The loss of synapses is a central event inneurodegenerative diseases. Synaptic proteins are often associated with disease neuropathology, but their role in synaptic loss is not fully understood. Of the many processes involved in sustaining the integrity of synapses, local protein translation can directly impact synaptic formation, communication, and maintenance. RNA-binding proteins and their association with RNA granules serve to regulate mRNA transportation and translation at synapses and in turn regulate the synapse. Genetic mutations in RNA-binding proteins FUS and TDP-43 have been linked with causing neurodegenerative diseases: amyotrophic lateral sclerosis and frontotemporal dementia. The observation that mutations in FUS and TDP-43 coincide with changes in RNA granules provides evidence that dysfunction of RNA metabolism may underlie the mechanism of synaptic loss in these diseases. However, we do not know how mutations in RNA-binding proteins would affect RNA granule dynamics and local translation, or if these alterations would cause neurodegeneration. Further investigation into this area will lead to important insights into how disruption of RNA metabolism and local translation at synapses can cause neurodegenerative diseases. Translational control occurs mostly by homeostatic responses that alter general protein synthesis. However, gene-specific translational control depends on regulatory elements in the mRNA, such as upstream open reading frames, secondary structures or regulatory protein-binding sites . As suchThe importance of RNA-binding protein function at synapses is highlighted in patients with neurological and neurodegenerative disorders where genetic mutations or deletions of genes encoding RNA-binding proteins result in loss of synaptic plasticity and neuron function. Genetic mutations or deletions in disorders of autism, fragile X syndrome and Rett syndrome, which also correspond with a loss of synaptic plasticity and function, strongly suggest that disruption of RNA regulation is a central cause of synaptic defects in these brain disorders. Neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are related by overlapping clinical phenotypes, genetic links and rapid disease progression . WhereasNeurons are highly complex cells with polarized and elaborate processes that extend long distances in the central nervous system. The distance between the synapse and cell body creates a supply and demand challenge for neurons, particularly at synapses. Neurons have the challenge of regulating the local translation of proteins at the synapse in order to meet the rapidly changing environment of neuronal inputs. The solution to these demands requires a local mechanism for controlling transport of mRNA to synapses and regulation of translation to allow local synthesis of new synaptic proteins at a moment’s notice. This is achieved by having all of the necessary components for translation; mRNA, ribosomes and translation factors, present in dendrites and even in the dendritic spines, ready for local protein synthesis , 9. The Messenger ribonucleoprotein complexes (mRNPs) form when mRNA associates with protein complexes. In higher eukaryotes, mRNPs comprise more than ten thousand different RNA sequences and hundreds of different RNA-binding proteins. mRNPs can also assemble into more complex structures known as RNA granules (or RNP granules). There are many types of RNA granules, for example, transport RNP granules (tRNP), stress granules, processing bodies, germ granules, and nuclear paraspeckles . The claTransport RNP granules (tRNP granule) are ribonucleoprotein particles that function in transport and storage of mRNA and can contain miRNA , 18. SevStress granules are cytoplasmic aggregates composed of RNA binding proteins, RNA and stalled translation initiation complexes that usually form in a reversible manner upon cellular stress . In someProcessing bodies are sites enriched with factors involved in mRNA degradation, mRNA surveillance, translational repression, RNA-mediated silencing, and may also be involved in storage of select RNAs and recycling/modification of decay factors. Many processing bodies exchange rapidly with cytoplasmic proteins and contain only a few stable components. Processing bodies have also been shown to interact with tRNPs  and are mRNP granules form in the nucleus. