Retroperitoneal lymph node (RLN) metastasis is an important indicator of endometrial cancer (EC) prognosis. Because vascular endothelial growth factor c (VEGF-c) is known to influence lymphangiogenesis and thereby lymph node metastasis, this study assessed the relationship of VEGF-c mRNA expression with RLN metastasis in EC.The uterine muscularis mucosae of New Zealand white rabbits were inoculated with a VX2 tumor cell suspension after which they were sacrificed at 15, 18, 21, 24, 27 and 30 days. Control groups consisted of those receiving no treatment or an injection of saline. EC and metastatic RLN tissues along with peripheral blood samples were collected, and VEGF-c mRNA expression was evaluated using fluorescence real-time quantitative PCR.P < 0.001). In addition, increased VEGF-c mRNA expression was observed in peripheral white blood cells of rabbits with RLN metastasis (P < 0.002).The establishment of an in vivo model of EC with complete RLN metastasis was pathologically confirmed at day 21 post-injection with VX2 cells. As compared to the control groups, VEGF-c mRNA expression increased significantly over time in the tumor site, RLN, and peripheral white blood cells of EC rabbits. Significantly higher VEGF-c mRNA expression was observed in metastatic RLNs as compared to those without metastasis (Injection of a VX2 cell suspension is a simple method of establishing an in vivo EC model. VEGF-c may play an important role in the development of EC and its metastasis to RLN and may be useful marker to predict RLN metastasis. Endometrial cancer (EC) is one of the most common malignancies of the female reproductive system . WhereasAnalysis of a panel of angiogenic factors, including vascular endothelial growth factors , matrix metalloproteinase-2 (MMP-2), and basic fibroblast growth factor (bFGF), in 16 gynecological cancer cell lines revealed that VEGF-c expression was significantly correlated with cell migration as well as MMP-2 levels . VEGF-c In the present study, an in vivo model of RLN metastasis of EC was established by injecting a VX2 tumor cell suspension into the myometrium of New Zealand white rabbits. Fluorescence real-time quantitative PCR (RT-PCR) was employed to detect VEGF-c mRNA expression in EC tissues, RLNs, and peripheral white blood cells at various time points after injection. These data may provide a theoretical basis for further studies to evaluate VEGF-c as a marker of RLN metastasis in EC.A total of 49 female New Zealand white rabbits weighing 2-2.5 kg were purchased from the Huadong Xinhua Experimental Animal Center in Guangzhou (License No: 0098816). One rabbit was used as the source of VX2 tumor cells while the remaining animals were used to establish the in vivo model. The animals were individually housed, allowed free access to standard laboratory food and water, and subjected to daily 12-hour light and dark cycles. The animal protocol used in this study was approved by the Center’s Animal Welfare Committee of Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.10 cells/mL, 0.2 mL of the cell suspension was injected into the quadriceps femoris of one rabbit. After 21 days, a solid mass was removed from the injection site, washed in normal saline, and placed in RPMI 1640. Areas with active growth were selected, and the tissue was cut into pieces of 0.5-1 mm in diameter. After vortexing, the solution (1 × 1010 cells/mL) was transferred to a syringe with a lumbar puncture needle.VX2 cells were kindly purchased from the Cell Bank of the Sun Yat-sen University. Stocks of VX2 cells were mixed in 5 mL RPMI 1640, resulting in a VX2 solution of approximately 1 × 1010 cells/mL) was injected into the muscularis mucosae of the myometrium 1 cm away from the cervix. The injection site was sutured, and the wound was closed with a 1-0 suture.Animals in the experimental group were anesthetized with 3% pentobarbital sodium at 1 mL/kg via the ear vein and then placed in a supine position. After sterilization, a mid-line incision was made on the lower abdomen. After the uterus was exposed, 0.5 mL of the VX2 cell solution  and stored in liquid nitrogen and then at -80°C, and the other half was fixed in 10% formalin and embedded in paraffin. Sections were obtained and stained with hematoxylin and eosin (H&E). The stained EC and metastatic RLN tissues were independently observed under a light microscope by two pathologists, who were blinded to the treatment conditions.At 15, 18, 21, 24, 27 and 30 days post-injection of the VX2 cell solution, rabbits were sacrificed by aeroembolism (injection of air into the ear vein) (n = 6 per time point). At 30 days, animals in the normal control and saline groups were sacrificed by the same method. EC and RLN tissues were observed macroscopically, and the long diameter (a) and short diameter (b) were measured to calculate EC and RLN volume using the following equation: V = a × bAt the time of sacrifice, a blood sample was also collected, incubated with a blood cell separation solution, Histopaque-1107 , and centrifuged at 3500 r/min for 10 min. The white blood cells were collected and stored at -80°C for extraction of RNA.As previously mentioned, metastasis was confirmed by histopathology of the RLNs after different time points (n = 6 per time point). A group of six rabbits at a particular time point was considered “non-metastatic” if no metastasis was observed in all six animals. A group was considered to have “partial metastasis” if some animals had RLN metastasis while others did not. Finally, in those groups with “complete” metastasis, all six rabbits had RLN metastasis.-ΔΔCt.RNA was extracted from 50-100 mg of the tissue samples and 100 μg of the white blood cells using Trizol. cDNA was obtained using the RNA samples (2 μg) and a RT-PCR kit  following the manufacturer’s instructions, which produced a reaction mixture of 20 μL. The conditions for reverse transcription were 70°C for 5 min and 42°C for 60 min. The diluted cDNA (2 μL) was then used for real-time PCR using the Platinum SYBR green q-PCR Super Mix–UDG  along with the following primers, which were designed with Premier 5.0 and synthesized by the Shanghai Yingwei Jieji Co., Ltd : VEGF-c: 5′ CCCCAAACCAGTAACAATCAGT 3′ (forward), 5′ CTGGCAGGGAGCGTCTAAT 3′ (reverse); and GAPDH: 5′ AGAGCACCAGAGGAGGACG 3′ (forward), 5′ TGGGATGGAAACTGTGAAGAG 3′ (reverse). The conditions for the fluorescence real-time quantitative PCR were as follows: 95°C for 2 min; 45 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s; and 95°C for 1 min, 58°C for 30 s and 95°C for 30 s. The relative expression of VEGF-c was calculated as follows: ΔCt (target gene) = Ct (target gene) – Ct (GAPDH). ΔΔCt=ΔCt (target gene) - ΔCt (standard) mean of target gene. The relative copies of the target gene were determined as 2Continuous variables among more than two groups were compared by one-way analysis of variance (ANOVA). When a significant difference between groups was apparent, multiple comparisons of means were performed using the Bonferroni procedure with type-I error adjustment. Differences in VEGF-c mRNA expression between non-metastasized and metastasized RLNs were determined using an independent two sample t test. Data are presented as means ± standard deviation (SD). All statistical assessments were two-sided and evaluated at the 0.05 level of significant difference. Statistical analyses were performed using SPSS 15.0 statistics software .P < 0.05). A representative image of the normal and tumor endometrium after 21 days is shown in Figure As shown in Figure The presence of RLN metastasis was also assessed. RLN enlargement was not observed macroscopically at 15 days post-VX2 cell injection; however, pathological examination confirmed the absence of RLN metastasis . Analysis of the peritoneal cavity revealed that primary tumors were restricted to within the uterus as peritoneal dissemination of the VX2 cells was not observed.VEGF-c gene is mapped to chromosome 4q34 [Tumor angiogenesis is related to its invasion and metastasis , and VEGome 4q34 , and in ome 4q34 . Straumeome 4q34  also repome 4q34 -24, whicome 4q34 . Furtherome 4q34  will be In the present study, differences in VEGF-c mRNA expression were observed among rabbits without and with RLN metastasis; differences were also noted between rabbits with full and partial metastasis with the highest expression found in rabbits with metastasis to all RLNs. These results suggest that the increased VEGF-c may arise from the EC during its development and progression, which may subsequently promote lymph node metastasis . This daThe current study was limited in that it did not confirm whether VEGF-c protein levels were correlated with those obtained for mRNA. Further studies will evaluate the VEGF-c protein levels, as well as markers of epithelial-mesenchymal transition (EMT), in tissues as well as serum samples using immunohistochemistry and enzyme-linked immunosorbent assays (ELISAs). In addition, although previous studies have reported a relationship between VEGF-c and lymphangiogenesis with metastasis -8, no meThus, an animal model of EC with RLN metastasis was established, which revealed an increase in VEGF-c mRNA expression in peripheral white blood cells with tumor growth and metastasis. Should further studies determine that VEGF-c levels are reflective of tumor load and metastatic state, examining serum VEGF-c levels may be useful for predicting RLN metastasis in EC patients and further inform clinicians as to the most effective treatment option, including intra-operative RLN dissection.ANOVA: Analysis of variance; EC: Endometrial cancer; ELISAs: Enzyme-linked immunosorbent assays; RT-PCR: Real-time quantitative PCR; RLN: Retroperitoneal lymph node; SD: Standard deviation; VEGF-c: Vascular endothelial growth factor c; VEGFR-3: VEGF receptor-3.The authors declare that they have no competing interests.YWH: study concepts; study design; definition of intellectual content; literature research; clinical studies; experimental studies; data acquisition; data analysis; statistical analysis; manuscript editing. LQX: study concepts; literature research; experimental studies; data acquisition; data analysis; statistical analysis; manuscript preparation. RZL: experimental studies. XH: clinical studies. TH: experimental studies. YNZ: guarantor of integrity of the entire study; study concepts; study design; definition of intellectual content; clinical studies; manuscript review. All authors read and approved the final manuscript.


