Due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. A growing database of proteins with RNA chaperone activity has been established based on evaluation of chaperone activity via the described assays. Although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. This possible mechanism and which proteins were found to possess RNA chaperone activity are discussed here.Proteins with RNA chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. They prevent RNA from misfolding by loosening misfolded structures without ATP consumption. RNA chaperone activity is studied  Among all biological macromolecules, RNAs represent one of the most functionally versatile players in the cell. RNA molecules fulfill many different tasks such as coding and transfer of genetic information; they play regulatory functions in various cellular processes and catalyze chemical reactions (like cleavage and ligations). In addition to its functional versatility, RNAs are also able to fold into countless different structures, many of which have similar stabilities as the native structure and therefore compete with the native fold. Furthermore, RNA molecules often undergo transition states during their folding pathways before they reach the native and active structure. These transient structures can represent traps along the folding pathway from which the molecules might have a hard time to escape and which then end up being long-lived intermediates. The reason for this structural versatility is the fact that RNA consists of only four different bases which are easily capable of forming stable helices, that are not necessarily the native structure. The threshold for RNA molecules to be able to perform their functions is usually the accomplishment of reaching its native and active structure. in vitro, the term RNA chaperone is reserved to describe proteins whose RNA folding activity has been verified on its natural target RNA in vivo. Therefore, most of the proteins in this paper will be referred to as proteins with RNA chaperone activity if their RNA folding activity was only determined in vitro and/ or on nonnatural RNA targets. In the cellular environment, RNA molecules do not appear as “naked” nucleic acids but always are found in conjunction with proteins. In some cases, the RNA molecule helps the protein partner to fold correctly; in others, the protein stabilizes the RNA structure. And last but not least proteins with RNA chaperone activity aid during the folding process of RNAs. Proteins with RNA chaperone activity open up misfolded RNA structures and do not require ATP . FurtherThis paper will focus on the diversity of proteins with RNA chaperone activity and which experimental assays are in use to determine whether a protein has RNA chaperone activity. I will present examples for proteins with RNA chaperone activity and discuss possible mechanisms of RNA chaperoning. The list of proteins with possible RNA chaperone activity is growing constantly. Proteins with different activities that support RNA folding are classified in this group. The definition of a protein with RNA chaperone activity is that the protein prevents RNA from misfolding by opening up misfolded structures. Proteins with RNA chaperone activity do not require ATP, which distinguishes them from RNA helicases, another group of proteins that facilitate RNA folding  . E. coli proteins S12 and StpA [Neurospora crassa, is a “stabilizer” for the mitochondrial self-splicing group I intron: its presence is required to keep the native structure of the intron which otherwise unfolds readily. Proteins with RNA chaperone activity interact only transiently with RNA molecules and are supposed to be dispensable once the RNA has been folded correctly. This was shown for and StpA , 4. A trand StpA . In thatIn the growing database of “proteins with RNA chaperone activity”, there exists an increasing number of proteins that simply possess RNA annealing activity. A prominent and intensively studied member of this group is the bacterial host factor Hfq that showed annealing activity on random substrates. Hfq in addition is an RNA chaperone as it was further demonstrated that Hfq does possess unwinding activity upon its native substrates , 7.In brief, the group of proteins with RNA chaperone activity includes proteins that, first, open up misfolded structures without requirement of ATP and that, second, are dispensable once the RNA has been folded. in vitro and are usually prevented from misfolding in vivo. RNA basically encounters two folding problems: a kinetic folding problem, where the RNA molecule has to surmount kinetic barriers during the search for its native structure. Secondly, RNA molecules meet a thermodynamic folding problem as the final native structure often has to compete with alternative folds that have similar energetic stabilities [RNA molecules are prone to misfold bilities . RNA folTertiary structures are higher order structures that are built by assembling the secondary structure elements into a more complex collapsed fold. They can also involve formation of helices. This is the case in pseudoknots where either a loop region interacts with a distant single stranded region or with another distant loop. Pseudoknots possess similar stability as secondary structures. But tertiary structural elements involve also other non-Watson-Crick interactions where for example, not only the Watson-Crick site of the nucleotide interacts with another nucleotide but also the Hoogsteen edge or the sugar edge of the nucleotide is involved in hydrogen bonding . An ofteThe first studies on RNA structure and folding were done in the 1960s with yeast tRNA molecules. Already then it was demonstrated that tRNAs are able to adopt two distinct conformations of which only one is the native structure which can be aminoacylated , 11. The RNA folding problem becomes even more prominent in the case of large RNAs such as group I introns or in the context of large protein-ribonucleic acid complexes such as RNase P and the ribosome. Tetrahymena thermophila ribosomal RNA: a subset of molecules misfolds and accumulates into an inactive population [It was demonstrated that the self-splicing group I intron of the thymidylate synthase gene of phage T4 misfolds in the absence of translation: when the ribosome does not prevent base-pairing between exon and intron sequences, the intron is not able to fold correctly and cannot perform the splicing reaction . A similpulation . MisfoldIn vivo, however, some group I introns require the assistance of proteins to splice efficiently and prevent misfolding. For an example, the Cyt-18 protein in Neurospora crassa mitochondira is a tRNA synthetase which stabilizes the P4-P6 domain of group I introns and recruits Cyt-19, an RNA helicase, which then unwinds folding traps and promotes splicing [splicing , 14. in vivo. For large RNP complexes such as the ribosome, a growing body of evidence suggests that several additional factors such as helicases exist that assist during the folding process in vitro and in vivo assays that use either simple oligonucleotide annealing or displacement reactions or that measure catalytic activities of correctly folded ribozymes. As proteins with RNA chaperone activity are very heterogeneous concerning their structure and their way to resolve the folding of RNA molecules, there are various RNA chaperone assays available to measure different activities. In principle, the assays can be divided into in vitro assay  might differ in sequence requirements or structure requirements from possible native substrates and might lead to negative results. Strand unwinding assays might give positive results in the case of single-strand binding proteins. Furthermore, in vivo assays to measure RNA chaperone activity can be negatively influenced by possible toxicity of the putative RNA chaperone when overexpressed or can lead to secondary effects in the cell that give false positive results. Therefore, it is recommended to measure the RNA chaperone activity at least in more than one chaperone assay to be certain that no non-specific activity is measured. Measuring an activity that might be targeted more specifically to a certain subset of substrates in the natural environment of the putative RNA chaperone makes it difficult to evaluate RNA chaperone activity using a single assay. The substrates in theIn this assay, two complementary oligonucleotides (oligos) present in concentrations above their dissociation constant are incubated together in the absence and in the presence of the protein to be evaluated for RNA chaperone activity. An increase in the rate of duplex formation is observed when the tested protein has RNA annealing activity. In principle two complementary short RNA oligos are used in this assay, although it has been a habit to choose DNA oligos instead. In order to measure RNA chaperone activity, however, I think that RNA oligonucleotides should be the preferred choice. Detection methods of duplex formation include native gel electrophoresis where the two complementary RNA strands can either be visualized by radioactive or fluorophor labelling and has been used in (besides many many other publications) –17. RNA The ability of a protein to open up and unwind an already formed RNA duplex is measured. To measure RNA chaperone activity in contrast to helicase activity, this assay is performed in the absence of ATP or an alternative energy source. Analogous to the annealing assays, melting activity can be detected by using native gel electrophoresis or by measuring loss of fluorescence energy resonance transfer (FRET) that occurs upon dissociation of the complementary fluorophor labelled RNAs. In addition to the above-described detection methods, new approaches to detect folding or unfolding of single molecules emerge and include time-resolved NMR, which becomes a powerful tool to study folding of small RNAs. The hammerhead ribozyme cleavage reaction and folding of the ribozyme-substrate 3-way helical junction have been studied in a great detail. Therefore, this assay represents a suitable tool to study RNA chaperone activity upon folding of the hammerhead ribozyme-substrate construct. Using the hammerhead cleavage assay, both annealing and strand displacement can be studied independently of each other . The advin vitro is tested with and without chaperones.Self-splicing of the thymidylate synthase group I intron (td intron) of bacteriophage T4 has been characterized, and the td intron has been used lengthily to monitor RNA chaperone activity of various proteins. Splicing of different group I intron constructs that do not fold readily into the splicing competent structure td intron construct both, 5′ and 3′ exons are shortened for the upstream exon down to 27 nucleotides and the downstream exon shortened to only 2 nucleotides. This short exon construct (td shosho) splices at 37°C but RNA chaperones increase folding and as a consequence the splicing rate of the short-exon construct is increased as well [In this in vitro transcribed construct contains 549 nucleotides of exon1 and 131 nucleotides of the 5′-part of the intron. The downstream construct consists of the remaining 147 nucleotides of the intron and 23 nucleotides of exon2. Correct and efficient folding of the trans-intron-constructs is significantly impaired at 37°C but works fine at elevated temperatures (55°C), which is monitored through splicing [Here, the td intron is split into two halves in the center of loop L6 in the P4-P6 domain, where in the wild type group I intron an open reading frame for an endonuclease is present. The upstream splicing . Chaperosplicing .  