The link between non-enzymatic RNA polymerization and RNA self-replication is a key step towards the “RNA world” and still far from being solved, despite extensive research. Clay minerals, lipids and, more recently, peptides were found to catalyze the non-enzymatic synthesis of RNA oligomers. Herein, a review of the main models for the formation of the first RNA polymers is presented in such a way as to emphasize the cooperation between life’s building blocks in their emergence and evolution. A logical outcome of the previous results is a combination of these models, in which RNA polymerization might have been catalyzed cooperatively by clays, lipids and peptides in one multi-component prebiotic soup. The resulting RNAs and oligopeptides might have mutualistically evolved towards functional RNAs and catalytic peptides, preceding the first RNA replication, thus supporting an RNA-peptide world. The investigation of such a system is a formidable challenge, given its complexity deriving from a tremendously large number of reactants and innumerable products. A rudimentary experimental design is outlined, which could be used in an initial attempt to study a quaternary component system. One of the major steps in the RNA world scenario of the origins of life, which remains unsolved, is the transition from random nucleotides to ribozymes capable of self-replication. Recently, major advances in the polymerization of nucleotides in various environments have been reported ,2,3, but4, H2O and CO2, would have contributed to a substantial greenhouse effect. Even in the absence of greenhouse gases, heavy bombardment by bolides may have generated sufficient heat to melt the ice [2, compared to present day values between 60–100 mW/m2 [The early Earth period relevant to the origins of life extends from the formation of the Earth at 4.56 Ga to ~3.8–3.5 Ga. The latter boundary is based on the discovery of the first body fossils of bacteria in rocks of 3.5 Ga in age ,8,9,10.  the ice . Heat fl00 mW/m2 . Oxygen 00 mW/m2 . There i00 mW/m2 ,16, but 00 mW/m2 . It is i00 mW/m2 . The earetc. [Experimental models for the bottom-up assembly of a protocell require the consideration of multi-component systems. It is unrealistic to use pure compounds as unique reactants, and minerals that constitute the entire Earth cannot be excluded either. Indeed, clays have been found to enhance self-assembly of biomolecules, such as RNA , peptideetc. . A heterAt least three different catalysts have been found to enhance the non-enzymatic polymerization of RNA, namely montmorillonite surfaces , lipid sIn this review, the major focus is on the emergence of the first RNA oligomers. An introductory section to define the main steps of non-enzymatic RNA polymerization and the RNA world hypothesis are described. The methods used for synthesizing activated nucleotides, essential for non-enzymatic RNA polymerization, are summarized. Major advances in non-enzymatic RNA oligomerization and some of their limitations are then presented. Finally, we outline a new model based on a multi-component prebiotic soup.The process of non-enzymatic polymerization of RNA, as generally accepted in the literature, can be divided into three smaller steps, namely 1) the emergence of the first RNA oligomers, which would have provided the basis for (2) template-directed RNA polymerization, resulting optimistically in robust and functional RNA polymers capable of self-ligation and self-catalysis, finally leading to (3) a non-enzymatic RNA replication . In the  the emerIn the RNA world hypothesis, it is suggested that the earliest cellular life was driven by RNA polymers, which could have simultaneously played the roles of biocatalysts and carriers of the genetic code . To datecis-acting manner. Riboswitches are now recognized as important and widespread elements in the control of gene expression in numerous evolutionarily distant Bacteria, Archaea, Plantae, Fungi and Algae [RNA has also been shown to possess a remarkable diversity of structural and metabolic functions. One of the most significant discoveries is that of riboswitches ,40,41, Rnd Algae . Furthernd Algae . Indeed,nd Algae ,45,46, wnd Algae ,48, a land Algae . Finallynd Algae ,51,52,53However, there is still a missing piece of the puzzle in experimental models of RNA polymerization and replication, which would link the evolution of the products of non-enzymatic RNA polymerization to self-sustained RNA replicases. A major reason for this gap is the insufficient production of large oligomers and in high enough yields to adequately sample polymer structure and function. This obstacle has been partially addressed by recent reports on improved polymerization reactions ,2,3, but0 > 0). Moreover, most RNAs are unstable, being prone to hydrolysis at high temperatures or extreme pHs. A prerequisite of the RNA world hypothesis, however, is that early systems should have been robust to maintain evolution and transmission of genetic information. Thus, it has been proposed in the literature that RNA, like DNA, is also a product of evolution and that an “RNA-like” polymer was used by the earliest forms of life  and use the free energy released to drive the enzyme-catalyzed polymerization . Prebiotiewed in ). In theAttempts to oligomerize nucleotides in the absence of enzymes is a challenge; hence, nucleotides activated with various groups were synthesized to increase the rate of phosphodiester bond formation. These nucleotides are not utilized in modern organisms, but might have formed under primitive Earth conditions and promoted early RNA oligomerization ,61,62,63etc., has been reported [The synthesis of phosphoroamidates under prebiotic conditions with the use of different amino group containing molecules, such as urea, ammonia, imidazole, glycine, ethylenediamine, histamine, reported ,65, but reported ,66,67,68reported ,70,71,72reported ,73,74 (Sreported . Furtherreported . However2+2(PO4)(OH), ludjibaite, Cu2+5(PO4)2(OH)4, reichenbachite, Cu2+5(PO4)2(OH)4, cornetite, Cu2+3(PO4)(OH)3, and hydroxylapatite, Ca5(PO4)3(OH), can promote nucleoside phosphorylation [Mineral surfaces could have played a role in the activation reaction. For example, it was found recently that phosphate minerals, such as libethenite, Curylation . More inrylation . These frylation ,79,80,81rylation ,83; theyrylation ,85, and rylation ,87.In this section, we describe four of the models that have been proposed in the literature for RNA oligomerization in the absence of a template.de novo forming oligomer had a preference, in the order of A > G > C > U [d,l-imidazolide of adenosine with d,l-imidazolide of uridine were reacted together in the presence of Na+-montmorillonite for three days. Homochiral products were formed, and their selectivity increased with increasing chain length of RNA oligomers up to pentamer [Minerals have long been hypothesized to play a key role in concentrating the products of prebiotic organic chemical processes leading to the origin of living systems . The cat > C > U . Regiosp > C > U . The effpentamer ,103. The2 and pH 8 (0.1 M HEPES or PIPES buffer) at room temperature. A wider range of conditions was subsequently examined, including the effect of salts, temperature and pH [2 is the most efficient catalyst, some catalytic effect can be achieved by a high concentration (1 M) of NaCl, even in the absence of Mg2+. More recently, Joshi and Aldersley [+ > Na+ > K+ and Cl− > Br− > I−, respectively. Thus, LiCl resulted in a maximum detected length of 12-mers, while pentamers were the maximum length detected in the presence of KI.Since the discovery of the catalytic role of montmorillonite in RNA oligomerization , the stae and pH . The autldersley  investigvs. polymerization declined from octamers at 4 °C to hexamers at 50 °C [Within a temperature range of 4–50 °C, the net rate of activated nucleotide hydrolysis at 50 °C . Kawamurat 50 °C . Howeverat 50 °C .5’ppA) were obtained, and activated nucleotides remained mostly intact, even after 50 days of incubation at pHs >10 [Oligomers can be obtained in a pH range of 6 to 10 with an optimum at pH 7–8 . At pH 1 pHs >10 . These r pHs >10 ,109.A wide range of clay minerals and of varied provenance were investigated for their catalytic activity, but only a few montmorillonites were deemed excellent (generating heptamers or longer); and no apparent trend in clay structure or provenance was found ,109. In +-, Li+- or Ca2+-montmorillonite are catalysts, but Mg2+-montmorillonite, smectite, nontronite, allophane, imogolite and sepiolite are not. However, other properties of the minerals, such as their bulk chemical composition, partial ionic substitutions, crystal structure, surface charge and particle size, should also be examined in detail. Interestingly, nucleotides were recently found to adsorb more strongly on nontronite than on montmorillonite and mainly at the edge sites, rather than the interlayer sites [The potential effect of other minerals, such as olivine, galena, calcite, magnetite, rhodochrosite, sphalerite, magnesite, siderite, hematite, brucite, chalcocite, talc, dolomite, pyrrhotite, pyrite and the carbonaceous chondritic meteorites, Murchison, Yamato-791717 and Yamato-86751, were also tested. None of the tested surfaces was found to promote RNA oligomerization at rates better than or comparable to those of montmorillonite ,112. Eveer sites ; adsorptet al. [Models of prebiotic RNA oligomerization on surfaces other than clays have been reported. Rajamani et al.  demonstret al. . The RNAet al. . In a seet al. . TogetheBecause it is still difficult to produce reasonably large yields of activated nucleotides in prebiotically plausible conditions, Di Mauro and coworkers chose to investigate the condensation of RNA cyclic nucleotides monophosphate (cNMP) as starting monomers in their model for non-enzymatic RNA oligomerization. 