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP complexes  or be permanently disassemble and degraded (reviewed in ). The trThe role of the protein components of tRNP granules is an ongoing question. There is evidence to suggest that RNA-binding proteins which associate with tRNP granules have the ability to regulate mRNA repression and/or translation. The RNA-binding protein, ZBP1 associates with and transports β-actin mRNA to synapses . Once inα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and enhanced long-term depression following mGluR activation [One well-known regulator of translation is the RNA-binding protein FMRP, which regulates translation and mRNA transportation to dendrites. Mutations in the gene encoding FMRP are associated with a loss of function and cause one of the most common inherited forms of autism, Fragile X syndrome . FMRP-astivation . Consisttivation , 66 and tivation , 68. At tivation , 69. In Several studies indicate that FMRP plays a critical role in regulation of mRNA translation by serving as a link between transport tRNP granules and polyribosomes. This is based on the distribution of FMRP to these fractions. For instance, FMRP is present in polyribosomes and acts to stall ribosomal translocation during elongation of its target mRNAs . The phoNeuron dendritic branching, synapse formation, and stabilization play significant roles in the structural and functional plasticity of the brain. Precise synapse development and formation is important for accurate neuronal network activity and normal brain function. Therefore, it is not surprising that alterations in dendrite morphology or defects in neuronal development, including changes in dendrite branching patterns, fragmentation of dendrites, retraction or loss of dendrite branching, and changes in spine morphology and number, contribute to disease. In particular, these changes have been observed in several neurodegenerative, neurodevelopmental, and neuropsychiatric disorders, such as, ALS , FTD 7575, AlzheAnimal models of neurodegenerative diseases also show changes in dendritic branches and abnormal spine morphology including animal models of ALS , 84 and In the last 10 years more RNA-binding proteins have been identified through genetic studies to be linked with causing neurodegenerative diseases. Genes that encode SMN –94, ATXNFUS is part of the FET family of RNA-binding proteins that include EWSR1 and TAF15 . FUS binThe majority of FUS is localized to the nucleus of cells; however, it can localize to different cellular compartments and RNA granules in response to various stimuli. This is facilitated by its non-classical proline-tyrosine nuclear localization sequence (PY-NLS) and nuclear export sequence (NES) . For exaThe majority of familial ALS mutations that are identified in the gene encoding FUS occur in its C-terminal PY-NLS , alterinD. melanogaster [C. elegans [R. norvegicus [D. melanogaster demonstrates that replacement of the endogenous FUS homolog with the human FUS R521C mutation causes defects in synapse structure and function that precedes motor neurodegeneration [Animal models expressing various FUS mutations and wild-type FUS reproduce aspects of the human diseases. For instance, nogaster , C. eleg elegans , and R. rvegicus , models rvegicus . Transgervegicus . Howeverrvegicus , which hrvegicus . These trvegicus . These oneration , which ineration . These oneration . It is pA potential significance of FUS aggregation comes from the finding that several stress granule markers are also deposited in the aggregates . This haFUS and TDP-43 have often been reported as having a similar role in disease and biology, but there are some distinctions and similarities, which we will point out in this section. TDP-43 is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins . Like FUTDP-43 can localize to different cellular compartments and RNA granules via a classical NLS and NES . It too TDP-43 is a major component of ubiquitinated inclusions found in the brain and spinal cord of the majority of ALS patients , 157. InD. melanogaster results in reduced lifespan and locomotor defects due to alterations in dendritic branching and synapses [D. rerio, both overexpression of human TDP-43 and knockdown of TARDBP result in swimming behavior defects caused by defective neuronal axon formation, and premature and excessive branching [M337V transgenic rats expressing mutant proteins in motor neurons recover their motor function when TDP-43M337V expression is turned off [Animal models of TDP-43 demonstrate that altering endogenous levels of the wild-type protein or expressing ALS-TDP-43 mutations is highly toxic and reproduces aspects of ALS and FTD , 162–166synapses –170, ovesynapses . In D. rranching . Similarranching . TDP-43Mrned off . This strned off . Conditirned off . Depletirned off . Overexprned off . In knocrned off , a positAnother property of ALS-TDP-43 mutations and wild-type TDP-43 is that they are actively recruited to cytoplasmic stress granules in response to stress . MoreoveMuch of the field has focused on stress granules; however, TDP-43 mutations have been shown to affect tRNP granule formation and migration. TDP-43 is associated with RNA granules that are prevalent throughout the dendritic arbor in neurons. “Aggregation” of TDP-43 is also important for the formation of these neuronal tRNP granules, and it is reasonable to assume disease-linked mutations might alter granule formation. Indeed, ALS-TDP-43 mutations are shown to increase the size of neuronal TDP-43 granules in the dendritic arbor of rat hippocampal neurons under basal conditions . DepolarThe role of RNA-binding proteins in translational control and how it relates to neurodegeneration is gaining interest in the field. This has occurred for a number of reasons: 1) identification of genetic mutations