in vivo. Though, when splicing and translation are uncoupled by introducing stop codons in the upstream exon, splicing is significantly reduced. This is due to alternative base-pairing of exonic and intronic sequences which prevent the formation of the intron's native fold [in vivo. Overexpression of RNA chaperones in the presence of the tdSH1 mutant is used to evaluate if the RNA chaperone is able to rescue the misfolded intron and restore splicing [Splicing of the group I intron within the thymidylate synthase gene of phage T4 occurs efficientlyive fold . The mutsplicing . E. coli  [Transcription read-through of the chloramphenicol acetyl transferase gene (cat) is inhibited due to the preceding transcription terminator stem. The stable hairpin secondary structure of the terminator inhibits the polymerase to transcribe the cat gene and as a consequence no chaloramphenicol resistance is achieved. The cells are chaloramphenicol (Cm) sensitive. Proteins with RNA chaperone activity are able to melt the terminator stem and as a consequence read-through occurs and the cells become chloramphenicol resistant. The transcription antitermination assay was used for assaying cold shock proteins or IF1 from E. coli  –26. http://www.projects.mfpl.ac.at/rnachaperones/index.html [Proteins that possess RNA chaperone activity are very divers and span from viral to bacterial and human proteins that are involved in many different cellular processes. The list of RNA chaperones is permanently growing. Recently, a database for proteins with RNA chaperone activity was established where every lab is able to contribute their data for a newly identified chaperone dex.html . The folThe first viral RNA chaperone activities were reported in the early 1990s and showed that nucleocapsid protein 7 (Ncp7) of HIV increases hammerhead ribozyme cleavage significantly , 28. OnlFlaviviridae core proteins were also monitored and shown to possess RNA chaperone activity in a hammerhead cleavage assay and/or RNA strand annealing activities , 31. IntNucleocapsid proteins from two members of the Coronaviridae family have been investigated and hammerhead ribozyme cleavage was shown to be enhanced in their presence , 33. AndA growing body of evidence suggests that there exist many more viral proteins that possess RNA chaperone activity. The list here is not complete but proteins that were shown to have only DNA annealing activities were left out in this list. The majority of these small viral proteins show strong propensity for disorder which suggests that disorder might be a mechanistic requirement for chaperoning.in vitro but also are required for strand annealing and strand displacement activities on their target RNAs in vivo [in vivo  [Interestingly, studies on Nc proteins demonstrated that these proteins not only possess RNA chaperone activity  in vivo . Recentlin vivo  .E. coli transcriptional regulator StpA, a 15 kD basic protein, was isolated as a repressor of a splicing-deficient group I intron in thymidylate synthase of phage T4 [in vivo in the folding trap assay [in vitro in a strand-annealing and strand-displacement assay and exhibited strong activities in both tests [in vivo DMS modifications to the RNA, in vivo folding of the group I intron was evaluated in the absence and presence of StpA [The ap assay . The proth tests . More de of StpA : Schroed of StpA . Ribosomal proteins are required within every cellular organism to build up the bacterial 70S or the eukaryal 80S ribosome. Many ribosomal proteins further regulate transcription or translation of their own operons. In addition, ribosomal proteins are also involved in various very different cellular processes and fulfill extraribosomal functions , 40. RibE. coli showed that 1/3 of the tested proteins possesses strong RNA chaperone activity in vitro in the trans-splicing assay [ in vitro [in vivo has not yet been provided. Recently, it was demonstrated that E.coli ribosomal proteins L15, L16, L18, and L19, that showed RNA chaperone activity in vitro, further possess protein chaperone activity comparable to other protein chaperones such as Hsp90 [The first observation that ribosomal proteins are capable of chaperoning RNA folding came from the Belfort lab: screening for cellular factors that increase trans-splicing of the thymidylate synthase group I intron revealed that many ribosomal proteins possess chaperoning activity, with ribosomal protein S12 from the small ribosomal subunit having the strongest activity . Furtherng assay . In addiin vitro . Althougas Hsp90 . It was Cold shock proteins (csps) are conserved throughout bacteria and plants. They are expressed during cold-shock, when misfolding of RNAs becomes a major problem for the organism and function as transcriptional antiterminator at low temperature. Many experiments that have been performed to describe chaperone activity of cold-shock proteins utilize DNA helices (and only sometimes in addition RNA duplexes) and refer to the activity as nucleic acid melting activity. However, it has to be mentioned that there are no elaborate studies on whether there is a difference between RNA duplex and DNA duplex melting and whether DNA melting activity automatically is the same as RNA melting. E. coli contains nine members of the csp family and CspA, the major cold-shock protein and CspE were identified to interact non-specifically with RNA molecules and to possess nucleic acid melting activities [tivities –46. Arabidopsis thaliana have been monitored for their nucleic acid melting activity, and it was shown that GRP7 (glycine-rich protein) and CSDP1 (cold shock domain protein) possess RNA chaperone activity [Oryza sativa), which are likely to be involved in adaptation to cold-shock, three of them exhibit RNA- (and DNA-) melting activities suggesting that GRPs in plants fulfill a chaperoning role during low temperatures [Cold-shock proteins in higher plants are highly conserved. Glycine-rich and Zn-finger containing proteins from activity . A receneratures . E. coli translation, initiation factor 1 (IF1) is a small 71 amino-acid long peptide, which contains 5 rigid β-barrels and belongs to the OB (oligomer-binding)-fold proteins such as the cold shock proteins. N- and C-termini of IF1 are highly flexible. It was demonstrated that E. coli IF1 is capable of complementing for a cspB and cspC double knock-out in Bacillus subtilis suggesting that IF1 and csps have at least partially overlapping activities [E. coli IF1 exhibits RNA chaperone activity in various assays including RNA annealing of complementary oligonucleotides, trans-splicing, in vivo folding trap assay, and transcription anti-termination in vivo and in vitro [In tivities . E. coliin vitro , 50. Heteronuclear Ribonucleoproteins encompass a group of about 20 polypeptides that are predominantly nuclear in localization and are involved in RNA processing. in vitro [in vitro in the trans-splicing and the cis-splicing assay [The first observation that hnRNPs possess RNA chaperone activity came in the early 1990s when fractionated HeLa nuclear extract was tested for annealing activity of an mRNA and its antisense partner. Three proteins, hnRNP A1, C1 and U, were identified and hnRNP A1 was further shown to enhance hammerhead ribozyme cleavage in vitro , 20. Latng assay . hnRNP Ing assay . It regung assay . PTB belng assay . It was ng assay . Membersng assay , human Lng assay .HnRNP K is also a multifunctional protein that is a transcriptional factor for c-myc and c-src –58; it ein vitro in the cis-splicing assay and in vivo in the folding trap assay [La proteins primarily bind RNA polymerase III transcripts and protect them from nuclease attack . They alap assay . β replication [The bacterial protein Hfq was first discovered in the end 1960s as a host factor for bacteriophage Qlication . The baclication –67. Hfq Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus Hfq proteins showed that Hfq forms homohexameric ring structures with a central cationic pore that forms the RNA binding site [Hfq encompasses an Sm-domain, which is highly conserved among various species and usually is found in eukaryotic spliceosomal RNPs. Crystallographic studies of ing site –70. Using strand annealing and strand melting assays to measure RNA chaperone activity of Hfq, only strand annealing activity was observed . HoweverThe prion protein is a misfolded isoform of the essential component of prion diseases such as Creutzfeldt-Jakob disease in humans—one of several neurodegenerative diseases. The function of the human prion protein is not clearly understood. It was demonstrated that the prion protein has RNA (and DNA) annealing activity ; howeverThe fragile X mental retardation protein (FMRP) is linked to the fragile X syndrome as the disease is due to transcriptional silencing of the gene. FMRP possesses RNA binding activity and its interaction partners include a large number of mRNAs, microRNAs, siRNAs, and small noncoding RNAs as well as a multitude of different proteins –76. It wEscherichia coli show RNA chaperone activity in vitro). Ribosomal proteins are usually small proteins many of which have long unstructured domains and are highly basic proteins.Chaperones provide a critical cellular activity. Proteins with RNA chaperone activity are very divers in structure as well as in function: StpA, a transcriptional activator and repressor of a multitude of bacterial genes, is a small (15 kD) bacterial protein with intrinsically unstructured regions. StpA has strong RNA chaperone activity. On the other hand the bacterial protein Hfq is a large multidomain protein complex (60 kD) and folds into a compact ring-like structure. Among ribosomal proteins, many were shown to possess RNA chaperone activity  are tested for folding in the presence of 1-2 μM protein. It was shown that E. coli ribosomal protein L1 displays maximal RNA chaperone activity starting from 400 nM up to 2 μM protein concentration [E. coli. Another indication that a crowding effect might play a role at least to some extent during RNA annealing is the following: when RNA chaperone activity is measured ntration . This me in vivo chaperone assay, which uses the folding trap of a misfolded group I intron in the thymidylate synthase gene of phage T4, it is always necessary that the measured protein is overexpressed and available in higher concentrations [E. coli protein StpA, which is found constitutively expressed in the bacterial cell, only shows its RNA chaperone activity in vivo when StpA is additionally over-expressed from an expression vector, thus showing that the cellular concentration of StpA is not sufficient to increase folding of the misfolded group I intron. Certainly, this observation might be due to the engagement of StpA in other regulatory functions in the bacterial cell; however, it also points to the direction that more than one molecule of StpA is required to assist folding of the td group I intron. As a consequence the question rises if and how it is possible to distinguish between RNA chaperone activity and a nonspecific single-strand RNA binding activity of the protein that might both prevent misfolding. Using the in vivo folding trap assay, however, not only proteins with possible RNA chaperone activity like StpA had been tested but also a viral single strand binding protein from Influenza virus (NP) was tested and did not show any increase in splicing suggesting that single strand RNA binding might not be sufficient for chaperoning. Furthermore, a detailed study on StpA wild type and mutants demonstrated that only the full-length StpA was able to show RNA chaperone activity by simultaneously interacting with two RNA molecules [In this line, it also has to be mentioned that in thetrations . For exaolecules . in vivo and in vitro RNA chaperone activities. In a mechanistical in vivo study of StpA, Schroeder and coworkers demonstrated that StpA loosens tertiary contacts within the thymidylate synthase group I intron [Neurospora crassa tRNA synthetase Cyt-18 that also increases group I intron splicing of td stabilizes tertiary interactions. But how is the opening of tertiary structure elements accomplished without the hydrolysis of ATP? This strand unwinding activity is more difficult to explain as the question remains of how a protein can actively open up hydrogen bonds when no apparent source of energy is required. RNA chaperone activity of StpA has been studied for more than a decade. It was shown that StpA has strong I intron . In contIn the protein world, it became more and more visible that the classical structure-function paradigm does not necessarily hold for many proteins and their activities. A growing body of evidence suggests that a multitude of proteins do not fold into compact domains but are fully or at least partially unstructured . In eukaThe idea of disordered RNA chaperones is especially attractive because there are many advantages of proteins with disordered regions over compact proteins: (1) the main advantage of a disordered region is that it can easily interact with a range of many different partners and is not limited to a single binding pocket or recognition element on a partner molecule. (2) The bigger surface of the unstructured protein might provide a “loosening effect” for the incorrectly folded RNA molecule. (3) The troublesome question of where the energy for the RNA unwinding might come from could be explained by the gain of compactness upon interaction with the RNA and a simultaneous loosening of the RNA structure see . As a coIn future the research focus on RNA chaperones will lie on the understanding of the molecular mechanism and how intrinsically unstructured regions in proteins might play a role in function. Interestingly, two very closely related ribosomal proteins L1 from Archaea, that encompass 70% aminoacid identity, possess opposite activities: the mesophilic L1 protein displays strong RNA chaperone activity whereas the thermophilic one inhibits ribozyme assays . A detaiin vivo targets of RNA chaperones. RNA chaperones are evaluated in assays for their broad specificity but in vivo they might be specialized to supervise folding of only a subset of RNA molecules. The specificity might possibly be conferred by a different domain than the chaperoning activity. The big challenge, however, will be to identify