3’,5’-cGMP and 3’,5’-cAMP oligomerization yielded 25-mers and up to octamers of RNA molecules, respectively. Other cNMPs, such as 3’,5’-cUMP, 3’,5’-cCMP or 2’,3’-cAMP, yielded only short oligomers not longer than pentamers . The conSubsequently, the authors proposed a molecular reaction pathway , involviRNA products of this system were shown to possess functional activity. The neo-formed oligo-G, which is the product of 3’,5’-cGMP oligomerization, could act as a ribozyme, which can self-ligate to its complementary RNA strand (oligo-C) through two routes . The disIn an attempt to further explore the extremely fast initial rates of oligomerization, the reaction of cGMP was considered by another group . It was The availability of cyclic monomers in prebiotic conditions is not impossible ,124. Addet al. [3)2 and Pb(NO3)2 at pH 6.9 in 31 days at −18 °C. This model of a microenvironment is supported by theoretical arguments and experimental results. For instance, some investigators have argued that much of the water on early Earth was frozen, but would have undergone periodic thaw/freeze cycles, due to bolide impacts or volcanic activity [Peptide-catalyzed RNA oligomerization in the water-ice eutectic phase was also found to be feasible. The eutectic phase of water-ice occurs at a temperature of −18 °C, where the majority of the solvent is transformed to the solid pure-water phase. The remaining part of the solution is liquid and contains the solutes at a much higher concentration. This particular environment could have led to the concentration of the monomers out of the bulk aqueous solution, thus favoring their oligomerization. The idea of such an environment emerged in the early 1990s ,127 and et al. , who shoet al. , can foractivity ,130,131.activity ,133. Furactivity . All of The eutectic-phase model may support the concept of synergism in the emergence of life. A dipeptide, serine-histidine (SerHis), was found to catalyze the formation of RNA phosphodiester bond in guanosine 5’-monophosphate imidazolide in the water-ice eutectic phase at pH 6.5 over 30 days at −18 °C . Althoug18O isotope analysis of the oldest mineral known [Four among the numerous chemical models that have been proposed to simulate prebiotic synthetic pathways towards informational RNA oligomers were presented here. These models represent significant advances in non-enzymatic RNA oligomerization, especially when long oligomers were obtained. However, several limitations still exist and bear further investigations. In the mineral-catalyzed model, clay surfaces were used to concentrate and protect organic compounds of the primitive soup, but plausible monomers capable of undergoing oligomerization are still a challenge. The temperature is also a critical parameter that needs to be re-thought. Early Earth’s temperatures are assumed to be extreme compared to current Earth’s temperatures. For example, temperatures were either high (80–110 °C), an assumption supported by al known  and by mal known , or veryal known . The temal known ,129,131.al known , it was etc., should also be included in the experimental system. Indeed, the more heterogeneous the soup, the more prebiotically plausible it is. Technically, this will lead us to a humongous number of starting conditions, which is very difficult to simulate in the laboratory, but it would be an improved approach.In the models reviewed above, it was shown that mineral surfaces, lipid surfaces and peptides can separately promote RNA oligomerization. Although it is very challenging and speculative to mimic a prebiotic environment, it is possible to arrive at a new model combining features of the previous studies. It is more logical to consider the co-evolution of all of these building blocks of life in one system. Other prebiotic molecules, such as aliphatic diamines, imidazole, nucleic acid bases, Here, we propose a quaternary system consisting of mononucleotides, amino acids, montmorillonite and single-chain amphiphiles, in which potential synergism between the catalysts and mutualism between RNA and peptides are anticipated in their co-emergence and co-evolution. This model recapitulates the RNA-peptide world  and can The RNA world hypothesis is widely believed to be the best available hypothesis for the emergence of life on Earth. However, there is still a gap between the products of non-enzymatic RNA polymerization and the self-sustained RNA replicases, due in part to the lack of consideration of feedback processes. To bridge this gap, a complementary hypothesis related to the RNA-peptide world was highlighted herein. In this hypothesis, RNA and peptides might have emerged and evolved simultaneously and probably in a mutualistic manner. RNA products would have played the role of the ribosome, and catalytic peptides would have been prototypes of the current polymerases. Finally, the emergence of functional RNA would have preceded an accurate replication of RNA. To test this hypothesis, we have outlined briefly an experimental model where different catalysts may interact synergistically to enhance RNA polymerization. Re-incubation of RNA and peptide products in a fresh soup could lead to their mutualistic interaction. If isolation of functional RNA from the pool of produced oligomers could succeed, such a model would contribute to determining one missing piece of the puzzle of the origins of life.


Currently, there is a debate about their biogenesis and function.This is the first meta-analysis of tRFs. Analysis of more than 50 short RNA libraries has revealed that tRFs are precisely generated fragments present in all domains of life (bacteria to humans), and are not produced by the miRNA biogenesis pathway. Human PAR-CLIP data shows a striking preference for tRF-5s and tRF-3s to associate with AGO1, 3 and 4 rather than AGO2, and analysis of positional T to C mutational frequency indicates these tRFs associate with Argonautes in a manner similar to miRNAs. The reverse complements of canonical seed positions in these sequences match cross-link centered regions, suggesting these tRF-5s and tRF-3s interact with RNAs in the cell. Consistent with these results, human AGO1 CLASH data contains thousands of tRF-5 and tRF-3 reads chimeric with mRNAs.tRFs are an abundant class of small RNA present in all domains of life whose biogenesis is distinct from miRNAs. In human HEK293 cells tRFs associate with Argonautes 1, 3 and 4 and not Argonaute 2 which is the main effector protein of miRNA function, but otherwise have very similar properties to miRNAs, indicating tRFs may play a major role in RNA silencing.The online version of this article (doi:10.1186/s12915-014-0078-0) contains supplementary material, which is available to authorized users. Small RNAs have been defined as 19 to 31 nucleotide long RNAs present primarily in metazoans and plants, classified as either a miRNA, siRNA or piRNA based on biogenesis, and found to regulate gene expression through association with an Argonaute family member . These nfection ,8, whilenfection ,10.Distinct from tRNA halves are the less well studied small RNAs known as tRNA derived RNA fragments (tRFs). There are three types of tRFs recognized, those derived from the extreme 5’ and 3’ ends of mature tRNAs (tRF-5s and tRF-3s), and those that map to the 3’ trailer fragment of precursor tRNA transcripts (tRF-1s). These classes were first observed in LNCaP and C4-2 cells, and one tRF-1 was found to promote cell proliferation . Soon afSince the initial classification of tRFs there have been multiple studies on tRFs in organisms ranging from archaea to humans, and these studies have been summarized in several recent reviews -16. ThesWe have taken advantage of the recent explosion of small RNA-Seq data and novel methods of mapping the miRNA interactome to perform a meta-analysis of tRFs and provide insight into their properties. Our analysis of publicly available data sets clearly shows that tRFs are DROSHA-, DICER1-independent precisely generated fragments present in organisms ranging from bacteria to humans. We find that tRF-5s and tRF-3s, but not tRF-1s, are very abundant in AGO1, 3 and 4 photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) data and use canonical miRNA seed rules to associate with mRNAs. Analysis of AGO1 crosslinking, ligation, and sequencing of hybrids (CLASH) data suggests tRF-5s and tRF-3s may interact with thousands of different RNAs in human cells.Despite the fact that tRFs are more evolutionarily conserved than miRNAs, are present in similar abundance, and are the only small RNA to display clear Argonaute sorting in humans, there is not a universally accepted nomenclature for tRFs or unique identifiers for different tRF sequences. As a result, it is possible for multiple labs to be working on the same tRF without noticing it, for example, cand45 in  is the sWe mapped small RNA reads from HEK293 cells to a collapsed tRNA gene [see Additional file Most of the tRF-1s observed in this data set are 15 to 22 bases long and always begin at the end of the tRNA gene sequence which becomes a mature tRNA, and end with a RNA polymerase III (RNA pol III) transcription termination signal  ,27. BecaIt is important to note that when looking at reads that map to a single tRNA, the peaks become much sharper Figure B. The prWe next wanted to compare read counts for tRFs in cell lines other than HEK293 cells. We can observe read counts for all three classes of tRFs for all cell lines in the  data setDrosophila melanogaster [elegans [pombe [Saccharomyces cerevisiae [Rhodobacter sphaeroides [Drosophila (approximately 500 Reads Per Million (RPM)) and very few in C. elegans, S. cerevisiae and R. sphaeroides, although about 7,000 RPM of tRF-1s were detected in S. pombe.To see if tRFs are present in other species we analyzed the publicly available small RNA data of mice , Drosophnogaster , Caenorh[elegans , Schizoss [pombe , Saccharrevisiae  and the aeroides . tRF-5s C. elegans and S. cerevisiae compared to human and mouse [see Additional file Drosophila has comparable numbers of 3’ trailers in the correct size range, and yet yielded fewer tRF-1, while S. pombe had fewer 3’ trailers in the correct size range and yielded a large number of tRF-1 clones. Thus, some factor other than the possible number of 3’ trailers in the correct size range, such as protein binding partners, helps determine how many tRF-1s are stable and identifiable in each species.The lower abundance of tRF-1s in the lower eukaryotes and absence in bacteria could be explained if the 3’ trailer sequences of pre-tRNAs were not in the 14 to 36-nucleotide range that was selected for cloning and sequencing. Indeed, 14 to 36 base-long pre-tRNA trailer sequences are ten-fold fewer in All the analyses of mammalian tRFs until now have been performed against RNA extracted from cell lines. To investigate if tRFs are also expressed in normal mammalian tissues, we analyzed the small RNA isolated from adult mouse ovary, testis and brain, and from mouse embryos and embryonic stem cells ,33. tRFsTo determine if all tRNA genes produce all three types of tRFs and, if they do, whether the tRFs are in comparable abundance, we selected those tRNA genes where a tRF-1 was detected in HEK293 cells at >20 RPM. A given tRF-1 has a unique sequence that can be assigned to a specific tRNA gene. When the 5’ and 3’ ends of more than one tRNA gene are identical in sequence, we classify them as a tRNA family. Thus, we compare the cloning frequency of a specific tRF-1 with that of the tRF-5 or −3 derived from the corresponding family of tRNA genes. A tRNA family represents a group of genes encoding the same tRF-5 or −3 sequences. These could include tRNA isoacceptors but are not necessarily so.TGA or tRF-3-GlyTCC or -LeuAAG are selectively absent