The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP. It is also the most common known cause of autism (reviewed in [FMR1 (summarized in: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part-fragilex). Fragile X syndrome is the most common inherited cause of mental impairment accounting for iewed in –6). Otheiewed in , increasiewed in , increasiewed in , macroceiewed in . In addiiewed in –14. ThisFMR1 gene encodes the RNA-binding protein FMRP [The ein FMRP , a negatein FMRP –18 and pein FMRP , 20 tranein FMRP –25 and tein FMRP –35.via interaction with the 5′ end of the small noncoding RNA, BC1, and bound and repressed FMRP target mRNAs [general model of FMRP-mRNA interactions, was only one of several possible models [In 2003, Zalfa et al. described a bridging mechanism in which the fragile X mental retardation protein (FMRP) et mRNAs . This moet mRNAs , 37–39, et mRNAs  rather tet mRNAs , 41, 42 et mRNAs –45. In re models .More recent investigations of some of the concomitants of the Zalfa model  found th2, 400 mM NaCl and 0.2% SDS [2, and 150 mM KCl, 1 mM EDTA, and 1 mM DTT [2.Z-buffer is 10 mM Tris-HCl pH 7.5, 2 mM MgCl0.2% SDS . The RNA1 mM DTT . StructuDra I linearized pBCX607 containing the entire BC1 sequence [AvaII linearized pBCX607 containing the first 65 b of BC1, Sac I linearized pMK-1 containing the last 60 b of BC1 RNA, a PCR fragment encoding a T7 RNA polymerase promoter and the first 75 b of BC1 RNA, Hind III linearized pTAR encoding an 85 b transcript that folds into a 57 b TAR element and a 28 b leader sequence and linearized pTri-XEF1 encoding eEF-1A mRNA (Ambion) were used to produce biotinylated RNAs via in vitro transcription (Ambion). Plasmids pBCX607 and pMK-1 were provided by Dr. Henri Tiedge (SUNY Brooklyn). Alcohol-precipitated RNAs were dissolved in 50 μL DEPC-treated H2O and quantified spectrophotometrically. RNA integrity was examined by agarose gel electrophoresis. For examining the effect of annealing on RNA-protein interactions, individual RNAs were heated in 1x transcription buffer (Ambion) at 65°C for 10 min and then allowed to cool slowly to room temperature for one hour prior to their use. Note. We obtained identical results using Z-buffer in the annealing reaction (not shown).sequence , AvaII l35S-FMRP, 35S-FMRP280, 35S-FMRP204, 35S-FXR1P, 35S-FXR2P, 35S-eIF4A, and 35S-luciferase and were produced from plasmids pET21A-FMRP, pND-L-mHisFMRP, pET9-FMRP280, pET9-FMRP204, pHA-FXR1P, and pET21b-FXR2P in an RRL-coupled transcription-translation system (Promega). Plasmid pHA-FXR1P was provided by Dr. Gideon Dreyfuss (University of Pennsylvania); plasmids pET9-FMRP280 and pET9-FMRP204 were provided by Dr. Darryl Spinner (IBR), plasmid pET21b-FXR2P was a gift from Dr. Jennifer Darnell (Rockefeller University) and plasmid pET-His6-eIF4A was a gift from Dr. Henri Tiedge (SUNY Brooklyn). Briefly, 35S-labeled- proteins were produced by combining twenty five microliters of TNT rabbit reticulocyte lysate (RRL) with 2 μL of TNT T7 RNA polymerase, 1 μL of 1 mM amino acid mix minus-methionine, 35 μCi of 35S-methionine, 1 μL of RNasin and 1 μg of plasmid DNA in a total volume of 50 μL. One microliter of a 50X Complete protease inhibitor cocktail was added to prevent proteolysis. Samples were incubated at 30°C for 90 min and then assayed for protein production by autoradiography.Affinity capture assays were performed as described previously ; the bouAutoradiograms were quantified using UN-SCAN-IT Gel 6.1 . The percent binding was calculated asμg), were treated at room temperature for 15 min with various amounts of ribonuclease V1 (cobra venom) or ribonuclease A in structure buffer as indicated. Reaction products were resolved on 1-2% TAE agarose gels containing 0.1 μg/mL ethidium bromide along with appropriate size markers. Gels were imaged using a Scion CFW-1308 M mega pixel camera and captured in inverted mode using FOTO/Analyst PC Image software version 9.04 (FOTODYNE). The resulting image files were digitized and analyzed using UN-SCAN-IT Gel 6.1.Annealed and unannealed BC1 RNA, BC1 fragment RNAs and control RNAs .RNA lowest energy secondary structures were determined using the Zuker algorithm, M-fold  nonspecifically bound to the avidin affinity column; however, addition of increasing amounts of NaCl decreased this nonspecific interaction so that at 125 mM NaCl the amount of bound FMRP was between 5%–10% of the total. As expected, tRNA also blocked the association of full-length FMRP to the avidin column, We chose to examine heretofore unstudied aspects of FMRP's interaction with BC1 RNA-using affinity capture, one of the older techniques used in FMRP RNA binding studies. We next sought a basic buffer to use. In several previous publications, we have used a buffer described by Schaeffer et al. , which uin vitro and purified by salt and alcohol precipitation; then, it was either heated briefly or left untreated. Each RNA  was then was titrated with a constant amount of 35S-FMRP, and the binding was assessed by affinity capture. Under the conditions used, unannealed BC1 RNA bound extremely weakly at all concentrations examined, in concert with previous obtained results [Having established this basic set of conditions, we applied them to study the FMRP BC1 RNA interaction. The weak binding between FMRP and BC1 RNA that occurs in protein excess  suggeste results , 64. On 35S-FMRP, unannealed eEF-1A mRNA evinced saturable binding at 90 nM RNA, while it took 100-fold more annealed BC1 RNA to achieve comparable binding; see Figure  To index the above results to a known standard, the interaction between eEF-1A mRNA and FMRP was also measured in parallel. Previous work had shown that this RNA binds strongly to FMRP, without prior annealing , 41. As 35S-FMRP was not markedly affected by annealing.To determine whether annealing affected the interaction of FMRP with eEF-1A mRNA, binding to annealed and unannealed forms of the message was assessed using a subsaturating concentration of eEF-1A RNA. As shown in Figure  +2 the annealing reaction was also carried out in the presence of 2 mM MgCl2. The results indicated that the magnesium did not alter the distribution of BC1 RNA conformer(s) or affect their resistance to RNase V1 (not shown).The above data implied that the conformer populations of annealed and unannealed BC1 RNA differ. To determine whether this was so, the annealed and unannealed BC1 RNAs used in These data unequivocally demonstrate that the conformer populations of annealed and unannealed BC1 RNA differ, and this difference is due to an increase in the amount of stable RNA duplexes in the annealed RNA. Moreover, the data support the hypothesis that the binding between FMRP and annealed BC1 RNA results from a unique conformer that is absent from the unannealed BC1 RNA conformer population.BC1 RNA contains several distinct higher order structural elements. Its 5′ end contains two cis-acting spatial targeting elements, DTE1 and DTE2 . The for35S-FMRP was incubated with 2 μM annealed versions of each RNA; binding was subsequently assessed by affinity capture and compared to that of annealed full-length BC1 RNA. We found that the 5′ 75 bases of BC1 RNA bound slightly less, than full-length BC1 RNA, P = .15, ANOVA). Truncation of this RNA by a 15 base deletion at its 3′ end further decreased binding . Significantly, this 60 base RNA is not expected to recapitulate the folding of the first 60 bases of full-length BC1 RNA, We next examined whether FMRP preferentially bound either of the dissected RNAs. Therefore, Ex15c to bind 2 μM annealed BC1 RNA. Previous studies showed that full-length FMRP is readily methylated in its RG-rich region, while FMRP15c, which lacks 25 amino acids upstream of the RG-rich region, is refractory to arginine methylation [15c was less than it was to full-length FMRP, but the difference did not rise to statistical significance.Different preparations or sources of FMRP has been posited as a potential explanation for the divergent BC1 RNA-binding data obtained by different laboratories , 60 and hylation .  FigureμM annealed BC1 RNA was incubated with equimolar amounts of fragile X family members, 35S-FMRP, 35S-FXR1P and 35S-FXR2P (full-length forms); subsequently, binding was assessed as previously described. As shown in We next examined whether the binding between annealed BC1 RNA and FMRP was unique. As an initial indicator, 2 1–280 [1–280. As shown in 35S-FMRP1–280. To determine whether 35S-FMRP1-280 lacked the ability to interact with RNA, we asked whether it could to bind to homoribopolymers. As shown in 35S-FMRP1–280 was unable to bind to poly(rA), poly(rG), or poly(rI : rC). These data suggest that 35S-FMRP1–280 does not significantly interact with ribonucleic acids under the conditions used. Interestingly, however, while a shorter NTD, that is, 35S-FMRP1–204, was also unable to bind to annealed BC1 RNA, It has been previously proposed that BC1 RNA interacts with the N-terminal domain (NTD) of FMRP . Neverth1–280 . To compDisparate data obtained using electrophoretic mobility shift assays have been published concerning the binding of FMRP to BC1 RNA , 40, 47.ds) [We specifically developed these assay conditions, which do not utilize tRNA as a general nonspecific binding competitor, because it had previously been demonstrated that both tRNA and BC1 RNA bind to FMRP in a low salt buffer with nearly identical dissociation constants (Kds) , and becds) . Thus, tds) , 38, trads) , and itsds) , 19, 27 ds) . Here, wds) . Neverthds) , 40, 47;2 domain, preannealed the RNA before binding was initiated [To try to understand the result, we first determined whether enhanced binding due to annealing was a general feature of FMRP's interaction with RNA. A search of the current literature produced mixed results. Darnell et al., working with kissing complex RNA a small double hairpin with additional loop-loop tertiary interactions that binds to FMRP's KHnitiated . Likewisnitiated , 70. Finnitiated . In contnitiated , the FMRnitiated , eEF-1A nitiated , and BMPnitiated  do not rWe next determined whether annealed BC1 RNA was structurally altered from unannealed BC1 RNA. Indeed, we showed that the annealed form exhibited an increased susceptibility to cleavage by RNase VI. These data support the hypothesis that a particular BC1 RNA conformer; that is, one containing a unique doubled-stranded RNA structure was the true FMRP interactor. Functional dissection of BC1 RNA into either a 5′ ID element or a 3′ element demonstrated that the major determinant that FMRP binds to is located in the 5′ 75 bases of the molecule. As FMRP also binds to tRNA and TAR RNA, these data imply that FMRP can weakly recognize hairpin-containing RNAs. E. coli [Spodoptera frugiperda [E. coli [the determining factor in a putative FMRP BC1 RNA interaction. Precedence for this assumption may be seen in the work of Stetler et al. who showed that methylating FMRP decreased, but did not abrogate, the direct binding of two G-quartet RNAs to FMRP's RG-rich region [E. coli-purified FMRP [15c) was reduced compared to methylated FMRP, it was not a statistically significant reduction. Thus, methylarginine residues in FMRP's RG-rich region are not