Xenopus laevis embryogenesis, we established an in vitro system using cytoplasmic extracts prepared from embryos that recapitulates in vivo spindle size differences between stage 3  and stage 8 . We identified the kinesin-13 kif2a as a driver of developmental spindle scaling whose microtubule-destabilizing activity is inhibited in stage 3 spindles by the transport receptor importin α, and activated in stage 8 when importin α partitions to a membrane pool. Altering spindle size in developing embryos impaired spindle orientation during metaphase, but chromosome segregation remained robust. Thus, spindle size in Xenopus development is coupled to cell size through a ratiometric mechanism controlling microtubule destabilization.Early development of many animals is characterized by rapid cleavages that dramatically decrease cell size, but how the mitotic spindle adapts to changing cell dimensions is not understood. To identify mechanisms that scale the spindle during DOI:http://dx.doi.org/10.7554/eLife.00290.001 In the earliest stages of development, animal cells undergo multiple rounds of division without growth via a process known as mitosis. Over the course of just 12 rounds of cell division, a single fertilized egg is transformed into more than 4000 smaller cells.Before dividing, the cell must first replicate its chromosomes. A structure called the mitotic spindle then separates the members of each chromosome pair and distributes them evenly between the two daughter cells. Given that the daughter cells become smaller with each round of division, the spindle must also become smaller to ensure that the chromosomes are pulled apart an appropriate distance. However, it has been unclear how the cell achieves this.Xenopus laevis—a frog that produces large embryos that are easy to manipulate. They began by preparing extracts of cytoplasm from X. laevis embryos at two different developmental stages: one set of embryos contained 4 cells each and the other set contained ∼4000 cells. They found that the spindle, which is composed largely of microtubules—hollow filaments that can become longer or shorter through the addition or removal of tubulin building blocks—was almost twice as large in the four-cell embryos as in the more developed embryos.Now, Wilbur and Heald report insights into the mechanism by which spindle size is coordinated with cell size, using the model organism Moreover, spindle size was determined by the actions of two proteins: kif2a and importin-α. Binding of kif2a destabilized microtubules and caused them to shorten; importin-α blocked this process by binding to kif2a and preventing it from interacting with microtubules. Wilbur and Heald found that over the course of development, importin-α became increasingly localized to the cell membrane, meaning that there was less available to bind to kif2a in the cytoplasm. This freed up kif2a to interact with and destabilize microtubules, and led ultimately to a reduction in spindle size.Given that the overall ratio of cell surface membrane to cytoplasm increases as cells undergo division without growth, interaction between kif2a and importin-α could be the long-sought mechanism by which spindle and cell sizes are coordinated early in development.DOI:http://dx.doi.org/10.7554/eLife.00290.002 Cell size varies widely among different organisms and cell types, and changes rapidly during early animal development when cell division occurs in the absence of growth. The mitotic spindle, a dynamic bipolar structure composed primarily of microtubules (MTs) and organizing proteins , must adXenopus laevis typifies the developmental scaling problem, and both cell and spindle sizes have been measured –containing cargos  arrested meiosis II extract derived from unfertilized X. laevis eggs, which has facilitated fundamental discoveries pertaining to the cell cycle and spindle assembly  containing four smaller and four larger cells. By microinjecting recombinant kif2a just prior to this division, we could specifically alter spindle size and assess both chromosome segregation and spindle orientation defects.The identification of a mechanism contributing to spindle size control in embryos enabled us to test its functional significance during development. At the end of the four-cell stage (stage 3), the Xenopus embryo, but additional mechanisms operate to ensure properly positioned cleavage divisions.As in embryo extracts, excess kif2a decreased spindle size and kif2a ΔNLS possessed greater activity, reducing spindle length up to ∼32% in the developing embryo . DespiteX. laevis development through differential activation of the kinesin-13 kif2a. While a number of perturbations have been shown to alter spindle size, we have identified, out the hundreds of known spindle associated proteins, physiological effectors that operate to scale the spindle during embryogenesis. Considering our recent work identifying the microtubule severing enzyme katanin as an activity that scales spindle sized between related frog species, modulation of MT destabilization has now emerged as a general means of physiologically scaling spindle size in Xenopus  plus protease inhibitors  and cytochalasin D (20 µg/ml), packed by centrifugation in a 2-ml microfuge tube at 1000×g for 1 min followed by 2000×g for 10 s. All buffer was removed, and embryos were crushed by centrifugation at 17,000×g in a swinging bucket rotor . Concentrated cytoplasm was removed from the tube and immediately placed on ice. Protease inhibitors, cytochlasin D, and energy mix were added as for egg extracts and UbcH10 C114S was added at 0.2 mg/ml final concentration. Cyclin B delta 90 (0.05 mg/ml) was added at the start of spindle reactions or prior to utilizing the extract for biochemical analysis. Purified stage 8 nuclei were generated by pelleting endogenous nuclei from stage 8 extracts multiple times through ELB buffer .escribed . Xenopus embryos . For staXenopus importin α  was then incubated for 15 min to retrieved MBP or fusion proteins. Resin was washed with a total of 25 bed volumes of phosphate-buffered saline with 0.01% Tween and eluted in SDS-PAGE loading buffer. Immunoblotting utilized 1:1000 dilution of all antibodies or antisera and was performed as described .2). A homemade pneumatic injection apparatus was utilized with microcapillary needles and calibrated by injecting dye into mineral oil. The total volume injected was approximately 10 nl.Embryos were injected at either the one-cell stage with in vitro transcribed RNA coding for importin α or into one blastomere at the two-cell stage with recombinant protein in injection buffer . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 2 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for choosing to send your work entitled “Mitotic spindle scaling during The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments based on the reviewers' reports.Your manuscript has been read and reviewed by two experts, who agree that your work is of general interest as scaling of cellular structures is a topic of foremost importance in modern cell biology. Both reviewers make the point that the first part of the paper (extract systems and the implications for changes in the spindle throughout development) is profound and very important, while the data presented in the second part (kif2a and importin α are responsible for the difference between spindle length) are not strong enough to provide extensive, mechanistic insights. However, we do believe that your work is worth publishing but for resubmission you would have to improve the second part as proposed below.A major claim in the paper is that at stage 8, spindle size is reduced upon release of an inhibitory interaction between the kinesin kif2a and importin α. This occurs through redistribution of importin α from the cytoplasm to a membrane fraction. This point needs to be shown in a more direct and quantitative manner. We would ask you to measure the cytoplasmic levels of active importin α, data that can be collected either with (1) FCS, (2) FRET , or (3) quantitative pull downs. We suggest this because we expect that the reagents already exist in your lab, based on previous papers you have published. A major claim in the paper is that at stage 8, spindle size is reduced upon release of an inhibitory interaction between the kinesin kif2a and importin α. This occurs through redistribution of importin α from the cytoplasm to a membrane fraction. This point needs to be shown in a more direct and quantitative manner. We would ask you to measure the cytoplasmic levels of active importin α, data that can be collected either with (1) FCS, (2) FRET , or (3) quantitative pull downs. We suggest this because we expect that the reagents already exist in your lab, based on previous papers you have published.We have addressed the concern with a quantitative pull down experiment. Tagged recombinant kif2a was added to stage 3 or stage 8 extracts and then retrieved, revealing the amount of active importin α in the each extract that is free to bind kif2a, using a kif2a NLS mutant as control . We founIn addition, we attempted to further quantify the kif2a-importin α by other approaches suggested by the reviewers. FCS experiments were performed upon addition of fluorescently labeled kif2a motor domain to either stage 3 or stage 8 extract at low concentrations. Fluorescence intensity was measured at 500 frames/sec and processed as by the PACI method developed by Needleman et al. (Biophysics J. 2009). Correlation curves indicated slower diffusion of kif2a motor domain in stage 3 compared to stage 8 extracts, indicating the interaction with other proteins and consistent with the formation of importin α-containing complexes. However, curve fits to this data were underdetermined for some parameters and therefore, despite qualitatively supporting our pulldown results, we feel the data are not strong enough to warrant addition to the manuscript.Experiments utilizing FRET or other methods that require a fluorescently labeled version of importin α would require addition or overexpression of importin α and therefore perturbation of its levels. Given the small changes in importin α partitioning that affected spindle size, we were unable to interpret data from the system perturbed in this way since any additional importin α protein is expected to affect its steady state distribution.Overall, the addition of the quantitative pulldown data strongly supports our model that partitioning of importin α out of the cytoplasm can decrease its interaction with kif2a, leading to a change in spindle size.


Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA), which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking. Ho. Ho23]. To increase the signal-to-noise ratio associated with linear probes, some groups have utilized Förster resonance energy transfer (FRET)-based systems ,9. In thUnlike linear probes, molecular beacons form stem-loop structures. The 5’ and 3’ ends of the beacon form a self-complimentary stem, while the central loop region is complimentary to the transcript of interest . The 5’ i.e., non-engineered) viral RNAs. As a result, molecular beacons can be used to visualize viral RNAs in clinical isolates . Wh. Whet alsitivity . Interessitivity . Because . Ea. EaN-GFP = 22 nM . As withN-GFP was localized to the nucleus as expected  construct containing the MMTV Ψ packaging sequence ; the Remexpected B, left. P signal B, middleMMTV RNA B, right omponent . Living One of the major drawbacks to the RNA-binding protein-based systems discussed to this point is that all of these systems require the insertion of specific stem-loop structures into the RNA of interest. On the other hand, although endogenous transcripts can be visualized using the hybridization-based techniques discussed earlier, these techniques are technically challenging. These limitations have resulted in the development of a genetic probe based on the RNA-binding protein PUMILIO1 to track endogenous RNAs in living cells . The PUMet al. created two PUM-HD mutants that recognized adjacent sequences on the mitochondrial NADH dehydrogenase 6 RNA [To visualize endogenous RNA molecules in living cells Ozawa se 6 RNA . The PUMse 6 RNA . Due to se 6 RNA . The PUMse 6 RNA . As a re9 different transcripts [As with all RNA tracking systems, however, the PUM-HD system has some limitations. Background fluorescence may result from non-specific complementation of the split GFP fluorophore, reducing the signal-to-noise ratio . Also, bnscripts . Accordiet al. described a series of RNA aptamers called “Spinach” that fluoresce when bound to GFP-like fluorophores  transcripts in the same cell. However, care must be taken to ensure that insertion of stem-loops into the target transcript does not affect RNA localization. Studies in yeast have indicated that insertion of MS2 stem-lops into the 3’ untranslated region just downstream of the stop codon does not affect transcript localization [RNA binding protein-based approaches to live cell RNA tracking including MS2-GFP, Bgl-mCherry, and λlization , althouglization ,68, althN-GFP system to study MMTV RNA trafficking in living cells. We found that MMTV RNA transiently interacts with cellular RNA processing factors, including components of P-bodies. Proteins associated with P-bodies repress the translation of cellular mRNAs and target these transcripts for long-term storage or degradation [Despite the limitations and caveats of RNA binding protein-based systems for RNA visualization, MS2-GFP and Bgl-mCherry have provided valuable insight into retroviral RNA trafficking, interactions with host factors, mechanisms underlying genome encapsidation, and the dynamics of particle assembly. Additionally, we have adopted the λradation . Additioradation . Accordiradation . Furtherradation ,73,74, aradation . Our obs


Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in  Axons extend great distances to make precise synaptic connections in the developing nervous system. Axons are guided to their targets by the growth cone, a dynamic structure at the axon distal tip that senses extracellular cues telling the axon where to go. In response to guidance cues, growth cones alter their shape and motility resulting in outgrowth and turning. The cytoskeleton (actin and microtubules) underlies growth cone motility and guidance. The signaling mechanisms linking guidance receptors to cytoskeletal change remain mysterious. Here, we define a new signaling mechanism downstream of the guidance receptor UNC-40/DCC involving the GTPases CDC-42 and Rac, which have long been known to control growth cone protrusion. We show that CDC-42 and Rac act in a linear pathway in axon guidance; CDC-42 acts upstream of the GTPase regulatory molecule TIAM-1, which is a GTP exchange factor specific for Rac and which activates Rac signaling. We also show that TIAM-1 acts with UNC-40/DCC signaling in protrusion and axon guidance. Our results imply that Rac GTPase function in axon guidance is complex and that distinct GEFs (TIAM-1 and UNC-73/Trio) might control Rac GTPases in different aspects of axon guidance. In a developing nervous system, axonal growth happens via extension and migration of the growth cone, an actin-rich neuronal structure from which filopodial and lamellipodial structures emanate in order to sense the surrounding environment for appropriate guidance cues Drosophila melanogaster and the nematode Caenorhabditis elegansC. elegans growth cone development and axon pathfinding, the two redundant Rac GTPases CED-10/Rac1 and MIG-2/RhoG act via two distinct pathways to activate the Arp2/3 complex: CED-10/Rac1 acts with WVE-1/Wave, while MIG-2/RhoG acts with WSP-1/Wasp The intracellular signaling mechanisms linking guidance receptors to cytoskeletal alterations are an intense area of study, and many remain to be elucidated. Rac GTPases, members of the Rho subfamily of small GTPases , have been well established as modulators of the actin cytoskeleton C. elegans genome encodes 19 predicted Dbl-homology (DH) GEF proteins (Wormbase). UNC-73/Trio is a well-characterized GEF for MIG-2/RhoG and CED-10/Rac1 in C. elegans axon pathfinding rh40) disrupts axon pathfinding mig-2; ced-10 rac double mutants are more severe than unc-73(rh40)Although much is known about the downstream effectors of the Rac GTPases in axon development, less is known about pathways upstream of Rac GTPases that control their activity in vivo. Rac GTPases switch between an inactive, GDP-bound state, and an active, GTP-bound state. Rac GTPase activation can be reached with the aid of a Guanine-nucleotide Exchange Factor (GEF), which catalyzes the exchange of GTP for GDP Here we show that TIAM-1 is a Rac GEF similar to mammalian T-cell lymphoma Invasion and Metastasis Factor 1 (Tiam1) C11D9.1 gene is predicted to encode a molecule with DH and Pleckstrin homology (PH) domains most similar to mammalian T-cell lymphoma Invasion and Metastasis Factor 1 (Tiam1) and Drosophila Still Life (Sif), but lacks an apparent EVH1 domain and PDZ domain found in Tiam1 and Sif C11D9.1 were obtained and sequenced (courtesy of Yuji Kohara). One of the cDNAs contained sequences complementary not only to C11D9.1, but also to the predicted upstream gene T21E12.2  T21E12.2/C11D9.1 locus collectively as tiam-1.A search of the additional predicted sequence encoded by  and Sif . While ttiam-1 cDNAs are shown in tiam-1 used here (ok772 and tm1556) remove at least part of the DH domain , but when in a double mutant combination (mig-2(mu28); ced-10(n1993M+); M+ indicates that the genotype has wild-type maternally-supplied gene function), most PDE neurons were affected  substitution in the Rac-specific DH1 domain) mig-2(mu28); ced-10(n1993M+) (p<0.001). mig-2(mu28); unc-73(rh40) and ced-10(n1993); unc-73(rh40) doubles display pathfinding defects equivalent to mig-2(mu28); ced-10(n1993M+)The ted 84%, . UNC-73/C. elegans genome encodes 19 predicted DH-containing GEFs including UNC-73/Trio . We screened mutant alleles or RNAi of the other 18 DH-GEFs alone or in combination with ced-10(n1993) and mig-2(mu28) for PDE axon pathfinding defects . The two deletion alleles of tiam-1 described above, ok772 and tm1556, caused low-penetrance defects in PDE axon pathfinding  (mig-2(mu28) and ced-10(n1993), these defects were synergistically enhanced (tiam-1(tm1556); mig-2(mu28) displayed 15% misrouted PDEs (p = 0.001), and tiam-1(tm1556); ced-10(n1993) 12% (p = 0.008). tiam-1 mutations slightly but significantly enhanced unc-73(rh40) (tiam-1 gene rescued the PDE axon pathfinding defects of tiam-1(tm1556); ced-10(n1993)  (tiam-1(+) expression in a wild-type background caused a low level of PDE axon pathfinding defects ; ced-10(n1993) rescued animals. Thus, TIAM-1 transgenic expression caused defects in axon pathfinding.The ctively) . In doubenhanced . For exa= 0.028) . We noteects 5%; , equivaltiam-1(ok772); mig-2(mu28) was significantly weaker than that of tiam-1(tm1556); mig-2(mu28) (p = 0.02) and other tiam-1 doubles with mig-2 and ced-10 ; mig-2(mu28) is still significant compared to tiam-1(ok772) alone (p = 0.046).The phenotype of d ced-10 . This mitiam-1 mutants alone also displayed weak defects in other neurons. In the AVM neurons, tiam-1(ok772) displayed ventral axon pathfinding defects and 6% ectopic neurite formation, defects not seen in wild-type (tiam-1(ok772) and tiam-1(tm1556) also displayed weak defects in VD/DD commissural motor neuron pathfinding  but not significant for tiam-1(ok772)) ((ok772)) .tiam-1 enhances mig-2 and ced-10 could indicate that TIAM-1 acts in parallel to these GTPases or that TIAM-1 acts in both of the redundant MIG-2 and CED-10 pathways in pathfinding. Studies below indicate that TIAM-1 acts with both MIG-2 and CED-10. However, the mig-2; ced-10 double mutant phenotype is much stronger than tiam-1, suggesting that another molecule might regulate MIG-2 and CED-1 in parallel to TIAM-1, possibly another GEF.These studies indicate that TIAM-1 displays a genetic interaction with the Rac GTPases CED-10 and MIG-2 and the UNC-73/Trio GEF in PDE axon pathfinding. That tiam-1 gene might by expressed, we