even though tRF-1 were detected in all three cases.The sequencing frequencies of these matched sets of tRFs were plotted Figure A. Not alTAA produces a tRF-1 that is nearly 40- to 50-fold more abundant than the tRF-5 or −3 generated from the LeuTAA tRNA family, and the Pearson correlation coefficients between tRF-5s and tRF-3s (R = −0.088), tRF-3s and tRF-1s (R = −0.224) and tRF-5s and tRF-1s (R = −0.099) are very low  did not significantly decrease the expression of any of the three classes of tRFs in mice  be applicable to a small RNA whose parental RNA is one of the most abundant RNAs in the cell?These questions are too much for any one group to answer, but it is tempting to speculate as to the possibilities. tRNAs are heavily modified and it is known that some of these modifications affect tRNA stability. Indeed, lack of a specific tRNA modification has been shown to increase tRNA half generation . This suThe matches to PAR-CLIP clusters and the large number of CLASH chimeras point to a role for tRFs in RNA silencing. In fact, it is known that a large number of CLIP-Seq clusters are not able to be assigned to miRNA seeds . Thus faIn addition, although miRNA-Argonaute complexes are traditionally thought to function in the cytoplasm, increasingly diverse functions for Argonautes in the nucleus continue to be discovered (reviewed in ). For exAlthough there has been a steady increase in tRF literature since their discovery, our current knowledge of tRFs clearly pales in comparison to other small RNAs. We have shown that tRFs display similar properties to miRNAs, and given the importance of miRNAs in processes ranging from development to cancer, it is not far-fetched to imagine equally important functions for tRFs.tRFs are a newly discovered class of small RNA that are highly abundant in different human cell lines, mouse tissues and organisms ranging from bacteria to humans. Individual tRFs show a narrow size distribution, suggesting that the fragments are precisely generated and not degradation products of tRNAs. Mutation of different components of the miRNA biogenesis pathway does not have an effect on tRF levels, and tRFs are seen in organisms that do not contain miRNAs, indicating tRF generation is distinct from miRNA biogenesis. In human HEK 293 cells tRF-5s and tRF-3s are associated with Argonautes 1, 3 and 4 as evidenced by PAR-CLIP data. These tRFs contain seed sequences, which match the central portion of large numbers of CCRs. This observation, along with the finding of thousands of tRF-mRNA chimeras in CLASH data, indicates tRF-3s and tRF-5s can target RNAs in a manner similar to miRNAs.The data analyzed in this manuscript were downloaded from either the GEO database  or NCBI Information about the tRNA genes for each species  was downloaded from the ‘Genomic tRNA database’ . For eacWe included the mature miRNA  and mRNA sequences in our previously built human specific tRNAdb that was used to query the expression level of tRFs and miRNA. We investigated tRF and miRNA expression with human Argonautes by analyzing the human Ago1 (GEO ID = GSM545212), 2 (GEO ID = GSM545213), 3 (GEO ID = GSM545214) and 4 (GEO ID = GSM545215) PAR-CLIP data isolated form HEK293 cell lines [The miRNA, tRF-5, tRF-3 and tRF-1 analyses were performed separately. For the miRNA analysis, reads were found that started with a mature miRNA allowing no mismatches, giving preference to longer miRNAs. For the tRF-5 analysis, reads were found that started with a sequence that mapped to the first 14 to 33 nucleotides of a tRNA, allowing no mismatches, giving preference to longer tRF-5s. For the tRF-3 analysis, reads were found that started with a sequence that mapped to the last 17 to 23 nucleotides of a mature tRNA, allowing no mismatches, giving preference to longer tRF-3s. For the tRF-1 analysis, reads were found that started with a sequence that mapped to the first 14 to 33 nucleotides of a tRNA trailer, allowing no mismatches, giving preference to longer tRF-1s. For the miRNA analysis, the reads were confirmed to not be pre-miRNAs by running blastn, word size 7, default scoring matrix, against a database composed of miRNA hairpins from miRBase. For the tRF-5 analysis, the reads were confirmed to not be longer tRNA fragments or full length tRNAs by running blastn, word size 7, default scoring matrix, against a database composed of mature tRNA sequences. All reads were checked to not be RNA fragments by performing a blastn search against the human Ref-Seq database using blastn, word size 7, default scoring matrix, 20 maximum hits. Reads that had a hit which either overlapped 6 or more bases of the small RNA, or 6 or more bases of an 18 base minimal small RNA sequence for the longer small RNAs, and had an e-value less than or equal to .001, were discarded. For all analyses the portion of the read following the small RNA sequence was searched against the human RNA Ref-Seq database using blastn, word size 7, default scoring matrix, 20 maximum hits. Because blastn is a local aligner, a conservative approach was taken to adaptor removal. Adaptor was removed if a perfect match was found for 12 or more bases of the adaptor, or 1 mismatch for 21 or more bases of adaptor, or 2 mismatches for 26 or more bases of adaptor. Reads were considered chimeras if a hit was found within four nucleotides of the end of the small RNA and had an e-value less than or equal to .01. Because the search space can affect the e-value, reads still possibly containing adaptor sequence and having a borderline e-value underwent adaptor removal with Biopython’s pairwise2 local aligner and were blast searched again to get an updated e-value. Many reads matched more than one transcript in Ref-Seq. To identify the most likely transcript, every read of the CLASH data was searched against the human Ref-Seq database using blastn, word size 7, default scoring matrix, 20 maximum hits. All hits with an e-value less than .1 were tabulated. For the chimeric reads, the most likely transcript was deemed to be the transcript which was most abundant in the data. If a tie still occurred, NM transcripts were given preference to XM transcripts, XM given preference to NR, and NR given preference to XR. All chimeras whose most likely transcript was either NM or XM were deemed to be a small RNA-mRNA chimera. All such tRF chimeras are reported in Additional file


This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork. We further discover that extensive backtracking provides access to a new 3′-end that allows for the  Escherichia coli RNAP pauses in a sequence-dependent manner during elongation RNA)  or anti-sense ((−)RNA) , or they can be double-stranded . Due to the high error rate of their RdRps , RNA virde novo on a free 3′ RNA end (de novo initiation (e.g. hepatitis C virus).The magnetic tweezers assay and the flow-cell preparation used here have been previously described in ,27,28.The construct employed here is made of four single-stranded (ss) RNA strands annealed to one complementary strand of 4 kb in length as described in detail previously . To link2, 2 mM MnCl2, 0.01% Triton X-100, 5% Superase RNase inhibitor (Life Technologies), 20 μg/ml bovine serum albumin, and an optimal concentration of nucleotides, [NTP]opt, of 1 mM ATP and GTP, 0.2 mM CTP and UTP, coming from HPLC-purified stock solutions. This concentration has been shown to be optimal for initiation of the RdRp on the 3′-end of an RNA template starting with 3 C residues followed by 15 U residues , 5 mM MgCl.0, 5 mM residues ,30.x, y, z) position . In theocument}  does notnt}  operates as a conduit for the plus-strand RNA release –43. By ade novo on an exposed 3′-end remains to be further investigated. A possible role for such reversal events could be the production of shorter RNA molecules .


Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity ( REases require divalent cations . This zinc finger protein engages the internal control region of the 5S RNA gene, as well as the 5S RNA transcript itself  as described by the manufacturer, where standard 10-μl reactions included 2 μl [α-32P]-CTP .DNA oligonucleotides were synthesized commercially (IDT) and purified by the manufacturer using gel filtration chromatography. The synthetic DNA template for the selection pool was 5′-TAinvitro selection and binding assays were obtained from New England Biolabs.Commercial REases and purified preparations of BamHI (E111A mutant), KpnI and PacI for 11 unique sequences. A 5 pmol sample contains about 10 copies of each unique sequence. PCR reactions (100 μl) contained 20 mM Tris pH 8.4, 50 mM KCl, 4 mM MgCl2, 200 nM dNTPs, 400 nM of each primer, 20 nM DNA template and 0.1 U/μl recombinant Taq polymerase (Invitrogen). The optimum number of PCR cycles was determined by qualitative appearance of the amplification product on an 8% polyacrylamide gel (29:1 acrylamide:bisacrylamide), electrophoresed in 0.5× Tris-borate EDTA (TBE) buffer and stained with ethidium bromide. PCR was achieved by 2 min at 95°C as initial denaturation, followed by 3 cycles of 30 s at 95°C for denaturation, 30 s at 52°C as annealing, 30 s at 72°C for extension, followed by 5 cycles of 30 s at 95°C for denaturation, 30 s at 65°C as annealing, 30 s at 72°C for extension and final extension for 5 min at 72°C. Products were separated by electrophoresis through 2.5% agarose gels in 1× TAE buffer and the major product purified using a QIAquick gel extraction kit (Qiagen). Initial RNA pools were transcribed from 6 pmol of DNA template (representing ∼1012 sequences) using an Ampliscribe T7 Transcription Kit (Epicentre) as described above. RNA was purified by denaturing 10% polyacrylamide gel electrophoresis , visualized by UV shadowing, excised and eluted from gel pieces overnight at room temperature with agitation in 2× PK buffer , 300 mM NaCl and 2% sodium dodecyl sulphate). The eluted material was extracted with an equal volume of phenol/chloroform  and RNA was precipitated by the addition of 2.5 volumes of ethanol. For initial selections, 23 pmol of pool RNA representing ∼43 copies each of 3 × 1011 DNA templates was used as a 0.46 nM solution in a volume of 50 ml in the presence of 2 nM target protein in the appropriate selection buffer (SB) such that REases are not catalytically active: BamHI(E111A): 10 mM HEPES pH 7.5, 400 mM NaCl, 1 mM MgCl2; KpnI: 10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM CaCl2; and PacI: 10 mM HEPES pH 7.5, 10 mM NaCl, 1 mM CaCl2. Prior to selection all RNA pools were incubated in SB at 70°C for 5 min and allowed to cool to room temperature to promote RNA folding. Selections were performed by incubation of RNAs for 45 min in SB buffer with or without protein followed by vacuum partitioning using nitrocellulose filtration . Unbound RNAs were washed from the filter using two aliquots of 10 ml SB. Captured