in genes encoding proteins that are involved in RNA regulation and linked to neurodegenerative diseases; (2) in vivo disease models do not recapitulate pathological aggregate phenotypes of the diseases and in vitro stress granule models do not fully explain the global impact on RNA misregulation; (3) at least in the case of TDP-43-FTD, cross-linking and immunoprecipitation of TDP-43 and transcriptome analysis reveal very few changes in RNA-binding profiles from control patients , and the identifiIf alterations of RNA granule dynamics, transport, and translation are important factors in maintaining the synapse, then how are RNA-binding proteins coordinating these processes? Dynamic exchange of mRNA between tRNP granules, stress granules and processing bodies is in part coordinated by specific RNA-binding proteins. There is evidence that supports RNA-binding proteins are essential in the shuttling of mRNA to and from these specialized granules until such time that the mRNA is translated in polyribosomes. The impact of deletion of RNA-binding proteins can lead to alterations in dendritic branches and spines as well as impact translation. Key questions that arise are: How do RNA-binding proteins target mRNA to each RNA granule? What cues initiate mRNA exchange between RNA granules? What post-translational modifications do RNA-binding proteins undergo to coordinate exchanges between RNA granules? It is clear that stress granule formation and the presence and numbers of processing bodies are affected by ALS-FUS and ALS-TDP-43 mutations. Recent work shows that tRNP granules are also affected by ALS-FUS and ALS-TDP-43 mutations. Putting all this evidence together would strongly suggest that mutations at least in these RNA-binding proteins would affect the dynamics of mRNP–RNA granule exchanges, which we defined here as the transfer of mRNA:Protein complexes between RNA granules in response to cellular stimuli and cues Fig. b. More cFinally, if RNA-binding proteins affect mRNP–RNA granule exchange, how does this affect local translation and synaptic function, and can the resulting effects lead to neurodegeneration? The strongest precedence by an RNA-binding protein has been set for FMRP in the autism disorder fragile X syndrome. In this case, loss of FMRP causes changes in synaptic morphology and function in fragile X syndrome, but this disease does not involve neurodegeneration. The challenge in the neurodegeneration field will be to mechanistically link dysfunction of mRNP-RNA granule exchange and local translation with initiation of neurodegeneration. This will be difficult to dissect given that RNA-binding proteins like FUS and TDP-43 regulate thousands of RNAs in both the nucleus and cytoplasm. A particular focus will need to be on target RNAs that encode proteins critical for synaptic function at both the steady state and in response to neuron stimulation. As observed in the FUSR521G transgenic model, defects in translation are more prominent at synapses in response to mGluR stimulation. The function of FUS and TDP-43 at synapses is not well understood, but the existing evidence point to a prominent role in maintaining the function and integrity of synapses: these RNA-binding proteins have been purified with synaptic tRNP granules; their expression is elevated upon neuron stimulation; genetic deletion results in altered dendritic spines and branches in primary cultured neurons. This in addition to their genetic link to ALS and FTD strongly implicate RNA-binding proteins as having a major role in causing alterations in RNA metabolism locally at the synapse, which would alter synaptic function and trigger neurodegeneration.


Further study indicated that IDH1/2 mutants showed significant ability to inhibit TET-promoted RNA5hmC. Consistent with this result, overexpression of IDH1/2 mutants also inhibited TET catalytic domain-promoted oxidation of RNA. In this study, we show not only the enrichment of 5hmC in mRNA, but also a regulatory mechanism of RNA 5hmC—IDH1/2 mutations inhibit TET-promoted RNA 5hmC, which suggests an involvement of IDH1/2 mutations in tumorigenesis through the deregulation of RNA biology.TETs (TET1/2/3) play critical roles in multi cellular processes through DNA demethylation driven by oxidation of DNA 5mdC to 5hmdC. Interestingly, recent studies indicated that TETs also oxidate RNA 5mC to 5hmC. However, little is known about the distribution of RNA 5hmC and the regulatory mechanism of RNA 5hmC in human. Here, we show that 5hmC is enriched in mRNA, and IDH1/2 mutants inhibit TET-promoted oxidation of RNA 5mC to 5hmC. Since IDH1/2 mutations have been described to block the DNA oxidative activity of TETs, we hypothesized that IDH1/2 mutations might also inhibit the RNA oxidative activity of TETs. To evaluate the role of IDH1/2 mutations in RNA 5hmC, TETs with/without IDH1/2 mutants were overexpressed in human HEK293 cells. Resultant DNA and RNA were digested and analyzed by triple-quadrupole LC mass spectrometer. DNA 5hmdC and RNA 5hmC modifications were quantified with external calibration curves of appropriate