studied expression of a tiam-1 transcriptional reporter transgene. We generated by PCR a 4793-bp region upstream of T21E12.2  (tiam-1 promoter region was placed upstream of the cyan fluorescent protein (cfp) coding region. Expression of the tiam-1promoter::cfp construct was primarily in neurons  , and thi neurons . Most ifC. elegans TIAM-1 is similar to the Rac GEFs Tiam1 and Sif. We tested if TIAM-1 from C. elegans acts as a GTP exchange factor in vitro using the mant-GTP RhoGEF exchange assay  S1216F missense mutation in the Rac-specific DH GEF domain of UNC-73 specifically abolishes Rac GEF activity unc-73(rh40) mutation S1216F. This mutation also abolished the ability of TIAM-1[DHPH] to induce ectopic protrusions in neurons as described below.The in vivo, we constructed transgenes that express constitutively-activated forms of the TIAM-1[DHPH] and UNC-73[DHPH1] Rac GEF domains in the PDE neurons. We based these experiments on previous results with Drosophila Trio (UNC-73 in C. elegans), in which a myristoylation sequence was added to the Rac-specific DHPH domain of Trio . The MYR::DHPH::GFP fusion proteins displayed a punctate, peripheral accumulation pattern consistent with localization to cellular membranes as seen in other MYR::GFP fusion proteins Based on these results, we created transgenes consisting of the TIAM-1 DHPH domain and both UNC-73 DHPH domains  fused inIn a wild-type background, the Rac-specific MYR::UNC-73[DHPH1] induced ectopic protrusions that emanated from the cell body and axons of PDEs . MYR::TIunc-73(rh40) S1216F mutation tiam-1(tm1556) background and observed ectopic protrusions similar to the wild-type background (data not shown), consistent with the idea that MYR::TIAM-1DHPH represents a constitutively-activated rather than a dominant negative molecule.To test the requirement of Rac GEF activity in ectopic protrusion, we introduced point mutations into the transgenes that abolish the Rac GEF activities of UNC-73 and TIAM-1  and ced-10(n1993) significantly suppressed the ectopic projections caused by MYR::TIAM-1[DHPH] and MYR::UNC-73[DHPH1] (mig-2(mu28) suppressed the ectopic lamellipodia and filopodia caused by MYR::UNC-73[DHPH1]  and MYR::TIAM-1[DHPH] . ced-10(n1993) suppressed MYR::TIAM-1[DHPH] strongly  and MYR::UNC-73[DHPH1] more weakly but significantly . The same integrated MYR::TIAM-1[DHPH] transgene (lqIs165) was used in both the mig-2 and ced-10 double mutants, yet upon outcrossing, the transgene showed significantly different effects (p<0.01). We do not know the nature of this variability, but it might be due to effects of different genetic backgrounds after outcrossing. Regardless, in both cases the effects increased significantly upon outcrossing from ced-10 and mig-2. We also scored integrated lines of the GEF dead constructs and the Rho-specific UNC-73 DHPH2 domain  and the osm-6 promoter (for PDE expression). The yk730h9 cDNA contained two errors relative to the reference gene models that led to frame shifts. These were repaired before proceeding  expression in In the PDE and VD/DD neurons, TIAM-1::mCherry localized to puncta primarily at the periphery of the cell bodies, consistent with plasma membrane localization, as well as in the axons . Consistmig-2/RhoG and ced-10/Rac1 transgenes tagged with gfp driven by the unc-25 promoter. MIG-2 contains an N-terminal myristoylation site and a C-terminal CAAX prenylation site. We placed gfp immediately after the Myr sequence in frame with mig-2, such that both myristoylated and prenylated forms of MIG-2 could be tagged with GFP. CED-10 was N-terminally tagged with GFP. GFP::CED-10 and GFP::MIG-2 accumulated at the cell periphery as previously described We constructed We analyzed the localization of N-terminally tagged mCherry::TIAM-1 and GFP::CED-10 in cultured NIH-3T3 fibroblasts. As expected, expression of GFP::CED-10 in fibroblasts caused marginal lamellipodial ruffling, a phenotype canonically associated with expression of vertebrate Rac1 in these cells . GFP::CEC. elegans, these data indicate that TIAM-1 and CED-10/Rac1 might interact in specific regions of the cell. In 3T3 cells, they co-localize where CED-10/Rac1 induces lamellipodial ruffles at the cell periphery   displayed ectopic lamellipodial and filopodial protrusions (44%) (cdc-42(G12V) transgenic lines, and each induced ectopic protrusions. We used one representative line for further studies.Evidence from cell and neuronal culture studies -1(DHPH)  [41]. Wens (44%) , as wellcdc-42(G12V) in tiam-1(ok772) and tiam-1(tm1556) mutants. In both cases, the ectopic lamellipodia and filopodia induced by CDC-42(G12V) were significantly suppressed  (unc-73(rh40) did not suppress CDC-42(G12V) (47% ectopic lamellipodia and filopodia in unc-73(rh40); cdc-42(G12V) compared to 44% in cdc-42(G12V) alone; p = 0.07)). These data suggest that TIAM-1, but not UNC-73, is required for the effects of CDC-42(G12V).To test if TIAM-1 was required for the effects of CDC-42(G12V), we assayed the effects of <0.0001) . These dosm-6p::tiam-1::mCherry, which produces full-length TIAM-1 tagged with mCherry in the PDE neurons but not surrounding cells, restored the effects of CDC-42(G12V) in tiam-1(ok772) and tiam-1(tm1556) mutants -induced ectopic lamellipodia and filopodia  were consistently enhanced compared to CDC-42(G12V) alone . This was also the case with fosmid rescue . Possibly, the excess TIAM-1 expressed from the transgene is activated by CDC-42(G12V), leading to increased defects. However, TIAM-1 expression alone causes ectopic protrusions  deletion removes the initiator codon and a large portion of the coding region and is likely a null allele (Wormbase). cdc-42(gk388M+) homozygotes, with wild-type maternal gene function, survived until late larval/early adulthood and were sterile. Alone, cdc-42(gk388M+) animals display 15% PDE axon pathfinding defects (tiam-1(ok772); cdc-42(gk388M+) animals showed no increase in pathfinding defects and resembled cdc-42(gk388M+) alone. However, unc-73(rh40); cdc-42(gk388M+) mutants displayed a synergistic increase in the number of PDE pathfinding defects (90%) compared to cdc-42(gk388M+) and unc-73(rh40) alone (p<0.001) (These findings were corroborated with loss-of-function studies. The  defects . tiam-1(p<0.001) . These lmig-2(mu28) and ced-10(n1993) suppressed the effects of CDC-42(G12V) (ced-10(tm597M+) deletion allele also suppressed CDC-42(G12V) (p<0.001). Suppression by ced-10(n1993) was significantly weaker than that of mig-2(mu28)  and ced-10(tm597M+) . ced-10(n1993) is a mutation in the C-terminal prenylation sequence and is a hypomorph, consistent with weaker suppression ced-10(n1993M+); mig-2(mu28) double mutant suppressed  slightly better than mig-2(mu28) or ced-10(tm597M+) alone . These data indicate that CED-10/Rac1 and MIG-2/RhoG are required for the ectopic protrusions caused by CDC-42(G12V).Our results suggest that TIAM-1 acts downstream of CDC-42 in ectopic lamellipodial protrusion. Given that TIAM-1 can act as a Rac GEF, we expect that MIG-2/RhoG and CED-10/Rac1 might also be required for the effects of CDC-42(G12V). Indeed, 42(G12V) ; p<0.001C. elegans. UNC-6/Netrin is a secreted ligand expressed in the ventral side of the animal that attracts axons that express its receptor UNC-40/DCC C. elegans Ephrin receptor We have shown evidence that the Rac GEF TIAM-1 regulates the Rac GTPases CED-10 and MIG-2 in response to CDC-42 signaling. We next sought to determine which dorsal-ventral axon guidance ligand/receptor pathway might be upstream of TIAM-1. Distinct parallel ventral guidance pathways control dorsal ventral guidance in tiam-1 with the loss-of-function alleles unc-6(ev400), slt-1(eh15), and vab-1(dx31), and scored defects in PDE axon ventral guidance (tiam-1(ok772) significantly increased the defects of vab-1(dx31) , and tiam-1(tm1556) enhanced slt-1(eh15) , suggesting that TIAM-1 might act in parallel to VAB-1/Eph and SLT-1/Slit. In contrast, neither tiam-1(ok772) nor tiam-1(tm1556) increased the amount of PDE pathfinding defects of unc-6(ev400) . slt-1(eh15) is a small deletion and is likely a null vab-1(dx31) is a complex rearrangement that deletes exons 1–4 and is also likely a null tiam-1 enhanced slt-1 and vab-1 but did not enhance unc-6 is consistent with TIAM-1 acting in the UNC-6/Netrin pathway in parallel to VAB-1 and SLT-1 in PDE ventral guidance.We constructed double mutants of guidance . tiam-1 did not suppress (tiam-1(ok772) significantly suppressed the ectopic lamellipodia incidence in these neurons . Furthermore, cdc-42(gk388M+) suppressed MYR::UNC-40 , suggesting that CDC-42 is also downstream of UNC-40. Consistent with this idea, cdc-42(gk388M+) did not enhance unc-6(ev400) in PDE ventral axon guidance (cdc-42(gk388M+) slightly suppressed unc-6(ev400) (p = 0.027). The nature of this possible suppression is unclear, but it is possible that cdc-42(gk388M+) might also normally attenuate UNC-6/Netrin signaling. While tiam-1 loss of function suppressed the ectopic protrusions driven by MYR::UNC-40, the axon guidance and branching defects were not suppressed significantly (data not shown), suggesting that MYR::UNC-40 perturbation of axon guidance and branching are not dependent upon TIAM-1 and might be due to effects distinct from ectopic lamellipodial protrusion.We tested if TIAM-1 might act downstream of MYR::UNC-40 in AVM. When expressed in the PDE neurons, MYR::UNC-40 did not result in ectopic lamellipodial protrusions. We do not know why this is the case, but possibly the PDE neuron is less sensitive to the effects