RNA was then quantitated by Cerenkov radiation detected on filters using the 3H setting of a scintillation counter. Bound RNA was eluted in 500 μl 2× PK buffer at 65°C for 30 min. The eluted solution was extracted with an equal volume of phenol:chloroform (1:1) and RNA precipitated from ethanol using 40 μg of glycogen as carrier. Before round 3 and subsequent odd rounds negative selections were performed by exposing the RNA library to nitrocellulose filter strips and processing the supernatant for selection. To prepare cDNA, one-third of the recovered RNA was reverse transcribed using primer LJM-4486 (5′-A5TATG2A2CGCT2CATGCAT). Prior to the addition of MMLV reverse transcriptase and dNTPs, the primer was annealed by incubating for 5 min at 65°C and 5 min at room temperature. Each 40-μl reverse transcription reaction contained 1.25 μM primer LJM-4486, 400 μM dNTPs, 50 mM KCl, 6 mM MgCl2, 50 mM Tris pH 8.0 and 2 μl 200 U/μl MMLV reverse transcriptase (Invitrogen.) Reactions were incubated at room temperature for 10 min and at 37°C for 30 min. One-half of the each reaction was used for optimization PCR. After PCR amplification of the remaining cDNA, DNA libraries were purified using a PCR Purification Kit (Qiagen). To prepare RNA pools for subsequent rounds of selection, 200 ng (4 pmol) purified DNA was transcribed as previously described. To monitor binding and recovery of radiolabeled RNAs at each round of selection, nitrocellulose filter radioactivity was compared to a fraction of total radioactivity to determine the fraction of RNA bound to the protein target. Twenty rounds of selection were performed for each enzyme with maximum RNA recovery for BamHI reached at cycle 14, for KpnI at cycle 15 and for PacI at cycle 19 , 15, 16 and 18 for KpnI, and 18, 19 and 20 for PacI were ligated into the pGEM-TEasy vector using a TA cloning kit (Promega). Clones with coherent sequences were aligned using multiple sequence alignment tool MUSCLE (EMBL).32P]-cytidine 3′,5′ bis(phosphate), , using T4 RNA ligase (New England Biolabs). Unincorporated nucleotides where removed by size exclusion chromatography using Chromaspin TE-10 gel filtration columns (Clontech). Binding reactions (25 μl) contained the appropriate SB, 4 nM RNA and 20 nM target protein. Reactions were incubated for 30 min at 37°C. Binding reactions were supplemented with glycerol loading dye and analyzed by electrophoresis through 8% polyacrylamide gels in 1× TBE at 3W for 1 h. Gels where then dried and exposed overnight using storage phosphor imaging technology (Typhoon FLA-7000).RNAs were 3′ end-labeled by ligation of -ATP , using calf intestinal alkaline phosphatase (CIP) and T4 polynucleotide kinase (New England Biolabs), followed by gel purification. All reactions contained 105 radioactive counts per minute of radiolabeled aptamer RNA. In-line probing reactions (10 μl) contained 50 mM Tris–HCl (pH 8.3 at 20°C), 20 mM MgCl2 and 100 mM KCl. Reactions were incubated at room temperature for 24 or 48 h. RNase T1 treatment was performed in 25 mM sodium citrate pH 5.0, 0.1 U/μl RNase T1, in the presence of 5 M urea and 0.75 mM EDTA (pH 8.0 at 20°C). Reactions were incubated at 55°C for 3 min. Partial alkaline hydrolysis reactions were performed in 50 mM Na2CO3 pH 9.0, 1 mM EDTA, incubated at 95°C for 5 min. All reactions were terminated by ethanol precipitation. Vacuum-dried pellets were then resuspended in 8 μl deionized formamide gel-loading solution and immediately analyzed by gel electrophoresis through 10% denaturing polyacrylamide sequencing gels  in 1× TBE buffer. Electrophoresis was at 48 W for 3 h. Gels were then transferred to blotting paper, dried for 1 h and signal detected by storage phosphor imaging. Signal analysis was performed using SAFA (RNA aptamers were dephosphorylated and labeled at 5′ termini using γ- . In invitro selection and characterization of RNA aptamers using REases BamHI, KpnI and PacI as targets. We identify RNA aptamers selected against KpnI that selectively bind (Kd ∼12–30 nM) and inhibit (IC50 ∼20–150 nM) this REase with high specificity. Evaluation of four representative anti-KpnI aptamers  by reverse titration and by competition studies suggests a 1 aptamer:1 KpnI homodimer binding stoichiometry. Binding appears to be at or near the KpnI DNA binding site, competing with DNA binding. Binding selectivity can be exquisite: mimicry of the KpnI DNA binding site by aptamer 20 does not confer inhibition of KpnI isoschizomer Acc65I. This suggests that aptamer 20 contacts KpnI surfaces beyond the minimal DNA recognition interface.Here we report the Secondary structure prediction, in-line probing experiments and CD spectra suggest that the selected aptamers evolved on the approximate intended hairpin. KpnI inhibition by a minimal version of aptamer 20 (aptamer 20s) confirms this prediction. Interestingly, none of the inhibitory anti-KpnI aptamers carries any obvious homology to the duplex DNA binding site 5′-GGTACC. This is not unexpected in that the previously-studied high-affinity anti-NF-κB p50 RNA aptamer also does not bear obvious primary sequence homology to the DNA sequence it mimics ,17,23.4 sequence within the N10 domain or the N9 sequence downstream from the tetraloop inactivated function even when an overall hairpin structure was roughly conserved . We also found that disruption of either the distinctive Ad Figure .Regrettably, there are no high-resolution structural data for KpnI, a member of the HNH superfamily. Homology analysis suggests that amino acid residues D148, H149 and Q175 compose the catalytic metal-binding active site of KpnI . FurtherOur prior selections of RNA aptamers against NF-κB family members ,35 sugge2+) and for PacI  are least like the actual conditions for enzyme activity. Finally, we note that BamHI selections employed catalytically inactive E111A mutant protein. It is possible that the selected aptamers do not bind as well to catalytically-active wild-type protein.It is intriguing that RNA aptamers selected against BamHI and PacI showed binding activity without enzyme inhibition. We hypothesize that three considerations are at play. First, these enzymes may have cationic surfaces that elicit RNA ligands lacking the ability to inhibit enzyme activity. Second, we found it necessary to identify different stringent salt conditions for selections with each enzyme. This means that the selection conditions for BamHI . However, it is possible that RNA cleavage might rapidly inactivate such aptamers so they do not function as competitive inhibitors.2+ chelation. Neither approach is selective, reversible or applicable invivo. Anti-REase RNA aptamers could prove valuable in studies of restriction enzyme mechanism. Moreover, these aptamers provide new examples in the growing list of natural and artificial RNAs that bind DNA-binding proteins by DNA mimicry. As such, they provide the raw material for future structural studies to understand in detail this DNA mimicry and to deduce principles by which an RNA scaffold might be designed to inhibit transcription factors with known DNA binding specificities.To our knowledge, the anti-KpnI RNA aptamers reported here are the first known selective restriction enzyme inhibitors. Current approaches to REase inhibition are limited to irreversible heat denaturation or Mg


The aim of this study was to assess the independent effect of radiographic measures of implant position, relative to pre-operative anatomical assessment, on the functional outcome of total hip arthroplasty according to change in the Oxford hip score (OHS) 1 year post surgery.A prospective cohort study was preformed to assess whether improvement in functional outcome (change in OHS at 1 year) and the relationship with femoral offset and length, and acetabular offset and height. After a power calculation 359 patients were recruited to the study and radiographic measures were performed by blinded observers. Regression analysis was used to assess the independent effect of the four radiographic measurements after adjusting for confounding variables.p < 0.001) decrease in acetabular offset  and increase in femoral offset . Hence there was no significant change in overall offset. Femoral offset was the only radiographic measure to be achieved statistical significance  in relation to clinical outcome, with increasing offset being associated with a greater improvement in the OHS. On combining femoral and acetabular offset increasing offset was associated with a greater improvement in the OHS .There was a significant (This study supports the long-held biomechanical theory of medialisation of the acetabular component with compensatory increased femoral offset results in improved functional outcome. Total hip arthroplasty (THA) is one of the most successful surgical procedures performed, being named as the operation of the century , and is There is conflicting evidence as to whether leg length is a predictor of functional outcome and patient satisfaction after THA –8. WhiteThere is, however, limited literature reporting the effect of implant position and reconstruction of centre of rotation and  offset on the outcome of THA. Despite the theoretical biomechanical benefits of medializing the acetabular component and increasing femoral offset to compensate for this resulting in a more favourable moment arm , 13, theThe primary aim of this study was, therefore, to assess the independent effect of radiographic measures of implant position, relative to pre-operative anatomical assessment, on the functional outcome of THA according to change in the OHS 1 year post-surgery. The secondary aims were to assess the effect of radiographic measures of implant position on non-hip specific functional outcome (SF-12 and EuroQoL) scores and patient satisfaction 1 year post-surgery.Ethical approval was obtained from the regional ethics committee  for collection, analysis, and publication of the anonymised data.During a 1 year period (2013) patients undergoing a THA at the study centre had functional outcome data recorded prospectively. Inclusion criteria for this study were: primary osteoarthritis, no deformity (precluding radiographic assessment), pre- and post-operative radiographs, and a cemented prosthesis. Patients undergoing revision during the first post-operative year were excluded. Patients undergoing consecutive bilateral THAs during the study period only had outcome and radiographic measures assessed for their first THA.The patient demographics, comorbidities, BMI, and patient reported outcome measures were recorded at the pre-operative assessment clinic. Categories of comorbidity included were: heart disease, hypertension, lung disease, vascular disease, neurological problems, diabetes, stomach ulcer, kidney disease, liver disease, depression, back pain, and pain in other joints, which were