absolutely required for this interaction either. Another difference between the published FMRP BC1 RNA interaction studies was that one group used full-length recombinant FMRP produced from baculovirus , 40, whiE. coli . A potenE. coli , 31. Howugiperda , the hos[E. coli , 74. Thu[E. coli . Again, h region . Consistied FMRP  and withied FMRP , we obseWe also probed the exclusivity of FMRP's interaction with annealed BC1 RNA. Here, we found that interactions of similar magnitude occur with fragile X paralogs FXR1P and FXR2P and to a lesser extent, eIF4A. These results demonstrate that BC1 RNA binds indiscriminately to RNA-binding proteins. Interestingly, contrasting results were recently obtained for kissing complex (kc) RNA , where i1–280, a construct comprising both N-terminal residues and the first KH domain (KH1). The results were consistent with previous work showing that this construct did not interact with unannealed BC1 RNA [1–280 did not interact with other nucleic acids under these conditions, we could not rule out the possibility that the protein was mis-folded although previous physical evidence suggests this is not the case [1–204, a construct that does interact with nucleic acids and is expected “to be at least partially folded and be monodisperse” under the conditions it was used [1 domain [1 domain (residues 205–280) did not interact with BC1 RNA [As all three paralogs have highly homologous N-terminal sequences but are more divergent in their C-terminal ends , we inqu BC1 RNA . Howeverthe case . Therefowas used . Neverthwas used , are notwas used . The add1 domain . While i BC1 RNA . HoweverAlthough we demonstrate an in vitro interaction between FMRP and BC1 RNA, its nature and its physiological significance remain elusive. For example, the structure of the unique BC1 RNA conformer that FMRP binds to has not been defined other than the fact that it contains double-stranded RNA and is primarily found in the 5′ half of the molecule. In fact, it is possible based on the rather high RNA concentrations used in the annealing reaction that FMRP may be interacting with a dimer of BC1 RNA. This would be analogous to the formation of intermolecular G-quartet RNAs such as MAP1B RNA . Clearly


Bacillus subtilis and will describe their established roles in virulence in several Gram-positive pathogenic bacteria that are imposing major health concerns worldwide. Implications on bacterial evolution through stabilization/transfer of genetic material (phage or plasmid DNA) as a result of ribonucleases' functions will be covered. The role of ribonucleases in emergence of antibiotic resistance and new concepts in drug design will additionally be discussed.It is widely acknowledged that RNA stability plays critical roles in bacterial adaptation and survival in different environments like those encountered when bacteria infect a host. Bacterial ribonucleases acting alone or in concert with regulatory RNAs or RNA binding proteins are the mediators of the regulatory outcome on RNA stability. We will give a current update of what is known about ribonucleases in the model Gram-positive organism  Bacterial pathogens predominantly respond to environmental changes, such as entry into a host, by adapting their physiology through altered gene expression. The gene products that give a pathogen an enhanced chance of survival within the host are termed virulence factors. Pathogens use a variety of different mechanisms to regulate virulence gene expression. Besides transcriptional control, several post-transcriptional mechanisms have been well documented in the literature , 2. In tEscherichia coli and Bacillus subtilis have given a detailed knowledge about the mechanisms of RNA decay and maturation for Gram-negative and Gram-positive bacteria [A variety of posttranscriptional regulatory strategies involve RNases. The cell can directly control global RNA decay by adjusting the levels of RNases , 5. Studbacteria , 7. For bacteria . The advbacteria –11. AlthIn this paper, we will primarily focus on RNase-mediated regulation of virulence gene expression in medically relevant Gram-positive bacteria. Different mechanisms will be presented showing that RNases can either activate or repress gene expression. The involvement of RNases in bacterial antiviral defense, transfer of mobile genetic elements and persistence will be presented. We discuss the genomic context within which RNases are embedded and how the conservation of patterns amongst several genomes can give us insight into their expression. Perspectives on the design of new generation antibiotics targeting several components of the RNA degrading machinery will be underscored.B. subtilis. An exhaustive overview of these is beyond the scope of this paper and we refer readers to some high-quality review articles that cover this topic [B. subtilis for specific RNases where homologues in pathogens have been associated with virulence. RNases are broadly divided into two groups: (1) exoribonucleases, which degrade RNA substrates from either the 5′ or 3′end and (2) endoribonucleases that cleave internally within an RNA molecule. The orchestration of mRNA decay in Gram-positive bacteria by the concerted action of several RNases is illustrated schematically in  Decades of research have resulted in the identification/characterization of several RNases within is topic , 7. InstB. subtilis (ymdA) was determined to be involved with RNA processing and was renamed RNase Y [E. coli, the major single-strand specific endoribonuclease which initiates RNA processing and degradation [B. subtilis resulted in altered stability of polycistronic mRNAs required for biofilm formation [ymdB, which has been shown to be required for biofilm formation [E. coli RNase E and B. subtilis RNase Y are not evenly distributed in the cytoplasm but that a fraction is localized at the membrane [in vivo [ Recently, an essential gene within  RNase Y , 13. RNaradation . Interesormation , but thiormation . RNase Yormation , 15. Recmembrane , 18. Alt[in vivo . TherefoB. subtilis RNase J1/J2 have been the subject of intensive research recently. RNases J1 and J2 were first identified and characterized in B. subtilis as the component that endonucleolytically cleaved the thrS leader mRNA [B. subtilis demonstrated that only RNase J1 is essential for growth [B. subtilis elutes as both a homodimer and a tetramer [der mRNA . Earlierr growth . Size extetramer . RNases tetramer , 22. Addtetramer .in vivo [The exoribonuclease activity of RNase J2 has been shown to be significantly less efficient compared to RNase J1 . The 5′–in vivo . It has in vivo , 25. Thein vivo , 26. RNain vivo , 27  ions were found in a cleft between the β-lactamase and β-CASP domains. However, this structure, bound to a UMP substrate, appeared to be in a closed conformation that could not explain the 5′ monophosphate substrate preference nor did it reveal the mechanism for the dual-enzyme activities [β-CASP and β-lactamase domains. These movements result in a widening of the narrow catalytic cleft between the domains creating a channel that is wide enough for a single-stranded RNA to enter. The cleaved nucleotide is discharged from the other side through a negatively charged exit tunnel [β-CASP domain where specific loop regions are displaced by up to 12 Å to accommodate the substrate. The RNA binding pocket consists of positively charged residues that extend beyond the cleavage site, giving a rationale for the binding of longer RNA transcripts and the endoribonucleolytic activity. The recently solved structure and modeling of B. subtilis RNase J1 has revealed a similar pattern of conformational changes upon substrate binding [The first structure solved for RNase J1 was from mophilus . The protivities . The strtivities . The bint tunnel . The bin binding .B. subtilis, the nonessential multifunctional PNPase (77 kDa) is the major exonucleolytic RNase, that forms a trimer to catalyze the 3′ to 5′ phosphorolytic degradation of RNA [B. subtilis transcripts and the processivity was inhibited by the Not I sequence (GCGGCCGC) [ In n of RNA –31. Addin of RNA . The 3′ CGGCCGC) . Thus, il PNPase 7 kDa is B. subtilis RecN and this complex was able to degrade single-stranded DNA (ssDNA) in vitro [B. subtilis was identified [B. subtilis PNPase catalyzes template-independent polymerization of dNDPs onto 3′ ends of ssDNA [ It is interesting to mention some of the remarkable new functions discovered involving this versatile protein. Recently, PNPase was found to copurify with in vitro . The exoentified . Extendiof ssDNA . This woof ssDNA . Bacterial RNase III belongs to the Class I RNase III family of enzymes, while Classes II and III include the eukaryotic Drosha and Dicer, respectively, which are involved in biogenesis of siRNA/miRNA in higher organisms 2+-dependent double-strand-specific endoribonuclease capable of cleaving dsRNA. It recognizes a variety of structures such as imperfect duplexes, helices interrupted by bulged residues, kissing loops, and stacked helices [E. coli and B. subtilis [B. subtilis [E. coli RNase III [ RNase III is a Mg helices –44. Clea helices –7). RNassubtilis  as well subtilis –7, 46. Msubtilis –49. No cNase III .B. subtilis, RNase P has been shown to cleave precursor tRNAs and tmRNA forming the mature 5′ end and to cleave the adenine riboswitch which stabilizes the downstream mRNA transcript [ RNase P is a ribonucleoprotein particle that contains at least one protein subunit and a single-ribozyme subunit . In B. sanscript , 53. Theanscript . Crystalanscript  or boundanscript  reveal tanscript .E. coli, RNA pyrophosphohydrolase (RppH) removes a pyrophosphate from the 5′ end of RNA, converting the triphosphate into a monophosphate, a reaction that occurs before RNA degradation by RNase E [B. subtilis [B. subtilis. Bioinformatic searches identified six putative homologues with canonical Nudix hydrolase motifs. To identify the functional RppH, these proteins were expressed and assayed for the deprotection event in vitro. They identified an RppH-dependent B. subtilis transcript by constructing a ∆rrpH strain and screening for mRNAs with altered half-lives [ In  RNase E . The finsubtilis  inspiredsubtilis  to searclf-lives . Detailelf-lives .E. coli [in vivo interaction partners of glycolytic enzymes within B. subtilis, several proteins known to be involved with RNA processing and degradation were identified [S. aureus has been characterized and evaluated using the bacterial two-hybrid approach [B. subtilis multienzymatic complex, different partner interactions were described and additionally, the association of the protein subunit of ribonuclease P (RnpA) with CshA was demonstrated [The degradosome has been first discovered and characterized in E. coli –63. A deE. coli . While sentified . The prientified . Furtherentified . This heentified . Recentlapproach . Althougnstrated .B. subtilis. Using the multigenome alignment tool (http://www.biocyc.org/), the genomic context was aligned for several Gram-positive organisms . Some of the most noteworthy features are highlighted below.  