of MYR::UNC-40 than is AVM, or the suppress ; 58%, coguidance . In factunc-6, unc-40, and tiam-1 (unc-6(ev400) and unc-40(n324) both displayed defective AVM ventral guidance as expected, but unc-6(ev400) was significantly stronger than unc-40(n324) . Both are predicted null mutations, suggesting that UNC-40 might not be the only receptor for UNC-6 in AVM ventral guidance. AVM defects in unc-6(ev400) mutants were scored previously by another group unc-6(ev400) (data not shown), similar to those previously described We assayed AVM axon guidance in single and double loss-of-function mutants involving d tiam-1 . unc-6(etiam-1(tm1556), tiam-1(ok772), and tiam-1(RNAi) significantly enhanced the AVM ventral guidance defects of unc-6(ev400)  caused defects in AVM guidance similar to unc-40(n324) that were not enhanced by tiam-1 mutations; and tiam-1(RNAi), which enhanced unc-6(ev400), did not enhance unc-40(n324) (unc-40(n324) and unc-40(RNAi) enhanced unc-6(ev400) in a manner similar to tiam-1. Enhancement of unc-6(ev400) by tiam-1 and unc-40 was also significant when only failure to reach the ventral nerve cord was considered (data not shown). Previous results showed that unc-40(e1430); unc-6(ev400) doubles had only slightly increased AVM guidance defects (∼25% to ∼33%) unc-40(n324) and unc-40(RNAi) and the previous study used unc-40(e1430)unc-6(ev400) that we report is due to different unc-40 alleles used.We found that 6(ev400) . This rece unc-6 , suggest40(n324) . These dunc-40 has a more severe phenotype than tiam-1 suggests that additional molecules might act in parallel to TIAM-1 downstream of UNC-40.In sum, these genetic interactions suggest that UNC-6 and UNC-40 act partially in parallel and also suggest that UNC-6 might not be the only ligand for UNC-40/DCC in AVM ventral guidance. They are also consistent with TIAM-1 acting in the UNC-40 pathway in AVM ventral axon guidance. That C. elegans molecule TIAM-1, which acts specifically as a GEF for Rac GTPases and interacts with them in axon guidance and neuronal protrusion. We show that TIAM-1 regulates neuronal protrusion downstream of the CDC-42 GTPase and upstream of the two redundant Rac GTPases MIG-2/RhoG and CED-10/Rac1. Genetic interactions of these molecules suggest that that they act in a common pathway in axon pathfinding. Furthermore, we provide evidence that TIAM-1 acts with the UNC-6/Netrin receptor UNC-40/DCC in axon guidance and downstream of UNC-40/DCC in neuronal protrusion. Previous studies have implicated Rac GTPases and the Rac GEF Trio as players in Netrin signaling and interaction with the Netrin receptor DCC In recent years, attention has focused on how guidance receptors engage cytoplasmic signaling mechanisms to control growth cone outgrowth and turning during axon guidance. In this report we present evidence of a signaling pathway  involvinGTP exchange assays indicated that TIAM-1 is a Rac-specific GEF . The actin vivo, whether or not their normal role is in protrusion. However, we found that transgenic expression of full-length TIAM-1::mCherry also induced ectopic protrusions in the PDE neurons similar to but at a much lower level than the MYR::DHPH construct, suggesting that regulation of protrusion is a normal cellular role of TIAM-1. This is consistent with tiam-1 suppression of protrusion caused by CDC-42(G12V) and MYR::UNC-40. In contrast, unc-73 suppressed neither of these, suggesting that UNC-73/Trio might not normally regulate protrusive events downstream of UNC-40. We speculate that UNC-73 might control Rac activity upstream of guidance receptors to control their cell surface trafficking  Our results and those of others How might our results fit with these known roles of Trio in Netrin signaling? Cell-specific and context-specific differences in Trio function downstream of guidance receptors are possible explanations. In other words, Trio might act with UNC-40/DCC in some neurons and not in others, or might display distinct, cell-specific interactions with guidance receptors.C. elegans, Rac GTPases engage the actin-nucleating Arp2/3 complex and the actin-binding protein UNC-115/abLIM to promote filopodial extension mig-2; ced-10 doubles are more severe than tiam-1 alone. Rac GTPases might affect multiple aspects of axon guidance, whereas TIAM-1 might specifically regulate them in downstream cytoskeletal effects.An alternative but not mutually exclusive explanation, consistent with previous results, is that Trio and TIAM-1 affect distinct aspects of Rac function in axon guidance. It is becoming apparent that the role of Rac GTPases in axon pathfinding is complex. Rac GTPases clearly regulate cytoskeletal events in the growth cone in response to guidance cues. For example, in C. elegansunc-73 did not suppress MYR::UNC-40. Thus, suppression of MYR::UNC-40 might target those molecules that act downstream of UNC-40/DCC but not those involved in it's trafficking . Our results provide experimental evidence for this idea, as we have shown that the TIAM-1 GEF but not the UNC-73/Trio GEF controls Rac function downstream of UNC-6/Netrin signaling in protrusion. UNC-73/Trio might act upstream of UNC-40/DCC and regulate its trafficking Rho-family GTPases control many cellular functions, and it has been assumed that upstream regulators confer specificity to Rho GTPase activity. Indeed, genomes encode many GTP exchange factors for Rho GTPases (e.g. the C. elegans handling and culture were performed using standard techniques tiam-1(tm1556) allele was provided by the National Bioresource Project for the Experimental Animal “Nematode C. elegans” (S. Mitani), and the tiam-1(ok772) allele was provided to us by the C. elegans Gene Knockout Consortium via the C. elegans Genetic Center (CGC). Polymerase chain reaction (PCR) was used to verify the homozygosity of tiam-1(ok772) and (tm1556) in strains. The following mutations and genetic constructs were used: LGI: unc-73(rh40), tiam-1(ok772) and (tm1556), lqIs37[osm-6::cdc-42(G12V)]; LGII: cdc-42(gk388), mIn1,vab-1(dx31), juIs76[unc-25::gfp]; LGIV: ced-10(n1993), zdIs4[mec-4::gfp], lqIs3[osm-6::gfp]; LGV: lqIs123[osm-6::myr::unc-73(DHPH1)]; LGX: mig-2(mu28), unc-6(ev400), slt-1(eh15), lqIs2[osm-6::gfp]; LG?: lqIs125[osm-6::myr::unc-73(DHPH2)], lqIs131[mec-7::myr::unc-40(cyto)::gfp], lqIs134[osm-6::myr::unc-73(DHPH1 S1216F)], lqIs165[osm-6::myr::tiam-1(DHPH)], lqIs216[osm-6::myr::tiam-1(DHPH T516F)]. Extrachromosomal arrays: lqEx504-506, osm-6::myr::unc-73(DHPH1); lqEx509-510, osm-6::myr::unc-73(DHPH)2; lqEx517-518, osm-6::myr::unc-73(DHPH1 S1216F); lqEx559-561, osm-6::myr::tiam-1(DHPH); lqEx571-572, lqEx584, osm-6::myr::tiam-1(DHPH T516F).C. elegans transformation was performed with standard methods by microinjection of DNA into the hermaphroditic syncytial germline All micrographs were obtained on a Leica DMRE microscope with a Qimaging Rolera MGi EMCCD camera or a Qimaging Retiga CCD camera. Openlab and IPlab software were used to obtain images.lqIs2 X and lqIs3 IV [osm-6 promoter::gfp] were used juIs76 II [unc-25 promoter::gfp] was used zdIs4 IV [mec-4 promoter::gfp] was used.Fluorescence microscopy was used to score axon pathfinding defects of fourth larval stage (L4) or young adult hermaphrodite animals expressing a green fluorescent protein transgene for specific cells. To score and visualize PDE axons, the integrated transgenes The cell bodies of the PDE neurons (PDEL and PDER) are situated in the posterior lateral post-deirid ganglion. PDEs extend an axon ventrally to the ventral nerve cord, which then bifurcates and extends anteriorly and posteriorly in the ventral nerve cord. If the axon failed to reach the ventral nerve cord or wandered beyond a 45° angle from a straight line ventrally from the cell body, it was considered mutant.A VD/DD commissural axon that failed to reach the dorsal nerve cord or that wandered laterally before reaching the dorsal nerve cord was considered mutant. The percent of animals with pathfinding defects was noted, and the percentage of defective axons was noted.The AVM cell body is situated laterally on the right side, in the anterior half of the body of the animal. The axon extends ventrally towards the ventral nerve cord, which then extends anteriorly until the first pharyngeal bulb. Similarly to PDE axons, if the axon failed to reach the ventral nerve cord or wandered beyond a 45° angle from a straight line ventrally, it was considered mutant. p-value significance was determined using Fisher Exact Analysis.Ectopic lamellipodial and filopodial protrusions were identified as any thin ruffle-like or finger-like protrusion from the cell body, which is normally smooth and round, or axon of the neurons.C. elegans expression plasmids utilized the standard Fire expression vectors with the unc-54 3′ UTR . Primer sequences used to amplify C. elegans genomic and cDNA sequences to construct these plasmids are shown in tiam-1 transcriptional promoter fusion plasmid was generated by amplifying by PCR the tiam-1 upstream promoter region, a 4793-bp region upstream of T21E12.2  (cyan fluorescent protein (cfp) coding region. For cell specific expression of full length TIAM-1, the open reading frame of the yk370h9 tiam-1 cDNA was amplified by PCR and fused to mCherry at the 3′ end to create a C-terminally tagged TIAM-1::mCherry molecule (tiam-1::mCherry expression plasmids contained the osm-6 promoter (to drive expression in the PDEs) unc-25 promoter (VD/DDs) All 21E12.3) , and plamolecule . tiam-1:gfp::mig-2 fusion plasmid was constructed by placing gfp in frame after the N-terminal