all recorded as dichotomous variables.The OHS , SF-12 sPatient satisfaction was assessed by asking the question “How satisfied are you with your operated hip?” 1 year after surgery. The response was recorded using a five point Likert scale: very satisfied, satisfied, neutral, dissatisfied and very dissatisfied. Patients who recorded very satisfied or satisfied were classified as satisfied.Radiographic assessment was performed by a blinded observer using a standardised protocol anterior–posterior radiograph of pelvis and hips pre-operatively and 1 year post-operatively. Radiographic measurement of offset and length for both the femoral and acetabular components were measured according to the methods described by Nunn et al.  and Jogg®) femoral stem (n = 327) or an Olympia  femoral stem (n = 32) using a Contemporary acetabular component (Stryker®). The surgical approach and technique were dependent upon surgeon preference with a higher prevalence of posterior approach . All surgeons aimed to maintain offset and restore equal leg length, with a stable hip at the end of surgery. All patients received three peri-operative doses of prophylactic antibiotics (cefuroxime). A standardised rehabilitation protocol as per local clinical care pathway was used for all patients, with active mobilisation and full weight bearing on the first day post-operatively. Patients were then reviewed at 6 weeks, 6 months, and 12 months post-operatively as per local protocol.During the study period twelve consultant orthopaedic surgeons performed or supervised all included THAs. All patients underwent a THA using a cemented Exeter V40 . A single measure intraclass correlation coefficient (ICC) was used for the quantification of inter- and intra-observer reliability of the radiographic measurements. The values greater than 0.75 indicate satisfactory reliability [p value of <0.05 was defined as significant.Statistical analysis was performed using Statistical Package for Social Sciences version 17.0 . Parametric and non-parametric tests were used as appropriate to assess continuous variables for significant differences between groups. A Student’s iability . A p valp = 0.001) correlation coefficient of 0.17 was demonstrated between the change in the femoral offset and the OHS. Using a bivariate normal model the required sample size using a two tailed analysis with an alpha of 0.05 and a power of 0.90 using the 0.17 correlation coefficient required 359 patients to be recruited.A power calculation was performed using data collected for the first 250 patients selected at random (pilot group). Using the OHS (primary outcome measure) and change in the radiographic measures, both of which demonstrated normal distribution, a significant (p = 0.87), age (p = 0.99), BMI (p = 0.92) or for the pre-operative functional measures (p > 0.80) between the study cohort and those patients not selected. The cohort demographics are presented in Table During the study period 806 THA were performed at the study centre, of which 530 had pre- and post-operative outcome data recorded. For this study a cohort of 359 were randomly selected from the 530 patients with complete data met the inclusion criterion. There was no significant difference in gender  and dissatisfied (n = 26) patients for change in femoral offset (p = 0.81 t test) and length (p = 0.80 t test) or acetabular offset (p = 0.28 t test) and height (p = 0.47 t test).The inter-observer variation for each of the radiographic measurements demonstrated satisfactory reliability . The intra-observer variation demonstrated excellent reliability .There were significant changes observed in femoral offset and length, and acetabular offset, but not for acetabular height Table . A similr = 0.198, 95 % CI 0.063–0.333, p = 0.004). Interestingly, on combing femoral and acetabular offset, increasing offset was associated with a greater improvement in the OHS , but was not as significant as femoral offset independently. Hence, it would seem that overall offset is not as important as femoral offset.Regression analysis was used to assess the independent effect of the four radiographic measures on change in the OHS when adjusting for confounding variables Table . FemoralA major limitation of this study was measuring offset using a digital radiograph of the pelvis. Measuring femoral offset using plane radiographic studies is limited by the precision of the technique and is dependent upon the patient position, magnification, and femoral rotation. It would have been of benefit to use Einbildröntgenanalyse (EBRA), which has been shown to have a measurement precision of around 0.8–1 mm , 25. ThiDuring THA it is estimated that on average the involved limb is lengthened by 2.5–6.2 mm , which iCharnley  and MullComputer navigation has been shown to enable quantitative control of offset, both femoral and acetabulum during THA, with the centre of hip rotation being maintained within 5 mm of the contralateral normal side . This teThis study supports the long-held biomechanical theory of medialisation of the acetabular component with compensatory increased femoral offset results in improved functional outcome, which has been demonstrated using a hip-specific validated outcome measure. The exact anatomic parameters of the femoral and acetabular components that relate to the optimal outcome of patients undergoing a THA remain to be identified. Potentially computer navigation may help improve implant positing and attain an optimal component orientation that achieves a stable THA with maximal functional outcome and longevity for each patient.


Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized  Escherichia coli, several types of toxin ribonucleases have been shown to catalyze intracellular RNA cleavage in a ribosome dependent  [Toxin-antitoxin (TA) systems are genetic modules composed of a stable toxin and a volatile antitoxin. They are widely distributed among archaeal and bacterial lineages and allow microbes to withstand environmental stresses –3. When nd YoeB) –11 or innd YoeB) –16 fashiMazF, which is one of the most investigated RNA interferases, is a part of a TA system along with its cognate antitoxin MazE . As withE. coli) RNA-seq was developed [E. coli and the 5′-end sequences of the extracted RNAs that correspond to the cleavage sequence were detected with SOLiD platform [The cleavage patterns produced by most RNA interferases are yet to be identified because of limitations from using classical methods. In most studies, primer extension analysis with MS2 bacteriophage RNA and chaperone protein CspA has been used ,26,27. Aeveloped . In thisplatform . This meE. coli MazF, ACA was identified as the cleavage sequence. Thus, to verify this approach in another microbe, an uncharacterized MazF homologue that is predicted in the Pseudomonas putida chromosome was also analyzed. The RNA-seq showed that UAC was the RNA cleavage sequence. This observation was further confirmed by a fluorometric assay [In our study, we also determined the cleavage sequence of RNA interferases using RNA-seq. The RNAs used in this study were designed with a computer algorithm that generates diverse sequences , and thuic assay , demonstE. coli strains were used in this study: DH5α  for the constructed plasmid propagation and BL21 (DE3) pLysS  for recombinant protein expression. Two plasmids were purchased from Takara Bio Service, pMD19 encoding the graA gene and empty vector pET21c .The following PCR primers were purchased from Tsukuba Oligo Service . All fluorescent-labeled oligonucleotides were purchased from Japan Bio Services . Barcode RNA was also purchased from Japan Bio Services . The sequence of PCR primers and a barcode RNA are listed in mazEpp) and the toxin (mazFpp) genes with Taq HS DNA polymerase (Takara) using genomic DNA of Pseudomonas putida as a template. These PCR products were purified by a PCR purification kit . pET21c and PCR products were digested with BamHI and EcoRI  and cleaned using phenol/chloroform extraction and ethanol precipitation. Digested mazEpp and mazFpp fragments were inserted into the corresponding pET21c multiple cloning sites with a DNA ligation kit (Takara). This generated the plasmids pET21c-mazEpp and pET21c-mazFpp, respectively. Each plasmid was then introduced into Escherichia coli DH5α and the colonies were grown overnight at 37°C on LB plate containing ampicillin (100 μg/ml). Plasmids were extracted using QIAprep Spin Miniprep Kit (Qiagen) and their sequences were determined using AB 3500 Genetic Analyzer  according to the manufacturer’s protocol.PCR was carried out to amplify the antitoxin (mazEpp and pET21c-mazFpp plasmids were introduced into E. coli BL21 (DE3) pLysS via heatshock. The E. coli harboring pET21c-mazEpp or pET21c-mazFpp were pre-cultivated in LB medium containing 100 μg/ml ampicillin at 37°C for overnight and then inoculated into the 0.5 L or 1 L of LB medium supplemented with 100 μg/ml ampicillin, respectively. MazEpp and MazFpp were induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside when OD600 reached 2.0 and 4.0, respectively. After 3.5 hours of incubation, cells were harvested by centrifugation at 7,000 g and then stored at -80°C for future use.The pET21c-E. coli cells containing MazEpp were thawed on ice and resuspended in lysis buffer . Cells were lysed by sonication for 15 minutes with a Handy Sonic UR-20P . The supernatant containing MazEpp was collected by centrifugation at 7,000 g for 15 minutes. Ni-NTA agarose (Qiagen) was added to the supernatant and rotated for 2 hours at 4°C to allow MazEpp to adsorb onto the Ni-NTA column. Non-specific proteins were eliminated with washing buffer  and 100 mM imidazole buffer . MazEpp was eluted with elution buffer . The molecular weight and purity were confirmed by SDS-PAGE. Protein concentration was determined using Qubit Protein Assay Kit (Life Technologies).E. coli cells containing MazFpp were thawed on ice and resuspended in binding buffer . Suspended cells were sonicated for 15 minutes with a Handy Sonic UR-20P and then centrifuged at 7,000 g for 15 minutes. The supernatant was then filtered with a 0.45 μm membrane . After the 1 mL His-Trap HP column  was equilibrated with 10 column volumes (cv) of binding buffer, the filtered supernatant was applied to this column and washed with 32 cv of binding buffer. Residual non-specific proteins and hexa-histidine tagged MazFpp were separated by gradually increasing the imidazole concentration using AKTA pure plus . The molecular weight and purity were confirmed by SDS-PAGE. Protein concentration was determined using Qubit Protein Assay Kit.graA and six artificial synthetic RNAs named 500–2, 1000–1, 1000–2, 1000–3, 1000–4, and 1000–5 [graA gene was digested with HindIII . pUC19 plasmids encoding synthetic RNA  and the pUC19 plasmid encoding 1000–5 synthetic RNA