The importance of genomic organization and operon structure for gene expression has been well documented . Often gpnpA, rnjB, rny) for the proteins involved in the degradosome are located in a relative close proximity to each other on the chromosome and genetic arrangements appear to have evolved in Gram-positive species  is the first gene of a bicistronic operon including ymdB which was recently shown to play a critical role in the bistable expression of genes involved in flagella and biofilm formation [rny, pgsA , cinA (competence-damage inducible regulator), and recA (member of SOS regulon) are relatively conserved among Gram-positive bacteria. The gene encoding CshA, the RNA helicase of the degradosome in B. subtilis [rnc) is transcribed as the first of a three-gene operon in B. subtilis, also encoding the essential smc (chromosome condensation and segregation ATPase) and ftsY  [The clustering of RNase genes, along with several genes for sporulation, cell wall peptidoglycan biosynthesis, motility, and cell division are also noteworthy. In al level . In B. sormation . Moreovesubtilis , is alsoarticle) . This orS. aureus [S. aureus RNases in stationary phase [Transcriptomic array data for . aureus , 75 revery phase , 75 appe Streptococcus pyogenes and Staphylococcus aureus. S. pyogenes  and S. aureus cause mild to systemic and life threatening diseases. The emergence of methicillin resistant S. aureus strains acquired both in hospitals and, in the community, has resulted in more deaths annually than HIV in the United States in recent years [ The role of several RNases in virulence has been recently studied in two major pathogenic bacteria,nt years , 77.sagA (streptolysin S), sda (DNase), and arc (arginine deiminase) have prolonged half-lives. It has been shown that the 3′–5′ exonucleolytic activity of PNPase is responsible for degrading substrates of class II after an elongated lag phase where mRNAs are stable [ GAS transcripts have been recently classified into two Classes, I and II, depending on their stability during stationary phase of growth . Class Ie stable . The inie stable , 80. It cvfA in Streptococcus and Staphylococcus, has been shown to affect virulence in both pathogenic bacteria in silkworm and murine models [S. pyogenes, a cvfA deletion affected expression of several virulence factors [S. pyogenes is strictly dependent on growth phase [S. pyogenes RNase Y interacts with the glycolytic enzyme enolase [S. aureus either through expression of the accessory gene regulatory locus, agr or independently [S. pyogenes and S. aureus [B. subtilis enzyme [The roles of ribonucleases in virulence gene expression are summarized in e models –83. In S factors . Moreove factors . These d factors  in agreeth phase . RNase Yth phase . Howeverth phase . S. pyog enolase  which mi. aureus , 83, sims enzyme .S. aureus to differentially express subsets of virulence factors according to stress and growth phase is largely attributed to mRNA stability [E. coli, Salmonella enteric, and several Yersinia species . In addiewed in . Intereson model .S. aureus RNAIII and other sRNAs [ska mRNA indicating that FasX-dependent stabilization is due to a limited access of RNases at the 5′-end [ska mRNA degradation, remains to be tested.The activation of an S. pyogenes mRNA encoding a virulence factor  depends on the presence of a sRNA, FasX . FasX bier sRNAs , 88. Dele 5′-end . WhetherClostridium perfringens, the VirR/VirS two-component system regulates several virulence genes including transcription of the 386 nts sRNA, VR-RNA [colA). Base pairing of VR-RNA with the 5′UTR of colA mRNA induces cleavage by an unknown RNase immediately downstream of the colA-VR-RNA duplex. This processing in turn leads to the formation of a shorter hairpin and increased mRNA stability in vivo. Mutational analysis of the RBS indicates that ribosome binding to the processed mRNA additionally stabilizes the colA mRNA [Within , VR-RNA . Recent , VR-RNA  has shedolA mRNA .ska and colA mRNAs [S. aureus, RNase III mediates transcript stabilization of cspA mRNA, encoding a cold-shock protein, through processing of a long hairpin structure in the 5′UTR. This processing leads to the formation of a short but stable hairpin that enhances the stability of the mRNA and its translation . Hence, the secondary structure of mRNA has an important role in controlling transcript stability as highlighted by the two examples for lA mRNAs , 90. IntEnterococcus faecalis is an opportunistic Gram-positive responsible for many nosocomial infections [E. faecalis rnjB, encoding RNase J2, is involved in the regulation of pilus gene expression and biofilm formation. Pili expression is important for pathogenicity of many Gram-positive organisms [rnjB results in destabilization of ebp operon transcript encoding the pilus proteins [fections . Recentlfections  showed trganisms . Inactivproteins . The mecS. aureus has been well documented. Specifically, in stationary phase, RNase III acts together with the agr encoded regulatory RNAIII [rnc strain was attenuated in a murine infection model, and that RNase III is involved in the regulation of extracellular protein secretion, such as the extracellular fibrinogen binding protein Efb in S. aureus [secY2 mRNA encoding a protein of the accessory secretory system. The effect of RNase III in virulence in y RNAIII  to reprey RNAIII , 71, 72.y RNAIII , 94–96, . aureus . This waListeria monocytogenes, translational regulation of lmo0850 mRNA mediated by the binding of LhrA sRNA is dependent on Hfq. The specific RNases involved in the degradation of the lmo0850 transcript still remain to be determined. Additionally, a lhrA deletion within L. monocytogenes altered the expression levels of approximately 300 genes, including chiA, a known virulence factor that encodes a chitinase [ In Gram-negative bacteria, Hfq has been shown to be a critical component of sRNA-mediated regulation . Evidenchitinase .S. pyogenes, which lacks three Cas proteins, Cse3 (CasE), Cas6, and Csy4. In these cases, the host-encoded RNase III mediates maturation of the pre-crRNA acting in concert with a trans-encoded RNA  and the endoribonuclease Csn1 [Listeria innocua, Neisseria meningitides, Streptococcus mutans, and Streptococcus thermophilus. Based on examination of several bacterial genomes, it was suggested that tracrRNA possibly co-evolved with the repeat sequences of crRNA [Recently, the role of RNase III in bacterial immunity against phages and plasmids has been demonstrated . CRISPR/ase Csn1 . Particuof crRNA .cis-acting regulatory RNA sequences that control expression of downstream genes by binding metabolites that induce structural changes to the transcript [Clostridium difficile. Binding of c-di-GMP to this RNA structural motif induces folding changes at the splice site causing a different splicing pattern. The binding of c-di-GMP results in the inclusion of a perfect RBS directly upstream of a nonconventional UUG translational start codon. Without the second messenger signal, c-di-GMP, the final spliced mRNA lacks the RBS and is not translated [Riboswitches are anscript . An inteanscript . In thisanslated . This woglmS ribozyme has demonstrated how the cell can sense nutritional status and modulate gene expression accordingly using catalytic RNA cleavage followed by RNase-mediated decay [B. subtilis, glucosamine-6-phosphate binds to the glmS ribozyme stimulating site-specific RNA self-cleavage in vivo. This cleavage results in transcripts that contain a 2′-3′ cyclic phosphate at the 3′ end and a hydroxyl group at the 5′ end. After cleavage, the downstream transcript is rapidly degraded. It was shown that targeting of glmS RNA for degradation was due to the 5′ hydroxyl end which was a substrate for the 5′–3′ exoribonuclease activity of RNase J1 [ The discovery of the mechanism for transcript destabilization induced by the ed decay . In B. sRNase J1 . This woE. coli hok/Sok and tisAB/IstR systems, RNase III is the key enzyme which degrades the mRNA-asRNA complex [E. coli MazF is an endoribonuclease cleaving mRNAs at a defined ACA consensus sequence independently of the ribosome, while RelE is a ribosome-dependent endoribonuclease that cleaves mRNAs positioned at the ribosomal A-site (reviewed in [in vitro and was capable of cleaving its inhibitory antitoxic sRNA [Toxin-antitoxin (TA) systems are continuously being discovered across bacterial species. Their involvement in phage resistance, plasmid maintenance, stress responses, and bacterial persistence is well documented . Th complex –114. Theiewed in , 114). Txic sRNA , 120. ThB. subtilis so far is ndoAI/ndoA where ndoA encodes the toxin , which cleaves at unpaired UACAU sequences, and ndoAI encodes the antitoxin [B. subtilis can be protective or lethal [B. subtilis was identified [txpA-RatA leads to degradation of the mRNA by an unknown RNase  isolated from patients [enterococci with plasmid stability. Moreover, the mazEF genes were located on the same plasmid with the vancomycin resistance gene cluster, vanA. Recently, the axe-txe system was identified in a plasmid from Enterococcus faecium, which also encoded multiple drug resistances. The system was expressed in clinical isolates and Txe was shown to have endoribonucleolytic activity in vivo [The identification of MazEF and to a smaller extent patients  demonstr in vivo . The pre in vivo  linking S. aureus and the unpaired UACAU recognition sequence for endoribonucleolytic cleavage was established [sraP mRNA which contains the consensus cleavage motif. The occurrence of the consensus sequence within coding sequences of other virulence factors was also demonstrated [spa and hla mRNAs [mazEF and sigB are transcriptionally linked, sigB expression is partially dependent on factors affecting transcription of mazEF such as heat-shock and antibiotic stresses [mazEF, therefore creating a feedback inhibitory loop that possibly affects its own expression [S. aureus [MazEF was also identified in ablished , 129. Inla mRNAs . As mazEstresses . SigB wapression . These f. aureus . Recentl. aureus . TherefoE. coli MazF cleaves preferentially ACA sequences in mRNAs located close by to the AUG codon thus generating leaderless mRNAs [Interestingly, it was recently shown that ss mRNAs . In addiss mRNAs . WhetherMycobacterium tuberculosis is a major worldwide health problem and in 2009 approximately 1.7 million people were estimated to have died from tuberculosis . Bioinformatic analyses revealed that M. tuberculosis encodes 88 putative TA systems, 30 of which were shown to be functional [M. tuberculosis complex (MTBC) while they were absent from closely related species. This implies that they were acquired after the speciation event and that they probably play a significant role in pathogenicity. The VapBC is the most abundant TA module in M. tuberculosis represented by 47 members. Subsets of VapBC modules were shown to be toxic when expressed and conversely this toxicity was counteracted by coexpression of the cognate VapB antitoxin [in vitro and this may account for the inhibition of translation observed in vivo which profoundly affects gene expression in response to different environments and stresses [nctional . Remarkantitoxin , 136. Vastresses , 136. Cestresses . Previoustresses ). Hence,fMet between the anticodon stem