myristoylation sequence of mig-2 and before the remaining genomic coding region, allowing for GFP tagging of both myristoylated and prenylated forms of MIG-2 (gfp::ced-10 was constructed by placing gfp at the N-terminus as previously described gfp::mig-2 and gfp::ced-10 expression was driven by the osm-6 or unc-25 promoter.A of MIG-2 . gfp::cecdc-42(G12V) genomic coding region under the control of the osm-6 promoter was constructed using site directed mutagenesis as described previously for ced-10(G12V)myr::unc-40(cyto) plasmid under the control of the mec-7 promoter was constructed as described in myr::GEF(DHPH) plasmids with the osm-6 promoter were used as means of overactivating the GEFs as previously described A plasmid containing an activated cmv::mCherry::tiam-1 N-terminal fusion construct was generated by placing the yk37h9 tiam-1 cDNA open reading frame, fused N-terminally to mCherry, behind the CMV promoter in the pTriEx-mCherry vector . cmv::gfp::ced-10 was constructed using the ced-10 cDNA open reading frame For expression in NIH 3T3 cells, a 2 at 37°C. Cells were plated onto coverslips the day before transfection at a density of 200,000 cells per well in a 6-well plate. For co-localization assays, pEGFP-C1 , pEGFP-C1 expressing CED-10 (“GFP::CED-10”), pTriEx-mCherry  and pTriEx-mCherry expressing TIAM-1 (“mCherry::TIAM-1”) were transiently transfected into NIH3T3 cells using GeneExpresso Transfection reagent  according to the manufacturer's specifications. To visualize subcellular protein localizations at high magnification, the NIH 3T3 cells grown on coverslips and transiently transfected were fixed in 3.7% formaldehyde for 1 h at room temperature. Coverslips were then washed three times in 1× Phosphate Buffered Saline (PBS), one time in ddH2O, and then mounted onto a glass slide using ProLong Gold antifade (Invitrogen/Molecular Probes) mounting medium. The cells were then visualized using compound fluorescence microscopy .NIH 3T3 mouse fibroblasts were grown in DMEM (Cellgro) supplemented with 10% Cosmic Calf serum (HyClone) and 1% penicillin-streptomycin (P/S) and maintained in 5% COWe amplified the DHPH domains of TIAM-1 (residues 488–856 relative to isoform A), placed it into the pET-28b vector (Novagen), which has a T7 IPTG-inducible promoter and a C-terminal 6HIS-tag, and transformed this construct into BL21(DE3) Gold competent cells (Agilent Technologies). For the TIAM-1[DH(T516F)PH] construct, we used site directed mutagenesis to make a point mutation in the wild-type construct using the Quikchange kit (Stratagene). We purified TIAM-1[DHPH]::6HIS using Ni-column based chromatography (Qiagen). For the mant-GTP assay, we used the RhoGEF Exchange Assay Biochem Kit  and followed instructions in the kit. We used 12.5 µM of TIAM-1[DHPH] in these experiments.N- methylanthraniloyl-GTP (mant-GTP) is a fluorescently labeled nucleotide analog that can be incorporated by small GTPases. Using a Varian Cary Eclipse fluorescent spectrophotometer, we measured the amount of mant-GTP incorporated by the GTPase due to the spectroscopic difference between bound and free mant-GTP . The GTPase  was incubated in buffer and mant-GTP for five minutes, after which time solvent (control) or the GEF (TIAM-1[DHPH]) was added. Experiments were recorded for one half hour.Figure S1tiam-1 locus. (A) Black boxes represent exons with coding region open reading frame, white boxes represent non-coding exons, SL1 indicates that an SL1 trans-spliced leader sequence was present in the cDNA sequence, and AAAAA indicates the presence of a poly-A tail in the cDNA sequence. The regions removed by the ok772 and tm1556 deletions are indicated. ok772 is an 838-bp deletion a coupled to an 18-bp insertion (CTGTAACTTAACTGTAAC), and tm1556 is an 851-bp deletion. The yk730h9 cDNA spanned the predicted gene models C11D9.1 and T21E12.2, indicating that these regions are part of the same transcription unit and thus represent a single locus, the tiam-1 locus. The dashed lines are an expansion of the 3′ end of the locus showing the structures of three cDNAs sequenced. yk730h9 is the longest cDNA and spans the entire locus. An arrow points to an intron that is spliced out specifically in yk730h9. An SL1 trans-spliced leader sequence is present at the 5′ end of yk730h9, indicating that it is a full-length cDNA. yk1684a10 is a shorter cDNA with splice variation. An in-frame stop codon is present in the 5′ region of the cDNA sequence, indicating that this cDNA contains the entire coding potential of this isoform. yk762f12 also encodes a shorter cDNA with splice variation and contains an SL1 trans-spliced leader, indicating that it contains the complete coding potential for this isoform. The sequences of these cDNAs have been deposited in Genbank. (B) The predicted molecules encoded by the three cDNAs. yk730h9 encodes isoform A, which contains a predicted EVH1-like domain and PDZ-like domain in addition to the DH and PH domains. yk762f12 encodes isoform B, which contains the PDZ-like domain and the DH/PH domains. yk1684a10 encodes isoform C which contains a truncated PDZ-like domain and the DH/PH domains. The regions removed by the deletions ok772 and tm1556 are indicated.A model of the (TIF)Click here for additional data file.Figure S2tiam-1 A. Open reading frame of tiam-1 A encoded by a repaired version of the yk730h9 cDNA is shown, with the amino acid sequence below. The yk730h9 cDNA contained errors relative to the reference gene models that resulted in frame shifts. These were repaired in the yk730h9 cDNA by site-directed mutagenesis. An extra G residue after base 1039 relative to the start codon in ok772 and tm1556 are shaded black. These do not represent the precise breakpoints of the deletions, as three of four breakpoints are within introns.The nucleotide and amino acid sequence of (PDF)Click here for additional data file.Figure S3C. elegans TIAM-1. (Ce) (this work), Still life/Tiam1 from Ascaris suum (As) (Genbank: ADY42014), and Still Life/Tiam1 isoform E from Drosophila melanogaster (Dm) (Genbank NP_001097519). While BLASTP and CCD did not recognize the EVH1 domain of Ce TIAM-1, they did recognize a conserved region in As TIAM-1 as an EVH1 domain. Identities are indicated. (B) An analysis of the putative PDZ-like domain as described for the EVH1 domain in (A). (C) ClustalW alignments of the predicted EVH1 and PDZ domains of the same molecules in (A and B). Conserved identical residues are indicated in red and by asterisks (*), and conserved similar residues are indicated by colons (:).BLASTP and ClustalW analysis of the TIAM-1 molecule. (A) BLAST alignments showing of the predicted EVH1-like regions of (PDF)Click here for additional data file.Figure S4tiam-1 mutants. In all micrographs, anterior is to the left and dorsal is up. (A) A fluorescent micrograph of an AVM neuron of a wild-type animal visualized with mec-7::gfp. (B) An AVM neuron from a tiam-1(ok772) mutant with an ectopic neurite. (C) A VD or DD motor neuron visualized with unc-25::gfp branched prematurely in a tiam-1(tm1556) mutant (arrow). The scale bars represent 2 µm.VD/DD and AVM axon defects in (TIF)Click here for additional data file.Figure S5in vitro, represented as “(DEAD)” on the X axis Click here for additional data file.Figure S6osm-6::gfp (arrows). TIAM-1::mCherry is excluded from the nucleus and localizes to the periphery of the cell body. (B) TIAM-1::mCherry accumulation at the periphery of VD/DD motor neuron cell bodies (arrows), driven by the unc-25 promoter. The outlines of cell bodies were traced (dashed line) to indicate peripheral localization of TIAM-1::mCherry. The scale bar represents 2 µm for all micrographs.TIAM-1::mCherry localized to the periphery of neuronal cell bodies. In all micrographs, anterior is to the left and dorsal is up. (A) two PDE neurons with TIAM-1::mCherry expression driven by (TIF)Click here for additional data file.Figure S7GFP::CED-10 and TIAM-1::mCherry accumulate to specific sites in NIH-3T3 cells. GFP::CED-10 and TIAM-1 localize to discrete regions of the peripheral plasma membrane (arrowheads in (A) and (B). GFP::CED-10 induced lamellipodial ruffles but TIAM-1::mCherry did not. Neither GFP nor mCherry alone localized to discrete regions of the cell ((C) and (D)) as did the tagged CED-10 and TIAM-1 molecules. The scale bar in (A) represents 10 µm for all micrographs.(TIF)Click here for additional data file.Figure S8tiam-1 genomic fosmid clone rescued suppression of CDC-42(G12V). (B) A graph charting the percentage of PDE axons with ectopic lamellipodial and filopodial protrusions (X axis) in different genotypes (Y axis). [cdc-42(G12V)] represents animals harboring a transgene that expresses activated cdc-42(G12V) driven by the osm-6 promoter. [tiam-1(+)] represents a transgene composed of the genomic fosmid clone WRM0633ch01 that harbors a wild-type copy of tiam-1. At least 100 PDE neurons were scored for each genotype, and p value significance was determined using Fisher's Exact analysis. Error bars represent 2× standard error of the proportion in both directions.A (TIF)Click here for additional data file.Figure S9C. elegans genomic or cDNA fragments to construct expression plasmids. The fragment of genomic DNA or cDNA amplified is underlined. (+) indicates the sense strand primer relative to the gene, and (−) indicates the anti-sense strand primer relative to the gene. Spacers and restriction enzyme sites added to the ends of the primers to aid in cloning are in italics.Primer sequences used to amplify (PDF)Click here for additional data file.