were digested with BbsI and BtgZI, respectively (New England Biolabs). Digested fragments were purified with a PCR purification kit and then in vitro transcription was carried out with MEGAscript T7 Kit (Life Technologies) according to the manufacturer’s instruction. Transcribed RNAs were purified with RNA Clean & Concentrator™-5  and their concentration was determined using Qubit RNA Assay Kit (Life Technologies).The transcript of d 1000–5  were used 1000–5 , and eacSubstrate RNA was prepared by mixing 300 ng of RNA 1000–1, 1000–2, 1000–3, 1000–4, and 1000–5. This substrate was incubated at 37°C for 15 minutes with two distinct RNA interferases: 2 U of MazF (Takara) in MazF buffer (Takara) containing 4 U of recombinant RNase inhibitor (Takara) or 300 ng of MazFpp in MazFpp buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% tritonX-100, and 4 U of recombinant RNase inhibitor). As the control experiment, the substrate RNA was also treated with 2.5 U of RNase III (New England Biolabs) in NEBNext RNase III Reaction Buffer (New England Biolabs) at 37°C for 15 minutes. These samples were cleaned with RNA Clean & Concentrator™-5 and then incubated with 20 U of T4 polynucleotide kinase (Takara) in T4 Polynucleotide Kinase Buffer (Takara) containing 1 mM ATP  at 37°C for 1 hour. They were purified using RNA Clean & Concentrator™-5. Subsequently, 125 pmol of barcode RNA and purified RNA fragments were incubated with 50 U of T4 RNA ligase (Takara) in the RNA ligation buffer (Takara) for 18 hours at 15°C. Samples were purified with RNA Clean & Concentrator™-5 and then the RNA concentration was determined using the Qubit RNA Assay Kit. Sequencing library was constructed according to the NEB Ultra RNA Library Prep Kit for Illumina protocols (New England Biolabs). For this study, the protocol for longer size RNA inserts was used. The constructed library quality was validated using the Agilent High Sensitivity DNA Kit . Sequencing was performed using the MiSeq platform with the MiSeq 500 cycles reagent kit v2  according to the manufacturer’s protocol.The output files configured in fastaq format for R1 and R2 reads were separately analyzed with CLC Genomics Workbench version 7.5.1 . Nucleotides with low quality or ambiguity were eliminated with the parameters limit of 0.05 and a maximum number of ambiguities equals to zero. Reads that included the 15-base sequence (CTGGCTTTGATGAAA) corresponding to the 3′-end sequence of the barcode were extracted from both strands via the following parameters: mismatch cost and gap cost equal five, while minimum scores for internal match and end match both equal 15. All the 5′ nucleotides upstream of this 15-base sequence were removed and the reads shorter than 15 bases were discarded. The resulting reads were mapped against five references . The parameters used for mapping were as follows: mismatch cost, insertion cost, and deletion cost equal three; length and similarity fraction both equal one. The fastaq files were deposited into the DNA Data Bank of Japan Sequence Read Archive (DRA004282).Purified MazFpp was incubated with RNA 500–2. Next, 3 pmol of MazFpp was pre-incubated with 0.3, 3, or 30 pmol of MazEpp at room temperature for 10 minutes. After the pre-incubation, 100 ng of RNA 500–2 was added and the mix was incubated at 37°C for 30 minutes in MazFpp buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% tritonX-100, and 4 U of recombinant RNase inhibitor). Samples were purified by RNA Clean & Concentrator™-5 and the gel loading buffer II (Ambion) was added to each sample. Samples were incubated at 95°C for 5 minutes and separated on a 10% polyacrylamide gel containing 7 M urea. RNA was stained using SYBR Gold (Life Technologies) and then detected with a Typhoon 9210 imager .A fluorometric assay  was usedgraA transcript and RNA 500–2 were incubated with 50 ng of MazFpp at 37°C in MazFpp buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% tritonX-100, and 4 U of recombinant RNase inhibitor). After incubation for 1, 5, 15, or 30 minutes, gel loading buffer II was added to each sample. Samples were incubated at 95°C for 5 minutes before electrophoresis on a 10% polyacrylamide gel containing 7 M urea. RNA was then stained with SYBR Gold and detected with a Typhoon 9210 imager.One hundred nanograms of mazEpp gene , mazFpp gene , graA gene , 500–2 , 1000–1 , 1000–2 , 1000–3 , 1000–4 , and 1000–5 .The GenBank accession numbers and GI numbers were as follows: In massive parallel sequencing, nucleotides are sequenced with significant depth, which generates reliable data about the frequency of the detected molecules . Indeed,Escherichia coli chromosome [E. coli MazF as a model endoribonuclease and then evaluated if our approach was consistent with previous results [MazF is one of the best-characterized RNA interferases, and it was originally identified in an romosome . Since t results ,36. To tth position divided by the coverage at the nth position. The relative coverage increase at each nucleotide position was arranged in decreasing order in each reference, and five nucleotides that showed the five largest relative coverage increases were selected from all references . Since this bacterium is widely used for production of chemical compounds and it is also a xenobiotic decomposer in environmental engineering [Having determined the MazF cleavage sequence as ACA, we next tried to characterize the RNA interferase with unknown cleavage specificity. For this purpose, we chose a putative MazF homologue encoded by the locus PP0771 in ineering , charactE. coli MazF, there is no evidence of its function. Hence, we isolated histidine-tagged MazFpp  was indeed stable even in the presence of MazFpp . As we anticipated, substitution of the first U to the other pyrimidine base C or T (DR-13-CAC or DR-13-AC) completely impaired the MazFpp-mediated cleavage . Howeverf MazFpp . Taken tRNA interferases, which compose TA systems, are the toxin endoribonucleases that disrupt the stability of intracellular RNAs by cleaving them in a ribosome independent –16 or deE. coli MazF cleavage as ACA  [P. putida MazF could cleave RNA at UAC triplets  , only ^Atriplets . Notablyrculosis , is low nt motif . SimilarThe five 1033 nucleotide synthetic RNAs employed in this study also facilitated this detection. When compared to the MORE RNA-seq, the sequence diversity of these RNAs was not rich, and thus, our method is not suitable for characterizing long base cutters such as MazF-hw, which recognizes seven bases in a strict manner ; indeed,P. putida genome and we found that 97 coding sequences lack UAC triplets. Interestingly, UAC triplets were also absent in the transcript of the antitoxin gene, encoded by locus PP1585. Recently, Tamman et al. demonstrated that this antitoxin composes a genuine TA pair that belongs to the HigBA family, and they named this TA pair GraTA (Growth rate-affecting toxin-antitoxin), consisting of GraT toxin and GraA antitoxin [graTA operon was efficiently repressed by GraA antitoxin, probably via its DNA binding domain. Since the graA transcript is tolerant of MazFpp  Graph of the coverage (blue bar) and relative coverage increase (orange line). (B) Graphical representation of the sequences around the base with increases in coverage. The nucleotide position with significant increases in coverage was numbered as zero. Twenty-five sequences were analyzed  and the (TIF)Click here for additional data file.S2 FigOne hundred nanograms of RNase A (blue) or MazFpp (green) was incubated with RNA oligonucleotides. The yellow plot represents a control reaction where no enzyme was added.(TIF)Click here for additional data file.S3 FiggraA transcript, which do or do not include UAC triplets, respectively, were incubated with MazFpp. Lanes 2 and 8, control reactions in which no enzyme was added; Lanes 3–6, 100 ng of MazFpp was incubated with RNA 500–2; Lanes 9–12, 100 ng of MazFpp was incubated with graA transcript.Synthetic RNA 500–2 or (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Allium cepa) or chromocentric  nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention.The localisation of poly(A) RNA in plant cells containing either reticular ( Cajal bodies (CBs) are multifunctional domains present within the nuclei of plant and animal cells. CBs are approximately 0.5–1.0 µm in diameter, but their size and number depend on the cell type, cell cycle, and metabolic activity Xenopus laevis oocytes temporarily concentrate in CBs CBs are associated with particular stages of snRNA biogenesis, such as transcription, those involving loud export factors, modifications and snRNP assembly The coilin protein is the key component and marker of Cajal bodies. The homolog of coilin in plants, Atcoilin shows a strong affinity for snRNA, but also for non-specific RNA Arabidopsis thaliana mutants, the growth phenotype is not observed in mutants lacking observable CBs de novo. In addition to the maturation of individual snRNAs, di- and tri-snRNP assembly also occurs in CBs. An in vivo kinetics analysis revealed the production of 3.8 tri-snRNPs per second, which is approximately 10-fold faster than in the surrounding nucleoplasm Although CBs are involved in many basic processes such as snRNA maturation, they have not been observed in all types of eukaryotic cells. Cajal bodies are absent from some adult cell types, including smooth and cardiac muscle, endothelial cells, dermal and epidermal cells, despite the presence of SMN and coilin in these tissues Larix deciduas Mill). In the present study, our purpose was to clarify whether this phenomenon occurs only in the generative line, or if it is also commonly observed in somatic cells. We localised poly(A) RNAs in reticular and chromocentric types of nuclei in the roots of plants via in situ hybridisation, and we evaluated whether these transcripts were protein coding and whether CBs may serve as a site for their synthesis. Measurements of the amount of poly(A) RNA were conducted in root cell nuclei under natural and stress conditions.In a previous study, Kołowerzo et al. Allium cepa L.  were placed on a wire mesh covering a container full of tap water so that only the root blastema was exposed to water. Two or three days after the culture was initiated, the bulbs possessed roots 1–2 cm in length. Lupinus luteus cv Zeus  seeds were soaked in water for 5 h and subsequently germinated at 21°C for 3–4 days on water-soaked tissue paper.Bulbs of Allium cepa and Lupinus luteus roots were excised under water and fixed in 4% paraformaldehyde in 50 mM Pipes buffer, pH 7.0, for 12 h at 4°C. The fixed roots were washed three times for 15 min in Pipes buffer and for 15 min in PBS buffer.Meristems of Arabidopsis thaliana Col-0 seeds were sterilised and sown in 2/3 Murashige and Skoog medium supplemented with 0.7% (w/v) agar. The growth conditions