool was designed to avoid highly structured sequences. Nonetheless, motifs determined for proteins known to prefer hairpin loops, such as yeast Vts1p and human SNRPA and LIN28A, were determined, and correspond to the single-stranded portions (e.g. the loop portion of a hairpin loop) of the structural motif. Significant preferences of many RBPs for ssRNA and, in a few cases, hairpin loops were identified . An alteTo study the kinetics of RBP binding to RNA sequences in a high-throughput manner, a recent study from the Greenleaf laboratory adapted an Illumina sequencing machine to measure binding affinity of the MS2 coat protein to ∼120 000 RNA sequences  represenin vivo by an RBP. The first genome-wide analyses were microarray based, and involved immunoprecipitation of RBP–RNA complexes using an antibody to the endogenous protein or to an epitope tag . Tenenbaum et al were the first to apply RIP-chip to the determination of the RNA fraction bound to HuB, HuA/HuR, eIF-4 E and PABP [A series of developments over the past decade have revolutionized determination of the RNAs bound and PABP . Since tand PABP  and reviand PABP ), leadinand PABP .in vitro after cell lysis, which has been observed under some conditions [in vivo contacts are maintained, as well as narrowing down the binding site [Although RIP allows identification of the target RNA molecules binding to an RBP, the data may include indirectly bound sequences, and precise locations of the binding site on the target mRNA may be difficult to determine. Additionally, RIP conditions must be calibrated to minimize reassociation of RBPs with mRNA nditions  but not nditions . Cross-ling site . UV crosing site , and so ing site  based oning site . The behing site . Preciseing site . In PAR-in vitro affinity measurements [C. elegans [C. elegans germ line [CLIP-seq and its variants are not without biases. UV cross-linking preferentially bonds certain nucleotides and certain amino acids , and noturements . UV crosurements , and who elegans  animals,erm line .in vitro are enriched in CLIP-seq reads [de novo [Recently, Friedersdorf and Keene demonstrated that a large fraction (up to 45%) of reads (including high-abundance sites) from published PAR-CLIP data sets overlap with binding regions observed in background data from FLAG-GFP immunoprecipitations . Backgroeq reads , often m[de novo . Backgro[de novo .Identification of protein-bound sites from CLIP-seq (and variants) data is nontrivial. Because transcript abundance varies, ascertainment of the protein’s preference for a target RNA through quantitation of the number of CLIP-seq reads mapping to a transcript sequence requires normalization to transcript abundance. Additionally, the choice of RNase and conditions chosen to fragment the cross-linked protein–RNA complex has significant impact on the base composition of the observed CLIP tags . To overin vivo methods discussed above to be protein-agnostic allows the identification of all the protein-bound sites in expressed transcripts.While the majority of recent efforts have focused on identification of the RNAs bound to a given RBP, mass spectrometry has allowed complementary approaches to discover new RBPs and determine all the RBPs binding an RNA. In addition, modification of the in vivo binding assay [in vitro or in vivo analyses are required to establish whether or not these novel RBPs simply bind RNA non-specifically as the PAR-CLIP data from many of the RBPs resembles background data derived from FLAG-GFP immunoprecipitations [Proteomics approaches have been applied to identify a more complete complement of RBPs in eukaryotic genomes, which is important because there are a number of RBPs without canonical RNA-binding domains , 92. In ng assay . Surprisitations .in vitro, and involve tethering the target RNA molecule to a solid support by chemical modification of the RNA [in vivo assembled RNPs has been accomplished using oligonucleotides complementary to the RNA sequence [Complementary to the RBP-centric approaches described above, development of mass spectrometric methods for determining the protein complement of diverse mixtures has allowed the direct ascertainment of proteins bound to an RNA sequence. Most approaches have been performed  the RNA  or using the RNA  and incusequence , coexpresequence , and delsequence . Althougsequence  and to csequence , demonstsequence .Several methods have been developed for discovering protein-bound sites on RNAs agnostic of the RBP, denoted protein occupancy (or interaction) profiling. These methods function similarly to PAR-CLIP, except that instead of immunoprecipitation of a target RBP, RNPs are purified using oligo (dT) beads  or chemiin vivo data will also be described.The goal of complete understanding of the functions of RBPs in post-transcriptional regulation requires computational analysis of RBP-RNA interactions to interpret experimental data and model how RBPs find and bind to their targets. A general strategy for predicting RBP binding involves (1) motif finding and (2) prediction of binding sites using the discovered motifs. Online databases collecting RBP motifs and Learning RBP binding motifs can be accomplished by applying DNA motif finders, which only consider the RNA sequence, or by considering RNA secondary structure either explicitly or as a layer on top of sequence preference. The basic approaches are summarized in Specialized methods that incorporate RNA secondary structure generally break down into two camps: the first is based on determining the linear structural context around a sequence motif. MEMERIS is an extension of the popular MEME algorithm that uses RNA accessibility as a prior probability to guide motif finding to single-stranded regions . SimilarThe second approach considers RNA secondary structure explicitly and includes stochastic context-free grammar (SCFG) and graphical approaches. CMfinder  and RNApThe fact that many RBPs have multiple RNA binding domains raises the interesting possibility of RBPs binding to bipartite or complex motifs. Gapped motifs have been described for PTB  and theIn vivo RBP binding sites are catalogued in three databases, starBase [Online databases that collect RNA–protein interactions are summarized in starBase , doRiNA starBase , and CLIstarBase . These dHigh-throughput identification of protein–RNA interactions has improved understanding of the targets of RBPs in diverse cellular contexts, and focus is shifting from cataloging RNA–protein interactions to understanding the individual and combined effects of RBPs on global RNA metabolism and gene expression. Methods to produce extensive functional data sets of RNA splicing , stabiliRBPs apply different strategies for binding RNA.For certain RBPs, RNA secondary structure is a key component of RNA binding, but widespread prediction and measurement of RNA secondary structure remains difficult especially due to the potential impact of other RBPs on mRNA structure.In vitro binding specificity of RBPs can be determined using microarray-based methods (such as RNAcompete) or high-throughput sequencing-based methods (such as RNA Bind-n-Seq).In vivo targets of RBPs are determined using CLIP-seq/HITS-clip, and precise binding sites are more easily defined using the PAR-CLIP and iCLIP variants.A general strategy for predicting RBP binding involves (1) motif finding and (2) prediction of binding sites using the discovered motifs.Canadian Institute for Health Research operating grant to Q.D.M. and T.R.H. MOP-125894.K.B.C. is supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship. This work was funded by