E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma) with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT) activity in human cells. Transcription regulator EWS (Ewing's sarcoma), which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG) repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs), resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in  Gene silencing has emerged as one of the major functions of short double stranded noncoding RNAs (ncRNAs) that are generated by specific processing machinery. The mechanisms by which small ncRNAs, siRNAs and miRNAs, participate in RNAi pathway involved in gene silencing, mRNA stability and translation arrest have been extensively studied ,2. In coE. coli . Ce. Ce24]. +, K+), which are positioned in the center of the structure and coordinated by the electron-rich carbonyl oxygens. The NMR structure of human telomeric repeat containing RNA (TERRA) r(UUAGGG)4 with K+ has been solved as the G-quadruplex structure in vitro and the structure of TERRA in living cells has been confirmed as G-quadruplex by a lights-switching pyrene probe , in vitro .,58.2C2 zn Figure  58,59]..2C2 zincN-methyltransferase (PRMT) 3 reduced the binding RGG domain of EWS to G-quadruplexes but increased its binding to single-strand DNA and RNA. PRMT1 (a homologue of PRMT3) reduced transcriptional activity of EWS through nuclear exclusion of EWS by methylation of arginine residues .,61.60,61residues . It suggresidues ,64. Thes4 which forms G-quadruplex in vivo. TLS might bind to TERRA and regulate transcription of TERRA.Folding of RNA into divergent structures is constrained by specific RNA sequences under physiological conditions. Recent analyses revealed that several ncRNAs form specific conformations in sequence-and structure-specific manners, mimicking an open promoter structures and G-quadruplex structures. In living cells, specific three-dimensional structures of these ncRNAs could contribute to regulation of transcription. It remains however unclear whether a role of EWS in transcription is fixed by its ability to target a specific RNA structure. Janknecht et al. reported that overexpression of EWS in RK13 and AKR cells leads to activation of the c-fos, Xvent-2, and ErbB2 promoters, while EWS did not bind to double-stranded DNAs in these promoters . These dncRNAs: noncoding RNAs; TLS: translocated in liposarcoma; EWS: Transcriptional factor Ewing's sarcoma; HAT: histone acetyltransferase; TERRA: telomeric repeat containing RNA.The authors declare that they have no competing interests.TO and RK made substantial contributions to conception, design and been involved in drafting the manuscript. RK has given final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content. TO and RK read and approved the final manuscript.


Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3′ end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10–13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity. Influenza continues to impose a substantial annual burden of morbidity and mortality. Current antiviral drugs approved for the treatment of influenza virus infections are limited to two viral targets, neuraminidase and M2 ion-channel protein. The viral RNA (vRNA)-dependent RNA polymerase (RdRP) that is central to the virus lifecycle is a promising target for the development of novel influenza antiviral compounds.7GTP) of a host pre-mRNA to the PB2 subunit, (ii) cleaving of a phosphodiester bond 10–13 nt downstream of the cap by the endonuclease activity in the PA subunit and (iii) initiating synthesis of viral mRNAs at the cleaved 3′ end of the capped segment by the PB1 subunit that harbors the polymerase active site [reviewed in . RNA sequences and nomenclature are shown in Unlabeled and 2-aminopurine (2-AP)-labeled uncapped RNA oligonucleotides were purchased from Thermo Fisher Scientific. The 5′-triphosphorylated RNAs were purchased from Fidelity Systems . Fifty-mer RNA was generated by T7 RNA polymerase mediated transcription of template DNA. Capping reactions were performed using the ScriptCap™ m2801% = 8.79 ] was expressed and purified as previously described, and the concentration of the protein was determined by UV spectroscopy, using ε% = 8.79 .260 nm of 1.0 = 60 µg/ml RNP as conversion factor (RNP complexes were isolated from purified Influenza virus A/PR/8/34 (H1N1) purchased from Charles River Laboratories , using an established protocol, and RNP concentration was determined by UV spectroscopy, using ODn factor .2 and 2 mM DTT. We note here, as also previously reported .Endonuclease activity of the PA-Nter domain was measured in buffer containing 20 mM Hepes (pH 7.9), 100 mM sodium acetate, 1 mM Mn(OAc)reported , that th2, 0.1 mM Mn(OAc)2, 10% glycerol and 2 mM DTT. The protein and RNA concentrations used in the assays are specified in the figure legends. Reactions were performed at 25°C. To generate radioactively labeled capped RNA, 5′ tri-phosphorylated RNA was capped at the 5′ end with α-P32-GTP using capping enzyme purchased from CellScript. Uncapped RNA was labeled at the 5′-terminus with γ-P32-ATP using T4 PNK purchased from Invitrogen. Following the endonuclease reactions, RNA products were separated in 20% polyacrylamide gels in 7 M urea and then visualized and quantified using a Molecular Dynamics PhosphorImager with ImageQuant software.Unless mentioned otherwise, the RNP-based endonuclease and primer extension assays were performed in buffer containing 20 mM Hepes (pH 7.9), 100 mM sodium acetate, 1 mM Mg(OAc)Fluorescence spectra were measured with a Jasco model 815 spectrophotometer equipped with fluorescence capabilities and temperature controller. The 2-AP modified RNA oligonucleotides were excited at 315 nm, and emission was recorded at 370 nm, at 25°C.0 is the initial populations of substrate RNA, At is the population at time t, kobs is the apparent rate constant, and t is time.All the kinetic traces from both fluorescence and gel-based assays were fit to the following single exponential equation to obtain the apparent rate constant for the endonuclease reaction,Previous observations of endonuclease activity associated with native influenza RNP had indicated different cleavage efficiencies for different RNA substrates and in particular high-efficiency cleavage of substrates that carried GC motifs ,13. To cGC–3′ sequence was inverted to 5′-CG–3′; and (iii) uc-AU20 that lacks any G or C within the sequence . PA-Nter generated three distinct products  from the uc-G20 substrate RNA . The uc-G20-P1 and uc-G20-P2 corresponded to 16- and 11-mer, respectively, indicating endonuclease cleavage at the 3′ end of G of the two GC motifs. The shortest product, uc-G20-P3, migrated faster than the 2-nt marker RNA denoted as uc-G20-2mer(M) , consistent with cleavage after the 5′G . The cleavage of this substrate was considerably slower relative to uc-GC-3  uc-G20, carrying two GC motifs 5′-AGCU-3′ and 5′-UGCA-3′, in a different sequence, (ii) uc-CG20, a derivative of the GC-3 oligonucleotide, in which the 5′–sequence . Figure  uc-GC-3 B. The di uc-GC-3 C. The af7GTP cap moiety (cap) at the 5′ end of the RNA (denoted as c-RNA) on cleavage by PA-Nter. We also examined the effect of the presence of the mNext, we determined the cleavage pattern for substrate RNAs by influenza A RNP complexes isolated from purified viruses. RNP complexes comprise the heterotrimeric influenza polymerase  bound to the vRNA template that is coated with viral nucleoprotein . RNP-medSupplementary Figure S3). RNP showed a cleavage preference for capped RNAs with a preferred distance of cleavage from the cap structure at ∼10–13 nt downstream from the 5′ cap, as reported previously . These results suggest that the influenza endonuclease cleavage specificity of RNP is determined by a combination of cap-binding-mediated distance measurement with an optimum at 12 nt from the 5′ end of the RNA and an intrinsic selectivity for cleavage after a G residue, which is conferred by the PA-Nter active site. As shown in Although the presence of cap did not affect the cleavage efficiency or RNA sequence specificity of PA-Nter (eviously . A caref2+) and RNP (1 mM Mg2+ and 0.1 mM Mn2+). PA-Nter did not show measurable endonuclease activity in the presence of 1 mM Mg2+ and 0.1 mM Mn2+ but required ≥1 mM Mn2+ concentration for detection of activity . To test whether the presence of 1 mM Mn2+ might impact the cleavage pattern of capped RNA by RNP, RNP-mediated endonuclease reactions were also performed in 1 mM Mn2+. 2+) or (1 mM Mg2+ and 0.1 mM Mn2+). As shown, for capped RNA, similar RNA products (c-G20-P1 and c-G20-P2) were generated by RNP in either buffer condition.We note here that the divalent metal ion concentrations were different for the endonuclease reactions performed with PA-Nter  is the major fluorescent product species contributing to the observed change in fluorescence intensity during the endonuclease reaction, and that this assay can be used to monitor the rate of formation of this dominant species.Of the products, uc-G20-2AP-P1 and the 3′ end fragment of uc-G20-2AP-P2 [denoted here as uc-G20-2AP-P2 (3′)] are the most likely to contribute to the overall fluorescence change. We therefore measured the changes in intensities of these two RNAs in the absence and presence of PA-Nter domain B. FigureWe next quantified and compared the rates of the endonuclease reactions from both the fluorescence and gel-based assays. The concentrations of RNA and PA-Nter domain used for the gel-based assay were 10 and 1 µM, respectively. All the kinetic traces from the fluorescence assay were fit to a single exponential to obtain the apparent rate constant for the endonuclease reaction . For the−1) was similar to the rate of formation of uc-G20-2AP-P2 observed in gel-based measurements (0.054 min−1), and that these rates are in the same order of magnitude as the rate of overall disappearance of the substrate RNA was 0.081 min−1 in the gel-based assay  obtained from fluorescence assay 7-8 -(A/U/G)-(A/U)-AGC-3′ -AGC-3′ , consist)-AGC-3′ , non-GC Here, we have also described a spectroscopic assay to elucidate the kinetics of RNA cleavage by PA-Nter of influenza endonuclease. Time-dependent changes in fluorescence intensity of 2-AP modified RNA oligos were measured to estimate the endonuclease reaction rates. We show that the kinetic parameters obtained from this method are consistent with the data from gel-based assay, confirming the validity of this assay. We further demonstrate that the catalytic efficiency of PA-Nter is dependent on the RNA sequence where GC containing RNA was cleaved with maximal efficiency while the activity was minimal for AU rich sequences.in vitro and in vivo studies will be required to fully understand the biological significance of such mRNA leader sequence preference in the lifecycle of the virus.Based on the aforementioned findings, we conclude that influenza endonuclease exhibits strong sequence dependence of RNA cleavage, which is an intrinsic property of the PA endonuclease active site. Further Supplementary Data are available at NAR Online.Hoffmann-La Roche, Inc. (to K.D. and K.K.); European Union Seventh Framework Programme (FP7) project FLUPHARM . Funding for open access charge: Hoffmann-La Roche Inc., Nutley, NJ.Conflict of interest statement. None declared.