involved continuous light with an irradiance of 60 µmol m−2 s−1 at 22°C. Meristems of 14-day-old Arabidopsis thaliana roots were excised under water and fixed in the same manner as the Allium and Lupinus roots.Allium and 25 min for Lupinus root cells at 35°C. Then, the sections were rinsed with PBS and treated with 0.1% Triton ×100 solution in PBS buffer, pH 7.2, for 10 min. To induce hypoxia, Lupinus seedlings were stressed by submersion in tap water for 3 h. After fixation, the roots were placed a citric acid-buffered digestion solution (pH 4.8) containing 9% cellulase (Onuzuka R-10) and 35 U/ml pectinase (Sigma) for 90 min. After rinsing with PBS and distilled water, the root tips were squashed onto slides.For double localisation assays, onion and lupine root tips were sectioned under water into 50 µm-thick sections using a Vibratome Leica VT1200. The sections were then placed in embryo dishes and treated with 2% cellulase (Onuzuka R-10) and 25 U/ml pectinase (Sigma) in 0.01 M citric buffer, pH 4.8, for 17 min for in situ hybridisation was performed. After a 1-h prehybridisation, hybridisation was conducted for at least 12 h at 26°C in hybridisation buffer (Sigma-Aldrich) with 30% formamide and antisense poly(A) DNA—5 Cy3 T(T)29 3 at a concentration of 50 pmol/ml. The slices were subsequently washed in 4× SSC, 2× SSC, and 1× SSC. For FISH double labelling (U2 snRNA-poly(A) RNA), the two probes were applied simultaneously in the hybridisation medium, and hybridisation was conducted at 30°C using an antisense DNA probe against U2 snRNA 5′ rhodamine green: ATATTAAACTGATAAGAACAGA TACTACACTTG.After rinsing with PBS, TritonX100-treated sections and protoplasts were incubated with the primary antibody mouse anti-PANA (1∶100) Arabidopsis thaliana U2B”:GFP and Atcoilin:mRFP plants were treated with TritonX100 (1∶1000 in PBS) for 14 min. Then, they were subjected to the same in situ hybridisation protocol applied for the sections, described above, using 5'-Cy3- or rhodamine green-labelled DNA probes for poly(A) RNA. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The control reaction was performed in the same manner, but using the hybridisation buffer without probes, and the primary antibodies were omitted.After fixation and rinsing in Pipes and PBS, the roots of in situ hybridisation was performed with DNA probes labelled with Cy3 (GenoMed Poland) for pectin methylesterase (5′TTCTCTGTATGTACCAGCTTTTATGTG3′), a cytokinin-specific binding protein (5′GAGTATGTAGCTCTCCTATTGCAGATAAT3′) cyclin B1 (5′CAATATCCTTAAGTACCCTCCTATTTCTT3′) and peroxidase (5′ ATCTTTGTAGTAATTATTGTCGAAACGAT3′) at a concentration of 50 pmol/ml in hybridisation buffer (Sigma-Aldrich) with 30% formamide. In case simultaneously in situ hybridisation with four probes at individual concentrations of 25 pmol/ml. In situ hybridisation was performed at 40°C for at least 12 h. The sections were then washed in 4× SSC and 2× SSC, and DNA staining was performed. The control reaction was conducted in the same manner as for double labelling with in situ hybridisation of poly(A).After digestion and Triton ×100 treatment, the sections were incubated with anti-Sm antibodies , then washed in PBS and incubated with a Cy2-labelled mouse anti-human antibody (Sigma-Aldrich). Next, The vibratome sections of roots fixed in 4% paraformaldehyde in 50 mM Pipes buffer, pH 7.0, for 12 h at 4°C after treatment with 0.1% saponin in PBS, were subjected to hybridisation using an LNA probe against poly(A) RNA labelled with digoxygenin at the 5′ and 3′ ends. For hybridisation, the probes were resuspended in hybridisation buffer (Sigma-Aldrich) with 30% formamide at a concentration of 20 nM. Hybridisation was performed for 12 h at 45°C. The sections were washed in 4× SSC and 2× SSC and post-fixed in 4% paraformaldehyde in Pipes buffer for 1 h at 4°C. Then, the sections were dehydrated, infiltrated, embedded and sliced according to Niedojadło et al. in situ hybridisation of poly(A) RNA was performed according to the protocol described for double localisation with poly(A) RNA. Control reactions were conducted without the primary antibody. DNA was stained with 4,6-diamidino-2-phenylindole .After 3–4 days of culture, lupine seedlings were transferred to Petri dishes with 5-bromouarcil in tap water  soaked tissue paper for 2, 4, 5 or 7 h. The roots were fixed and sectioned, and the sections were treated according to the above protocol. Then sections were incubated with anti-BrUTP antibodies  (diluted 1∶1000) overnight at 4°C, then washed in PBS and incubated with goat anti-mouse antibodies labelled with Alexa 488 . Next, in situ hybridisation of poly(A) RNA, 53 cells from three different experiments were analysed. Similar to all of the results presented in this report, these results were registered and analysed using a Nikon PCM-2000 confocal microscope and a fluorescence inverted Nikon Eclipse TE 2000-E microscope. The analysis was performed using NIS-Elements AR3.00. The obtained data were corrected for background autofluorescence, as determined based on the signal intensity in the negative control. The non-parametric rank-based Kruskal–Wallis test was used to compare multiple groups, and when significant differences were detected, the Mann–Whitney test was applied to compare the two experimental groups. The tests were performed using GraphPad Prism 5 software. P <_0.05 was considered significant.To calculate the fluorescence signal resulting from the 2), and images analysis were conducted using NIS software. Representative examples are shown in the results. For each experiment, the 10 Cajal bodies were analyzed. This analysis was repeated three times, and the average and standard error were compiled.For Pearson correlation analysis, rectangular regions of interest were drawn to cover a single CB and its surroundings (1–4 µmLupinus luteus) (Allium cepa) nuclei  RNA is present in the nucleus than in the cytoplasm (up to more than 80% in lupine) . In the  luteus)  and reti) nuclei . Some of) nuclei , B. Thes) nuclei .Lupinus luteus  RNA in splicing speckles in animal cells s luteus  or Alliuium cepa  roots. Tectively . Strong ectively . As a coectively .Lupinus luteus upon double labelling of mRNA and U2snRNA via in situ hybridisation  RNA signal was observed in Cajal bodies  mRNA colocalise with snRNA, which mainly occurs in the perichromatin region and Cajal bodies in plants disation . While Udisation . Similardisation –H. The rl bodies . Despite by U2B"  and Atco by U2B"  accumulaLupinus luteus cells, in which the Cajal bodies are large and easily distinguishable after staining with poly(A) RNA and other markers of these structures. The mRNA sequences of four protein-coding lupine genes were chosen for this analysis: a cytokinin-specific binding protein, cyclin B1, pectin methylesterase and peroxidase. Simultaneous localisation of the core spliceosomal Sm proteins and four mRNAs revealed that the CBs, which contain large amounts of Sm proteins, are also the structures that house mRNAs encoding proteins (Because poly(A) RNA was localised in plant CBs, we attempted to determine whether some of these RNAs were protein coding. Experiments were performed in proteins . In addiproteins , peroxidproteins , cytokinproteins , and pecproteins  mRNAs coproteins , while tproteins . The mRNNext, we tested whether these structures are associated with the transcription process, or with subsequent stages of mRNA maturation. Observations made using an antibody against the elongated form of RNA polymerase II with a phosphorylated serine 2 in the CTD domain revealed clusters of this antigen in the nucleus. The regions of RNA polymerase II localisation did not overlap with the CBs . We alsoImmersion of cut roots or whole seedlings in water leads to hypoxia, which often occurs during flooding We have shown that in the cells of plant roots, a considerably larger amount of poly(A) RNA is present in the nucleus than in the cytoplasm (up to more than 80% in lupine). The presence of similarly large amounts of poly(A) RNA in the nucleus has been described previously in many types of animal cells. In comparison with the cytoplasm, larger proportions of both protein-coding mRNA and the total pool of poly(A) RNA are localised in the nucleus in COS-7 cells as well L. luteus, A. thaliana Col-O) and reticular (A. cepa) types of nuclei in meristematic and differentiated root cells. To our knowledge this is the first report of the presence of poly(A) RNA in Cajal bodies in somatic plant cells. A similar phenomenon was described by Kołowerzo et al. The presence of poly(A) RNA in splicing speckles has been reported in animal cells Arabidopsis thalianain situ localisation of long non-coding RNAs, many of which are polyadenylated We have shown that at least a portion of the poly(A) RNAs found in CBs are protein-coding RNAs. However, stages in the metabolism of mRNAs can be carried out in the CB at different intensities or speeds for individual mRNAs encoding proteins, which may be indicated by the frequency of the colocalisation of the four different transcripts of CBs in situ run-on transcription assays demonstrated small amounts of transcribed RNA in CBs, but only after a long incubation period when partial transport of a new form of RNA from the nucleus to the cytoplasm was observed. This means that Cajal bodies are not sites of RNA transcription. Similar results have been obtained in animal cells The presence of mRNAs in the CBs of plants, prompts the question of whether Cajal bodies serve as a site of transcription. We have shown that in the CBs of lupine cells, there is a lack of active RNA polymerase II. However, Arabidopsis thaliana cells after heat and ethanol treatments, which represent two abiotic stresses Arabidopsis that are predicted targets of nonsense-mediated decay (NMD) escape this mechanism However, the possibility that CBs participate in the storage or retention of poly(A) RNAs is supported by our observations in hypoxia-treated cells. In cell nuclei after hypoxia, the amount of poly(A) RNA increases by 67%. A similar phenomenon has been observed in In conclusion, our results demonstrate the prevalence of a new Cajal body component, poly(A) RNAs, including protein-coding RNAs, in plant cells. The obtained results do not resolve the role of these structures in the metabolism of poly(A) RNA. However, it has been established that the CBs present in plant roots are not a transcription site but may be involved in the storage or retention of poly(A) RNAs.Figure S1Localisation of poly(A) RNA in protoplasts in lupine root cells: meristematic (A), differentiated (B). Arrows indicate CBs. Double labelling of poly(A) RNA and Sm proteins in: Allium (C-E), Lupinus (F-H) and hypoxia-treated lupin cells (I-K). Bar, 5 µm. N- nucleus, Nu- nucleolus.(TIF)Click here for additional data file.