and loop. Cellular depletion of tRNAfMet had a bacteriostatic effect on cultures and production of VapB allowed cells to resume growth. The depletion of tRNAfMet by VapC not only inhibited cell growth but was additionally found to activate initiation of translation at correctly positioned elongator codons. The authors further speculated that this mechanism has the potential to translate reading frames that were normally silent. This work in enteric bacteria has significant implications for virulence in Gram-positive organisms, particularly M. tuberculosis where VapBC modules are highly represented [2−) and singlet oxygen, which can be lethal to cells [fMet, would reduce the occurrence of toxic events and enhance survivability of the organism.Interestingly, Winther and Gerdes  have shoresented , 136. Thto cells . A mechaE. coli was recently reported [E. coli resulted in a dramatic decrease in formation of persisters [M. tuberculosis revealed overexpression of TA systems [ Bacterial persistence is a phenotype where part of the cell population enters a dormant nongrowing state which in turn confers resistance to antibiotics and other stresses . The invreported . The autrsisters . Consist systems .Erwinia carotovora but homologues are found in the genomes of several Gram-positive and Gram-negative pathogenic bacteria [hok/Sok (type I), MazEF (type II) systems have also been shown to confer phage resistance to their hosts (reviewed in [The importance of the CRISPR systems in antiphage resistance was discussed earlier. Abortive infection (Abi) is mediated by the type III ToxIN systems and constitutes another mechanism by which bacteria defend against phages . During bacteria . The hokiewed in , 118). TE. coli pnpA mutant cells in exponential phase of growth. It was proposed that PNPase protects sRNAs from degradation mediated by RNase E [S. aureus RNase III has been shown to initiate the decay of mRNAs repressed by the quorum sensing dependent RNAIII [RNases often act in concert with sRNAs and/or RNA binding proteins. Interaction with a sRNA can lead to occlusion of the ribosome binding to repress translation. Often an RNase is recruited to the site of interaction to degrade the mRNA making the regulation irreversible. It has also been proposed that in the absence of ribosome binding, the mRNA becomes more exposed to the action of RNases so that repression of translation can lead to rapid degradation , 5. Ther RNase E . While St RNAIII , little E. coli RNase E was recently reported where the enzyme acts at a distance cleaving further into the coding sequence [sodB mRNA through association with Hfq and the sRNA RyhB which in turn occluded ribosome loading onto the RBS. When the mRNA was stripped of translating ribosomes, RNase E cleavage sites present within the coding sequence were exposed to the endonucleolytic activity of RNase E. Whether a similar mechanism exists in Gram-positive bacteria, remains to be elucidated.In the cases described so far where regulation of mRNA stability occurs through the combined action of a sRNA and a ribonuclease, the initial cleavage site is located within the mRNA-sRNA duplex as it is the case for RNase III  or proxisequence . RNase ESinorhizobium meliloti, SMc01113/YbeY, which shares structural similarities with the MID domain of the Argonaute (AGO) proteins. Deletion of this protein induces pleiotropic effects as found for Hfq. Moreover, the protein regulates the accumulation of sRNAs and mRNAs similarly to Hfq [B. subtilis, three small basic proteins were proposed to act as RNA chaperones acting in concert with the sRNA FsrA, to promote degradation of transcripts encoding iron-using proteins under conditions of iron deprivation [Hfq, although a major player in sRNA-mediated degradation in Gram-negative bacteria , does noy to Hfq . This prrivation .Hence, these examples illustrate the important role of RNases in mRNA turnover and gene regulation and show that repression of translation mediated by sRNA  is often subsequently followed by mRNA degradation. All these regulatory events involve the catalytic activity of the enzymes. However, one cannot exclude that RNases might also regulate gene expression solely through their RNA binding activity. Notably, the dsRNA binding activity of RNase III has been reported to promote translation of lambda phage cIII RNA .csdA mutant [csdA, becomes incorporated in the RNA degradosome complex in E. coli after cold shock [Caulobacter crescentus, the Krebs cycle enzyme aconitase was found associated with the degradosome as opposed to the glycolytic enzyme enolase that is found in E. coli [E. coli [E. coli and that this enhances PNPase activity [ The activity of RNases or other enzymes of the degradosome can be modulated. The first evidence of a modified degradosome came during a screen for suppressors of a cold-sensitive phenotype in a A mutant . It was ld shock  and it c E. coli . Binding[E. coli . It was activity . Within activity . AltogetE. coli PNPase synthesis is autoregulated at the posttranscriptional level by an RNase III-dependent mechanism [E. coli RNase III [ Moreover, several examples have been highlighted where the activity of RNases can be cross-regulated. For instance, echanism . In factechanism –158. MorNase III . One canNase III , 78. Sur This paper highlights the importance of RNases in gene expression in several Gram-positive pathogenic bacteria. Furthermore, we have illustrated by several examples the roles of specific RNases in the regulation of the expression of mRNA targets involved in virulence. However, the full repertoire of targets for RNases, and the roles of accessory proteins  in gene regulation still need to be assessed, especially in Gram-positive bacteria. Without doubt our knowledge will greatly advance from current methodologies such as deep sequencing and tiling arrays where the full RNome of a bacterium can be evaluated in wild-type and mutant backgrounds. Moreover, high-throughput sequencing combined with crosslinking immunoprecipitation (HITS-CLIP) has proven valuable for identifying RNA-protein interactions , 161. ThS. aureus and against other Gram-positive pathogens [ The emergence of antibiotic resistances amongst pathogenic bacteria urgently necessitates the discovery of novel drugs. Several possibilities of targeting components of the RNA decay machinery in the design of novel drugs have been reported. Recently, an inhibitory compound of RnpA was identified that proved to be effective against several strains of MRSA, biofilm associated athogens . Despiteathogens , 163. TAathogens . The emeathogens  which caathogens . Since TS. aureus RNase III. The enzyme eliminates antisense RNA production by specific processing of sense-antisense complexes.A recent study by Lasa et al.  revealedrnpA, rnc, rnjA/rnjB, pnpA, rny as well as the pyrophosphohydrolase rppH and the helicase cshA is shown for several major Gram-positive pathogenic bacteria, namely, Listeria monocytogenes, Staphylococcus aureus, Mycobacterium tuberculosis, Enterococcus faecalis, Clostridium difficile, and Bacillus anthracis.In the supplementary material, the genomic context for the genes encoding the ribonucleases Click here for additional data file.


Kinesin-1 protein is able to cross-link anti-paralleled MTs in vitro. We propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain.In neurons, microtubules (MTs) span the length of both axons and dendrites, and the molecular motors use these intracellular ‘highways' to transport diverse cargo to the appropriate subcellular locations. Whereas axonal MTs are organized such that the plus-end is oriented out from the cell body, dendrites exhibit a mixed MTs polarity containing both minus-end-out and plus-end-out MTs. The molecular mechanisms underlying this differential organization, as well as its functional significance, are unknown. Here, we show that kinesin-1 is critical in establishing the characteristic minus-end-out MT organization of the dendrite in vivo. In DOI:http://dx.doi.org/10.7554/eLife.00133.001 Neurons, or nerve cells, are excitable cells that transmit information using electrical and chemical signals. Nerve cells are generally composed of a cell body, multiple dendrites, and a single axon. The dendrites are responsible for receiving inputs and for transferring these signals to the cell body, whereas the axon carries signals away from the cell body and relays them to other cells.Like all cells, nerve cells have a cytoskeleton made up of microtubules, which help to determine cellular shape and which act as ‘highways' for intracellular transport. Microtubules are long hollow fibers composed of alternating α- and β-tubulin proteins: each microtubule has a ‘plus'-end, where the β subunits are exposed, and a ‘minus'-end, where the α subunits are exposed. Nerve cells are highly polarized: within the axon, the microtubules are uniformly oriented with their plus-ends pointing outward, whereas in dendrites, there are many microtubules with their minus-ends pointing outward. This arrangement is conserved across the animal kingdom, but the mechanisms that establish it are largely unknown.Caenorhabditis elegans (the nematode worm) to conduct a detailed in vivo analysis of dendritic microtubule organization. They find that a motor protein called kinesin-1 is critical for generating the characteristic minus-end-out pattern in dendrites: when the gene that codes for this protein is knocked out, the dendrites in microtubules undergo a dramatic polarity shift and adopt the plus-end-out organization that is typical of axons. The mutant dendrites also show other axon-like features: for example, they lack many of the proteins that are usually found in dendrites. Based on these and other data, Yan et al. propose that kinesin-1 determines microtubule polarity in dendrites by moving plus-end-out microtubules out of dendrites.Yan et al. use the model organism These first attempts to explain, at the molecular level, how dendritic microtubule polarity is achieved in vivo could lead to new insights into the structure and function of the neuronal cytoskeleton.DOI:http://dx.doi.org/10.7554/eLife.00133.002 Neurons are highly polarized cells that usually elaborate two sets of morphologically and functionally distinct processes: a single long axon that sends out information to its targets, and multiple shorter dendrites that receive synaptic input from the environment or other neurons. Among the many differences between axons and dendrites, one distinct feature is that they have different microtubule (MT) organization. MTs, composed of strands of tubulin polymers, are dynamic structures with two molecularly and functionally distinct ends: a plus-end that favors polymerization and supports net growth and a minus-end that favors depolymerization . Since MDrosophila sensory neuron axons  that harbors the minus-ends, the majority of neuronal MTs have ‘free-floating' ends, raising the question of whether these microtubules are locally polymerized or transported from the cell body . Based oon axons . In dyne neurons . In unc-unc-116 (kinesin-1/kinesin heavy chain) mutants, we find that dendritic MT polarity is completely reversed and adopts an axonal-like plus-end-out organization. The consequences of this polarity reversal in the dendrite include ectopic accumulation of synaptic vesicles (SVs) and active zone proteins, and loss of dendritic enrichment of dendritic proteins. These results demonstrate that the proper polarized MT organization is essential for conferring neurite identity because it specifies the compartmental distribution of vital axonal and dendritic constituents such as SVs and neurotransmitter receptors in vivo. We also provide evidence that kinesin-1 is able to cross-link anti-parallel MTs in vitro. Combined with the structure–function analyses, we propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain.The signature pattern of MT polarity provides structural characteristics for axons and dendrites. In addition, the polarity pattern of MTs likely instructs the transport of motor-based polarized cargo and distribution of asymmetrical cellular material in the neuron. Thus, MT polarity might play an instrumental role in the establishment of neuronal polarity . Here, wC. elegans. The ventrally localized DA9 cell body elaborates two functionally distinct processes. An axon migrates to the dorsal nerve cord, where it extends anteriorly and forms presynaptic specializations, and a dendrite extends anteriorly along the ventral nerve cord  mutants, UNC-104::YFP also accumulates in the tip of dendrite in addition to its normal localization at the axonal tip   is viable, the e2310 allele likely represents a partial loss-of-function mutant. Two additional partial loss-of-function alleles of unc-116, wy270 and rh24sb79, show similar mislocalization of MT motors in the DA9 dendrite, suggesting that this phenotype is indeed due to loss of the protein activity (unc-116(e2310) mutants rescued the dendritic distribution phenotype of KLP-16, indicating that UNC-116 functions in the DA9 neuron (UNC-116 encodes the sole  or KHC) . Since cactivity . Consist9 neuron . Taken tunc-116 animals. In wild-type animals, DA9 axonal outgrowth and neuromuscular junction formation occur embryonically, whereas most dendrite outgrowth takes place postembryonically starting from the L1 stage (unc-116(e2310) mutants, the sequence and directionality of axonal and dendrite outgrowth as well as the length of the axon are indistinguishable from that of the wild-type strain. The length of the DA9 dendrite, however, is dramatically longer compared with wild-type animals at every stage of development (unc-116(e2310), we examined localization of synaptic vesicle (SV) markers. The UNC-104/Imac/kinesin3 motor traffics SVs to presynaptic specializations (unc-104(e1265) mutants, SV markers are completely absent from the axon, accumulating instead in the cell body and dendrite due to the activity of dynein (unc-116(e2310) mutants, both the axon and dendrite contain a similar number of vesicle clusters (unc-104(e1265);unc-116(e2310) double mutants, SVs are largely absent from both processes and trapped in the cell body. These results argue that UNC-116 is not directly involved in SV transport in the DA9 neuron; rather, it creates the minus-end-out MT polarity of the dendrite. As a result of this aberrant MT polarity, the plus-end motor UNC-104 promotes locomotion of cargoes into the dendrite. If our model is correct, the unc-116 mutations should not only affect RAB-3 but also other synaptic vesicle proteins and active zone proteins. Indeed, the active zone protein SYD-2/liprin and other SV markers like SNG-1/synaptogyrin show similar ectopic localization to DA9 dendrites in the unc-116(e2310) mutants, suggesting that the changes in MT polarity affect the entire presynaptic structure , a receptor tyrosine kinase (CAM-1), and a gap junction protein (FBN-1) are all reported to be dendritically enriched in DA9 (unc-116(e2310) animals, however, these dendritic proteins accumulate within the cell body and largely disappear from the dendrite, suggesting that unc-116 is required for dendritic localization of postsynaptic proteins , adaptors, and cargos . Interesribution . The lowThe ability of UNC-116 to slide anti-parallel MTs requires that the motor have two distinct binding sites for MTs. Indeed, the C-terminal domain of kinesin-1 has been shown to bind to MTs in addition to the N-terminal motor domain . To furtunc-116 mutants, all of which show MT polarity defects  might specifically lack its ability to bind to MTs with the C-terminal motif. We tested whether this form can still rescue the dendrite polarity phenotypes as well as phenotypes related to the canonical transport function of UNC-116. To assess the canonical organelle transport function of UNC-116, we examined localization of mitochondria in DA9. In wild-type animals, a mitochondrial marker TOM-20::YFP localizes to discrete puncta in both the axon and dendrite of DA9 (unc-116(e2310) mutants, the TOM-20 signal is completely absent from both processes and only found in the cell body  mutant, suggesting that this length phenotype is independent of the MT polarity.Second, we examined the molecular lesions in three  defects . The molg domain . Point mg domain . Therefoe of DA9 . In unc-ell body , suggestlization . This reklc-1 or klc-2 single mutants nor klc-1; klc-2 double mutants showed any defects in MT polarity markers  animals, axon expression of GFP::TBA-1 remains largely unchanged, whereas expression in the dendrite increases, with a dendrite/axon intensity ratio around 1.2, suggestive of excessive MTs in the unc-116 mutant dendrites  dendrite  III, RB1975 klc-1(OK2609) IV, and CB1265 unc-104(e1265) II. TV6687 unc-116(rh24 sb79) III was a kind gift from Dr. McNally at University of California at Davis, TV3706 unc-116 (wy270) III, carrying a single L129 to F mutation in motor domain,  was previously isolated from our laboratory.Worms were raised on NGM plates at 20°C using OP50 Podr1::RFP or GFP (40 ng/μl): wyEx3892 [Pitr1::dhc-1::GFP], wyEx 2559 [Pitr1:: unc-104::YFP], wyEx3128 [Pitr1::klp-16::YFP], wyIs674 (Pitr1::klp-16::YFP), wyIs309 (Pmig13::klp-16::GFP), wyEx4974 [Pida-1::unc-104::GFP], wyEx4980 [Pida-1::klp-16::GFP], wyEx4978 [Pida-1::GFP::rab-3], wyEx4828 [Pdes-2::ebp-2::GFP], wyIs349 (Pmig13::loxP-ebp-2::GFP), wyIs85 (Pitr1::GFP::rab-3), wyEx2505 , wyIs386 (Pitr1::acr-2::GFP), wyEx1902 [Pitr1::mCherry], wyEx403 [Pitr1::cam-1::YFP], wyEx2396 , and wyEx2709 [Pitr1::tom-20::YFP]. Detailed subcloning information will be provided on request.Expression clones were made in the pSM vector, a derivative of pPD49.26  with extra cloning sites . The plasmids and transgenic strains (1–50 ng/μl) were generated using standard techniques and coinjected with markers C. elegans using a Plan-Apochromat 63×/1.4 objective on a Zeiss LSM510 confocal microscope system. Visual inspections and some quantification were done using a Zeiss Axioplan 2 microscope with Chroma HQ filter sets for GFP, YFP, and RFP (63×/1.4NA objective). Worms were immobilized using 10 mM levamisole  and oriented anterior to the left and dorsal up.Images of fluorescently tagged fusion proteins were captured in live Dynamic imaging was performed on an inverted Zeiss Axio Observer Z1 microscope equipped with the highly sensitive QuantEM:512SC camera. A Plan-Apochromat 63×/1.4 objective was used for acquisition. L4 worms were cultured at 22°C for imaging. They were mounted onto 2% agarose pads and anesthetized with 6 mM levamisol (Sigma) for no longer than 20min. All videos were acquired over 25 s with 8 frames per second. Videos were then analyzed using the ImageJ software.Tubulin was purified from porcine brain and polymerized to microtubules by a previously described method, then stabilized by taxol . Convent4, 1 mM EGTA, pH 6.8) containing with 10 μM taxol. After the excess solution was blotted with a filter paper, the specimen was stained with an equal volume of 2% uranyl acetate for 40 s. The specimens were observed with a transmission electron microscope, Tecnai Spirit (FEI Co.). The microscope was operated at 120 kV. The magnification for overviewed images was ×21,000 and the nominal magnification was ×67,000. The defocusing values were usually −1 to −1.5 μm. The images were recorded using CCD  at 30 μm/pixel corresponding to 0.47 nm. The electron dose per single image was estimated as 10 e/Å2.Taxol-stabilized MTs (25 μg/ml) with/without kinesin-1 (85 μg/ml), NudC (50 μg/ml), and 2 mM AMP-PNP were mixed in a test tube according to the condition then sit for 10 min. The kinesin-1 and NudC were mixed at a molar ratio of 1:5. The mixed solution of 5 μl was loaded on a hydrophilized carbon grid  to sit for 60 s in a humid chamber. Unabsorbed protein was rinsed by BRB80 buffer  and blotted with filter paper to remove excess solution and make a thin aqueous layer. The grid was then plunged into ethane slush at −185°C to create a thin layer of vitreous ice. The frozen-hydrated specimens were examined at liquid nitrogen temperature using a cryo-holder  with an EF-2000 microscope  operated at 200 kV. The electron micrographs were recorded using a 2 k × 2 k CCD camera developed by TVIPS  with an The defocusing values of all obtained images were estimated by ‘ctfDisplay'. The images were then corrected by phase flipping after the phase contrast transfer functions (CTF) were determined from the defocusing values. Corrected images were processed by the band-pass filter (1/100 nm to 1/10 nm), and so their background noise was reduced. The polarity of microtubules was determined by checking their Moiré patterns. Most of the image processing was performed by Eos . review process). 