In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using  In metazoan cells, synthesis of mature messenger RNAs (mRNA) requires several steps of processing, which are accompanied by the deposition of proteins onto the mRNA to form messenger ribonucleoprotein complexes (mRNPs). Importantly, the composition of mRNPs influences gene expression both in the nucleus and in the cytoplasm and undergoes constant remodelling ,2. In euIn mammalian cells, splicing plays an important role in mRNP remodelling and thereby influences later steps of gene expression. Consequently, the deposition of export components occurs mainly during splicing –5. This According to current models, ALYREF is deposited onto mRNAs through its interaction partner UAP56 ,15. The in vivo and UIF was reported to influence export through binding with NXF1  . CHTOP eith NXF1 ,24. Finaith NXF1 ,26. Inteith NXF1 ,24. ThusShort-linear motifs (SLiMs), also referred to as ELM (eukaryotic linear motifs), are short (3–10 amino acid long), conserved sequences situated in intrinsically disordered regions, lacking secondary and tertiary structures. SLiMs mediate low-affinity interactions between proteins and also between RNA and proteins  –30. Althin vitro binding to spliced RNA and for efficient export of spliced mRNAs in vivo. Together, these findings expand our view on how nuclear mRNA processing during splicing orchestrates the recruitment of export factors to the RNA and provide insight in to the role of short linear motifs in this process.In this study, we investigated the interactions of mammalian export components with different RNA substrates. We find that export components UAP56 and ALYREF are recruited to spliced mRNA in an EJC- and cap-dependent manner. We identified four short, conserved, linear motifs present in proteins involved in mRNP processing and export, providing a systematic view into the family of export adaptors. Finally, we show that short linear motif in ALYREF (WxHD) is required for its DDX39 and UAP56 were obtained by reverse transcription (RT)-PCR using HeLa cells RNA. ALYREF was amplified by PCR from plasmid DNA (Origene). ALYREF deletion mutants: 1–106, 106-ter, 1–187, RRM, ΔN15, ΔC53 and ΔN15/ΔC53 were generated using PCR. ALYREF 87 WQHD/DQAK 90 was generated by site-directed mutagenesis. Expression vectors for FLAG CBP80, FLAG Y14 and FLAG SELOR were described previously  Figures.in vitro splicing experiments, constructs were inserted into the pCI-neo vector (Promega) with an N-terminal FLAG (DYKDDDDK) tag  of radiolabelled RNA were cloned into pGEM-4Z (Promega). MINX was described previously (Templates for eviously . AdML aneviously –3 and 5.Recombinant PYM (rPYM) was obtained as described previously . Brieflyin vitro splicing assays were obtained as described previously (HEK 293 cell lysates expressing FLAG-tagged proteins for eviously . For imaeviously . For luceviously .The Flp-In T-Rex System (Life Technologies) was used to generate tetracycline-inducible, stable mammalian cell lines. Transgene expression was induced by adding 1 μg/ml doxycycline to the cell culture medium 72 h prior to harvesting. For immunoprecipitation experiments, HeLa cells were cultured and transfected as described previously .In vitro splicing assays were performed as described previously  corresponding to amino acids 3–20 of human UAP56 and 241–257 of human ALYREF, respectively. The eIF4A3 polyclonal antibody was made by GenScript with an N-terminal peptide of eIF4A3 . The antImages were obtained at room temperature on an Olympus FV1000 confocal microscope using a 60x UPlanApo (NA 1.35) objective. Assembly, contrast and brightness adjustments of final image sets were performed using Photoshop CS4 Cells were seeded onto coverslips 24 h post-transfection, fixed with 3.7% formaldehyde (Carl Roth) in phoshate buffered saline (PBS) after 48 h and permeabilised for 5 min with 0.5% Triton X-100 (Sigma-Aldrich) in PBS on ice. Nuclei were counterstained with DAPI (Carl Roth). Coverslips were mounted in DABCO:Moviol (Carl Roth).Cells were harvested 72 h post-transfection using 300 μl Passive Lysis Buffer (Promega). Lysates (2 μl) were analyzed using the NanoGlo DLR Assay (Promega) on a Centro XS3 LB960 microplate luminometer (Berthold Technologies).P-value better than 0.01 for subsequent iteration cycles. An analogous procedure was used for finding sequence conservation in BTZ, BCLAF1, THRAP3, UIF, CHTOP, SKAR, LUZP4 and PHAX. When it became clear  that the sequence conservation was restricted to one or more short motif-size regions, the multiple alignments where cropped and (if necessary) split, continuing the iteration cycle with one multiple alignment per conserved motif. Iterative refinement was stopped when no new significant hits were found in the database. The sequence logo representation shown in Figure For computational motif discovery, a set of ALYREF orthologues from various metazoan species was subjected to multiple alignment by the L-INS-I method of the MAFFT software . The resin vitro splicing system, which has been shown to recapitulate the binding of different mRNP components to intron-containing (spliced) and intronless (non-spliceable) AdML and MINX RNA model substrates , indicating that it binds strongly to the spliced mRNA Figure ,42. Simis Figure , lane 6.s Figure . As expes Figure , lane 5.s Figure –17. Nota7GpppG) interferes with binding of UAP56 and ALYREF to spliced mRNA . As shown before for the MINX splicing substrate, DDX39, UAP56 and ALYREF only precipitated RNA fragments that contained both the cap and an EJC-binding site. Moreover, differential precipitation of cleavage products is also independent of the RNA length, as demonstrated by the use of DNA oligos that target different positions in the spliced RNA . Interestingly, binding of ALYREF was stronger on long cap-containing mRNA fragments. This is in agreement with previous studies suggesting that an RNA of sufficient length can enter the mRNA export pathway more readily than short and structured RNAs .It has been reported that the presence of either the cap or the EJC increases binding of export factors to mRNA. However, it was not known if the simultaneous presence of both EJC and cap confers cooperative binding. Our results suggest that 5′ cap and EJC-binding site have an additive effect on the interaction of export factors with spliced mRNA.In vitro, the displacement of EJCs can be recapitulated by the addition of rPYM to splicing reactions , two previously described UAP56-interaction domains , an NXF1-binding motif (15–37 aa) and two arginine-rich regions  ,22. Notain vitro ,48.We constructed three large truncation mutants to investigate the mode of ALYREF interaction with mRNA. We deleted either the N-terminal part (106-ter aa), the C-terminal part (1–106 aa), or both parts of ALYREF  Figure  and testTo correlate UAP56 interaction and RNA binding of ALYREF, we used mutants of ALYREF previously described to abolish binding to UAP56  in orderin vitro, opposing the prevailing model that UAP56 is required for the deposition of ALYREF on spliced mRNAs.In conclusion, we show that neither of the potential RNA-binding domains of ALYREF  can mediate RNA binding in isolation. Furthermore, the deletion of the UAP56 binding sites maringally reduced the capability of ALYREF to bind spliced RNA In living cells, the recruitment of ALYREF to mRNAs during splicing is presumably executed in nuclear speckles ,48–49. WRecently it was reported that many mRNA-interacting proteins are intrinsically disordered and enriched in repetitive amino acid motifs ,50–51. AAll proteins containing the SLDD/E motif have been previously described to be involved in mRNA export, i.e. ALYREF, SKAR, UIF and LUZP4 Figure . The motThe LDxxLD motif is present in ALYREF, PHAX and twice in CHTOP. In ALYREF this motif is part of the C-terminal UAP56-binding motif . CHTOP wThe SLDD/E and the LDxxLD motifs described above were previously reported to be variants of the same motif . AlthougAnother short conserved motif present in ALYREF (WxHD) was found in BTZ, a component of the EJC core ,42, and Since ALYREF binding to mRNA does not involve the RRM and is independent of the presence of UAP56-binding motifs, we hypothesised that the conserved WxHD motif could contribute to mRNA binding of ALYREF Figure .in vitro splicing  compared to NanoLuc . This effect was much less pronounced upon overexpression of the WQDH mutant of ALYREF, indicating that the WQHD motif plays an important role in the export of spliced mRNAs to the cytoplasm.7GpppG) dissociates UAP56 and ALYREF from spliced RNA  in ALYREF, which is also present in four other proteins involved in mRNP formation. In ALYREF, the WQHD motif is located at position 87–91 in an unstructured region between the N-terminal RGG-like domain and the central RRM. Mutation of this motif leads to reduced binding of ALYREF to spliced RNA and impairs the interaction with CBP80 and eIF4A3. In contrast the interaction with UAP56 is not affected , which might belong to the growing family of SLiMs, also referred to as ELMs ,59. SLiMNotably, the function of two motifs (HDxR and WxHD) in two additional proteins – BCLAF1  and THRAP3  has not been determined yet. BCLAF1 and THRAP3 were initially described as spliceosomal components , both weIt has been speculated that splicing and deposition of EJCs enhance recruitment of export components and thereby stimulate export of spliced mRNPs . The EJCIn vivo, the assembly of export complexes on mature mRNA may involve additional components of the mRNP, which have not been tested in this study. It has been shown, for example, that ALYREF interacts with the 3′ end processing factor Pcf11 . ALS leaThe recently published screening for hexanucleotide repeats expansions toxicity modifiers identified the components of export machinery as potential candidates. In fruit fly, the deficiency of ALYREF supressed the toxicity of G4C2 expansions. This phenomenon might be explained by our observation that EJC-containing mRNAs are exported more efficiently when ALYREF is overexpressed Figure . In cont