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  in the kinesin-1 mutant is due to the microtubule transport function of kinesin-1, which might selectively transport minus-end microtubules to the distal end of the dendrite. The reviewers propose that, based on the plus-end direction of kinesin-1 transport, an equally likely interpretation of the authors' data is that kinesin-1 may function to promote transport of plus-end microtubules out of the dendrite into the cell body. The authors conclude the reverse . The authors should consider this alternative interpretation of their results that kinesin-1 moves plus-end distal MTs out of the dendrites by using their plus-end directed motor activity to transport plus-end MT towards the cell body, that is out of the dendrite In the same vein, their conclusion schema  is a bit confusing in this regard: in most of the paper, the authors represent minus-end microtubules distal in dendrites and the circle represents the cell body. In Figure 6G the circle indicates the distal end of the dendrite. The authors should make sure this is not a mistake. They should try to make clearer the difference between ‘long minus end out' MTs and the fragment/transported plus-end out MTs (maybe using a different color). The authors must discuss how they envision that kinesin-1 can establish minus end-out microtubules. If this motor were to slide microtubules along each other in dendrites, all of the microtubules would eventually collapse into the cell body. Finally, can the authors comment on the localization pattern of kinesin-1 in DA9? Is it found in the dendrite as the model in Figure 6G predicts?Unc-116: the authors should be more accurate when they describe the e2310 allele of Unc-116 (kinesin-1). For example, they mention that this allele ‘…likely represents a partial loss of kinesin activity'. But then they mention the study from Patel and colleagues, suggesting that e2310 mutation affects a residue at the ‘…C-terminal end of kinesin-1 adjacent to the MT binding motif'. This is unlikely to affect kinesin-1 motor activity as suggested earlier. The authors argue that the C-terminal domain of unc-116 is likely to bind microtubules based on sequence similarity (but not identity) of the MT binding domain to homologs, but whether the C. elegans sequence binds to MTs is not known. The major model presented by the authors will be strengthened by these data if the resources are available to perform this work. Finally, does unc-116-MT rescue the dendrite elongation defect of unc-116 mutants? This experiment is relevant as the authors indicate in the Discussion that MT polarity appears to affect the length of the dendrite, when this could in principle be due to organelle trafficking roles.2. More experiments on 3. More quantification: while the data are generally high quality, certain arguments would be strengthened by additional quantification, for example quantification is lacking from main Figures 1, 4, and 5.Kapitein and Hoogenraad, Mol Cell Neurosci 2011. The authors should improve their Discussion and quoting of the literature. For example, there are several more recent reviews with more relevant information than the ones cited . This has been very nicely reviewed in unc-116 animals the dendrite takes on a longer, more axonal like configuration with reversed MT polarity.5. It seems that dendritic outgrowth of the DA9 neuron occurs during post-embryonic development, suggesting that microtubule polarity of the dendrite is set up at L1. One reviewer feels it would be appropriate to demonstrate microtubule polarity via (+) and (-) end markers and the establishment of longer dendrites at L1 through adult stages to further support their hypothesis that in [Editors'note: during revisions the editors made the following suggestion.]The reviewers agreed that it would be beneficial to re-include the data regarding the dendrite elongation phenotype that you removed during the first revisions:“[We] raised the issue of the basis for the dendrite elongation phenotype, which the authors have sorted out, but [we] felt that entirely removing the dendrite phenotype is not the best solution in the revision since the paper now refers to a lack of all other morphological defects , but awkwardly omits dendrite length. The basis of this phenotype appears complex, yet [we] feel the authors should find a way to include at least the basic observation.” We have followed the suggestions of the reviewers and performed additional experiments. The most constructive suggestion from the review was that our genetic data could be interpreted by two different models. One model is the one that we had originally presented: Kinesin-1 shuffles minus-end-out MTs into the dendrite. The alternative model is that Kinesin-1 shuffles plus-end-out MTs out of dendrite into the soma. We have performed two experiments to distinguish these two models. The new results favor the alternative model proposed by the reviewers. Accordingly, we have changed the manuscript to reflect these results. We have also included new in vitro data that directly support our model.Since the in vitro experiments we added were not requested by the reviewers, we will describe them first before providing a direct reply to all the questions.The genetic results we showed were consistent with the model that UNC-116/Kinesin-1 establishes the minus-end-out MT polarity in dendrites. Through structure-function analysis, we propose that kinesin-1 potentially binds to MTs with its C-terminal tail domain and slides anti-parallel MTs against each other. To directly test if Kinesin-1 can crosslink anti-parallel MTs, we collaborated with Dr. Hirotsune's laboratory, who performed in vitro MT cross-linking assays with the kinesin-1 complex. Consistent with previous literature, they found that kinesin-1 could cross-link MTs. They further used cryo-EM to determine the polarity of the cross-linked MTs. They found that the majority of MTs are of anti-parallel polarity . While these experiments were performed with vertebrate proteins, we feel that it provided valuable support for our model. We therefore included the data in the revised manuscript.We want to be upfront about the low case number for the EM reconstruction of the MTs in this analysis. Since the assay is incredibly time consuming, we could only complete the reconstruction of 13 pairs of MTs, out of which 9 pairs are anti-parallel and 4 pairs are parallel. While the anti-parallel MTs accounts for the majority of the cases, it does not reach statistical significance based on these numbers. We are open to not including these data if the reviewers and editors advise us to do so.Below is the point-by-pointreply to the questions raised by the reviewers.1) Interpretation of the data.We would like to thank the reviewers for coming up with this alternative model. Our original model is that “Kinesin-1 slides minus-end-out microtubules (MTs) into dendrites”. The alternative model states that “Kinesin-1 slides plus-end-out microtubules out of dendrites”.unc-116 mutants, the net number of MTs should decrease compared to the wild-type animals. On the contrary, the alternative model would predict that in unc-116 mutants, the net number of MTs should increase. We used two methods to measure the relative concentration of MTs in dendrites.The results we presented in the original manuscript are consistent with both models. Therefore, we performed two experiments to distinguish the two models. We reasoned that if the original model is correct, in unc-116 mutants show significantly higher MT abundance compared to the wild type .First, we used a GFP::tubulin (GFP::TBA-1) transgene to assess the overall level of MTs. Since the absolute fluorescence intensity is affected by the expression level of the transgenes, we used the ratio between the dendrite and axon fluorescence as a measure for the relative enrichment of MTs in the dendrite vs. axon. We found that unc-116 mutant dendrites have three times higher frequency of EBP-2::GFP comets compared to the wild-type controls. No difference was found in the axons between mutant and wild type.Second, we analyzed the frequency of the EBP-2::GFP comets as a measure of the number of dynamic MTs in DA9 dendrites. Interestingly, we again found that unc-116 mutants, plus-end-out MTs accumulate in the dendrite and lead to the erosion of polarity.Together, both experiments support the alternative model and suggest that Kinesin-1 regulates the minus-end-out MT polarity in the dendrite by specifically transporting plus-end-out MTs out of the dendrite. In the Regarding the conclusion schema , the symbol was indeed a mistake. We apologize for that and we have corrected it. We have also distinguished the longer MTs and shorter transporting MTs with two different colors as the reviewers suggested. We envision that the kinesin-1 slides plus-end-out MTs out of the dendrite to create the dominant polarity of minus-end-out MTs in the DA9 dendrite. We envision that this function must be regulated. From the EBP-2::GFP experiments, it is clear that a small percentage of MTs are plus-end-out even in the wild type dendrites.We have also created UNC-116::GFP to examine the localization of this motor in DA9. Similar to the UNC-104::GFP construct, we found that the vast majority of fluorescence is found at the tip of the axon, again supporting the plus-end-out MT polarity in the axon. We do not find a high level of UNC-116::GFP in the dendrite as expected. However, these experiments are not perfect since they detect transgenically expressed UNC-116. In this case, even antibody staining will likely not be useful, as it will lose the cellular specificity attained by the DA9 dendrite being tightly associated with the ventral nerve cord. It is therefore impossible to differentiate the potential signal from DA9 and those from the many other neurites. One related note is that kinesin-1 was found in the vertebrate dendrites .2) More experiments on Unc-116.e2310 allele and confirmed the previously reported TC5 transposon insertion. The transposon was inserted into the C-terminal coding region of unc-116, which results in a premature stop codon after residue 692, which truncates the potential MT-binding domain and the very C-terminal IAK motif of kinesin-1/UNC-116. The IAK motif was shown to be important for regulating the motor activity of kinesin-1 . So we anticipate that e2310 allele will cause impaired motor activity.We have sequenced the In order to test whether the putative MT-binding domain of UNC-116 is able to bind MTs, we expressed GFP-tagged UNC-116(aa521-863) in the HEK 293T cells and co-stained with MTs. As shown in the following figures, GFP-UNC-116(aa 521-863) co-localized with MTs (Author response image 1). Similar experiments were used to demonstrate that the vertebrate kinesin-1 counterpart can directly interact with MTs . Importantly, a mutant form of this domain that includes three R to A mutations showed no colocalization with MT markers. The same mutations when introduced in the vertebrate Kinesin-1 domains also diminish the interaction between MT and the C-terminal MT binding motif . Together, these results further support our model.unc-116. In our original submission, we showed that in unc-116 mutants, the DA9 dendrite exhibits axon-like MT polarity and is also significantly longer than the wild type dendrite. The reviewers asked us to test whether the increased length is caused by the change of MT polarity. Through structure-function dissection of UNC-116, we identified a mutant form of UNC-116 which is defective in the putative novel MT sliding function, yet appears to maintain other vesicular trafficking functions as measured by the localization of mitochondria . When expressed in DA9, this mutant UNC-116 (UNC-116 MT) failed to rescue the MT polarity phenotype, but fully rescued the dendrite length phenotype, indicating the longer dendrite is not a consequence of MT polarity change. Consistent with this notion, we examined the dendrite length of another mutant wy774, which we have isolated from our forward genetic screen. This mutant exhibits similar MT polarity phenotype as the unc-116 mutants but is mapped to a different genomic locus . Interestingly, this mutant shows normal dendrite length, further suggesting that longer dendrite in the unc-116 mutant is not a consequence of MT polarity change but likely due to other functions of UNC-116/kinesin-1. In the light of these new results, we have removed the data and discussion on dendrite length in the revised manuscript.We want to thank the reviewers for raising questions about the dendrite length phenotype in 3) More quantification.We have included quantifications in Figures 1, 4  and 5 .4) One reviewer felt the authors were potentially misleading in the title and abstract by not indicating that they have investigated this in worms, rather than in mammals […].We have clarified the organism in the title and abstract. We have modified our introduction on the dendrite MT polarity, cited a number of recent reviews, and included discussion on kinesin-2.5) [It] would be appropriate to demonstrate microtubule polarity via (+) and (-) end markers and the establishment of longer dendrites at L1 through adult stages […].unc-116 mutants. The results are summarized below and shown in Author response image 2.We agree with the reviewers that this is an important issue. Using +end and –end markers as suggested by the reviewers, we examined the DA9 dendrite polarity at different developing stages in both wild type and 1) In wild type L1s (10 hour after hatching) animals, (-) end marker KLP-16::GFP is localized to the dendrite, but does not show the dramatic enrichment at the tip of the dendrite found in adults. At the same stage, (+) end marker UNC-104::GFP avoids the dendrite and is dramatically enriched at the tip of the DA9 axon. These results suggest that in wild type young animals, the dendrite likely already contains mix-polarized MTs but the minus-end-out MTs are not as dominant as in adult dendrites. We looked hard for any evidence that would suggest that very young dendrites bear axonal-like polarity, a phenotype that was observed in the stage 2 neurons in dissociated vertebrate cultures. However, we did not see any UNC-104::GFP fluorescence in the dendrites of wild-type animals at any developmental stages. We therefore conclude that as soon as the dendrite grows out, it has adopted a mixed MT polarity.2) at 20 hours after hatching, wild type DA9 dendrite showed dramatic enrichment of (-) end marker KLP-16::GFP at the tip of the dendrite, suggesting that the minus-end-out MTs have become dominating species by this time point.unc-116 mutant dendrites showed the same distribution of (-) end marker and (+) end marker as in the wild type. In other words, unc-116 mutant did not show a phenotype at this very early stage of dendrite development.3) at 10 hour after hatching, unc-116 mutant dendrite completely fails to enrich the (-) end marker KLP-16::GFP at its tip; instead, it enriches the (+) end marker UNC-104::GFP at its tip, suggesting a polarity reversal starting at this stage.4) at 20 hours after hatching, the Taken together, these results suggest that the dendrite starts out as a process with a mixed MT polarity . With time, MTs becomes more dramatically polarized with minus-end-out being the dominating species.UNC-116/Kinesin-1 likely functions in the maturation stage to establish and maintain the polarity. These data are consistent with our MT gliding model.[Editors' note: during revisions the editors made the following suggestion: The reviewers agreed that it would be beneficial to re-include the data regarding the dendrite elongation phenotype that you removed during the first revisions.]unc-116 animals in the manuscript as a supplement to Figure 3.We have followed your suggestions and put back the DA9 dendrite length elongation phenotype of ReferencesJ Cell Biol, 162, 1045–55. doi: 10.1083/jcb.200302175Nakata, T, & Hirokawa, N. (2003). Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head. J Cell Biol, 117, 1263–75. doi: 10.1083/jcb.117.6.1263.Navone, F, Niclas, J, Hom-Booher, N, Sparks, L, Bernstein, HD, McCaffrey, G, et al. (1992). Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells. J Biol Chem, 285, 8155–62. doi: 10.1074/jbc.M109.068247Seeger, MA, & Rice, SE. (2010). Microtubule-associated protein-like binding of the kinesin-1 tail to microtubules. Biophys J, 96, 2799–807. doi: 10.1016/j.bpj.2008.11.069.Wong, YL, Dietrich, KA, Naber, N, Cooke, R, & Rice, SE. (2009). The Kinesin-1 tail conformationally restricts the nucleotide pocket.


Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5′-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. Tomato mosaic virus (ToMV), and that it forms a complex with ToMV replication proteins and another essential host factor TOM1 that is a seven-pass transmembrane protein. We further demonstrate that the replication proteins acquire the ability to synthesize negative-strand ToMV RNA and RNA 5′ cap only in the presence of both TOM1 and ARL8. The replication proteins of ToMV are multifunctional proteins that participate in RNA replication on membranes and RNA silencing suppression in the cytosol. Our results suggest that ToMV replication proteins are programmed to express their replication-related activities only on membranes through interactions with these host membrane proteins.Many important pathogens of plants, animals, and humans are positive-strand RNA viruses. They replicate via complementary RNA in replication complexes formed on host intracellular membranes. In the replication process, not only viral replication proteins but also host factors play important roles. Although many host factors whose knockdown affects the multiplication of positive-strand RNA viruses have been identified, the function of each host factor in virus multiplication is only poorly understood in most instances. In this paper, we show that a host small GTP-binding protein ARL8 is required for the multiplication of  Poliovirus and Hepatitis C virus, and most plant viruses, including Tobacco mosaic virus (TMV), Brome mosaic virus (BMV) and Tomato bushy stunt virus (TBSV) are positive-strand RNA viruses. These viruses have single-stranded, messenger-sense RNA genomes in virions. After infection, their genomic RNAs are released into the cytoplasm of host cells and are translated to produce viral proteins including those that are required for RNA replication . The replication proteins recruit their genomic RNAs onto intracellular membranes and synthesize complementary, negative-strand RNAs. The negative-strand RNAs are sequestered with the replication proteins in membranous compartments that are isolated from the cytosol, and are used as templates to synthesize positive-strand RNA , which are released into the cytosol Many animal viruses of medical and veterinary importance such as Flock house virus, TBSV, and/or Hepatitis C virus, presumably by modifying the structure of viral replication proteins Poliovirus and Coxsackievirus B3 (CVB3: a picornavirus) bind to GBF1, a guanine nucleotide exchange factor for a small GTP-binding protein ARF1, and modulates the function of GBF1-ARF1 to preferentially recruit phosphatidylinositol-4-kinase IIIβ over other effectors of ARF1 and to facilitate the formation of phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles, which are the essential binding site for 3D polymerase The multiplication of positive-strand RNA viruses depends not only on viral replication proteins but also on host factors. To date, a large number of such host factors has been identified Tobamovirus includes TMV, Tomato mosaic virus (ToMV), Youcai mosaic virus , and other related viruses. The genome of a tobamovirus is a non-segmented, single-stranded, 5′-capped RNA of 6.4 kilobases that encodes a replication protein of approximately 130 kDa (130K protein) and its read-through product of 180 kDa (180K protein), a cell-to-cell movement protein, and a coat protein (CP). The 130K protein has a methyltransferase-like domain that is involved in 5′ capping of progeny RNAs and a helicase-like domain, and the read-through region of the 180K protein contains a polymerase-like domain The genus TOM1 and TOM2A encode seven-pass and four-pass transmembrane proteins, respectively. In A. thaliana, simultaneous inactivation of TOM1 and its paralog TOM3 results in complete inhibition of multiplication of both TMV-Cg and ToMV TOM2A and its related genes results in incomplete inhibition of TMV-Cg multiplication but had little effect on ToMV multiplication In tobamovirus multiplication, several host factors including eEF1A, a subunit of eukaryotic translation initiation factor 3, TOM1, and TOM2A have been suggested to participate Nicotiana tabacum) BY-2 cell line in which infection of ToMV-180FS-GFP that encodes FLAG-StrepII-tagged 180K (180K-FS) (In a previous study 180K-FS)  could be180K-FS) .g supernatant of the extract of ToMV-180FS-GFP-infected and evacuolated protoplasts  and found that it was a small GTP-binding protein, NtARL8. NtARL8 co-purified with the 180K-FS protein from the solubilized membranes but not from the 15,000 x revisiae  [40]–GTP, and uncapped (5′-triphosphate) RNA that corresponds to nucleotides 1–30 of ToMV RNA , and RNA was extracted and analyzed by PAGE. 32P-labeled RNA, indicating capped RNA, was detected in the 180K-FS-purified fraction from infected plant cells and in 130K-FS-purified fraction from yeast coexpressing AtTOM1 and AtARL8a (32P-labeled bands disappeared after treatment with tobacco acid pyrophosphatase (TAP)  RNA was detected in the 130K-FS-purified fraction from yeast coexpressing AtTOM1 and AtARL8a, but not in the fractions from yeasts expressing 130K-FS alone or with either AtTOM1 or AtARL8a . Taken together, these results suggest that activation of the 130K protein’s capping functions depends on coexpression of TOM1 and ARL8.We further examined whether 130K-FS protein co-expressed with AtTOM1 and/or AtARL8a can be guanylated. Affinity-purified 130K-FS proteins from solubilized membranes of AtTOM1- and/or AtARL8-coexpressing yeasts were incubated with AdoMet and  GTP (10 µCi in 30 µL reaction mixture) and the uncapped RNA substrate (1 µg in 30 µL reaction mix), and incubated at 25°C for 1 h, followed by RNA purification by phenol extraction and alcohol precipitation. Treatment of the RNA with tobacco acid pyrophosphatase  was performed according to the manufacturer's instruction. RNA was separated with 8 M urea-9% PAGE and 32P signals were detected using a Fuji BAS-2500 bio image analyzer . For guanylyltransferase reaction, the replication protein-purified fractions were mixed with AdoMet (100 µM), GTP (1 µM), and [α-32P] GTP (10 µCi in 30 µL reaction mixture) and incubated at 25°C for 30 min. Protein samples were precipitated with 10% trichloroacetic acid and dissolved in standard SDS-PAGE sample buffer, then separated with NuPAGE 3–8% Tris-acetate gel (Invitrogen).Purification and solubilization of P15 membrane fractions from ToMV 130K-FS-expressing yeast cells and ToMV-180FS-GFP-infected BY-2 cells were performed as described previously Figure S132P in ToMV genomic RNA might be introduced by this enzyme.Effects of the H81A mutation in ToMV replication protein genes on RNA 5′ capping and RdRp functions. Wild-type and H81A ToMV RNAs (1 µg) were translated in membrane-containing BYL reaction mixtures (50 µl). Top panel shows an immunoblot of translation products detected by anti-ToMV replication protein antibodies. To eliminate excess GTP, membrane fractions of BYL reaction mixtures were collected by centrifugation at 15000 xg and subjected to protein guanylation and RdRp assays. The positions of ToMV genomic RNA (gRNA), subgenomic RNA (CPsg), and RF RNA are indicated to the right. Note that ToMV genomic RNA is a substrate of host CCA tRNA nucleotidyltransferase and a part of (EPS)Click here for additional data file.Table S1List of primers used in this study.(DOCX)Click here for additional data file.