standards. It was found that compared with total RNA (5hmC/C: less than 2 X 10 They found that Tet ortholog (CG43444) mutant had smaller brain size and decreased neuroblasts in fruit fly [There are three members in ten-eleven translocation (TET) family in human, namely TET1, TET2 and TET3. Facilitated by 2-ketoglutarate (2-KG) and Feruit fly , which iDrosophila[Despite the recent exciting findings about TET ortholog (CG43444)-promoted RNA 5hmC in rosophila, little rosophila, block trosophila, 20. IDHrosophila–23. IDH1rosophila, 21–23. rosophila, 20. Thirosophila, 20. Con−7), mRNA showed much higher 5hmC level (5hmC/C: ∼7 X 10−6) in human cells. It is also demonstrated that IDH1/2 mutants inhibit the RNA oxidative activity of TETs, which suggests an involvement of IDH1/2 mutations in tumorigenesis through the deregulation of RNA biology.In this study, it was found that compared with total RNA  as described in our previous studies [HEK293 and U2OS cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin and streptomycin at 37°C under a humidified atmosphere of 5% CO studies , 25.pS-Flag-SBP TET1, pS-Flag-SBP TET2 and pS-Flag-SBP TET3 were gifts From Dr. Yu at University of Michigan Medical School . pCDNA3BGenomic DNA (2 μg) was first denatured by heating at 100°C. Two units of DNase I , 2 units of DNA degrase , and 2 unit of Nuclease P1  were added in a total volume of 50 μL. The resultant mixture was incubated at 37°C for 10 hours. Then one unit of alkaline phosphatase  was added to the mixture, followed by 5 hr incubation at 37°C. Complete digestion into nucleosides was confirmed by visualization of the digest on a 1% agarose gel. The reactions were diluted 2 folds, and 10 μl of the solution was injected into LC-MS/MS. The nucleosides were separated by reverse phase ultra-performance liquid chromatography on a C18 column, with mass spectrometry detection using AB SCIEX QTRAP 5500 LC/MS/MS in positive electrospray ionization mode. In brief, the digested DNA was performed using the mobile phase of 92% water (containing 0.1% formic acid) and 8.0% methanol at a flow rate of 400 μL/min. The ionspray voltage was maintained at 5500 eV. The turbo gas temperature was set at 550°C. Multiple reaction monitoring (MRM) was used to monitor the nucleoside to base ion mass transitions: m/z 228 to 112 (collision energy (CE) 15 V; declustering potential (DP) 60 V) for 2’-deoxycytidine (dC); m/z 242 to 126  for 5-methyl-2’-deoxycytidine (5mdC); m/z 258 to 142  for 5-hydroxymethyl-2’-deoxycytidine (5hmdC). Both Q1 and Q3 quadrupoles were maintained at unit resolution. Analyst 1.6.1 software  was used for data acquisition and processing. Linearity in ionization efficiencies were verified by analyzing dilution series of authentic standards. External calibration curves for dC, 5mdC, and 5hmdC were used to create standard curves for subsequent normalization and quantification, respectively.25  following the manufacturer’s protocol. The resultant RNA  were digested by 12 U nuclease P1 in 60 μl of buffer containing 25 mM NaCl, and 2.5 mM ZnCl2 at 37°C for overnight, followed by the addition of 1 M NH4HCO3 (3 μl) and 3 U alkaline phosphatase. After incubation at 37°C for 2 hrs, 10 μL of RNA solution was directly injected into LC-MS/MS to analyze 5hmC. Then, RNA solution was diluted 10 times and 10 μL of the diluted solution was injected into LC-MS/MS to analyze C, 5mC and 5hmC. The nucleosides were separated by reverse phase high-performance liquid chromatography on an Agilent C18 column , with mass spectrometry detection using AB SCIEX QTRAP 5500 LC-MS/MS in positive electrospray ionization mode. The mobile phases (delivered at 0.40 ml/min) consisted of H2O for A and methanol for B. An isocratic elution (80% A and 20% B) was performed at stop time of 2 min. Multiple-reaction-monitoring (MRM) mode was used to monitor analyte parent ion to product ion: m/z 274.2 to 142.1 for 5-hydroxymethyl-cytidine (5hmC) (collision energy (CE) 12 V; declustering potential (DP) 104 V; Entrance potential (EP): 13 V; collision cell exit potential (CXP): 24 V); m/z 258.1 to 126.0 for 5-methyl-cytidine (5mC) ; m/z 244.1 to 112.2 for cytidine (C) . The following instrument settings were used: column temperature: 25°C; ionspray voltage: 5500 V; turbo gas temperature: 550°C. Both Q1 and Q3 quadrupoles were maintained at unit resolution.Total RNA was isolated using RNeasy Mini Kit  according to the manufacturer’s instruction. Polyadenylated mRNA was purified using Dynabeads Oligo (dT)2-hydroxyglutarate (2-HG) in metabolite extracts from cells was analyzed by LC-MS/MS. The Waters ACQUITY UPLC system and a Waters UPLC BEH C18 2.1x150 mm column (1.8 μm particles) were used in the HPLC analysis. The hydrolysates (10 μL) were injected onto the column. Formic acid (0.05%) in water and acetonitrile were used as mobile phases A and B, respectively. The elution profile was: 0–3 min, 90% A and 10% B. The mobile phase flow rate was 0.4 mL/min. Column temperature was maintained at 25°C. Mass spectrometric detection was achieved by AB SCIEX QTRAP 5500 mass spectrometer with an electrospray ionization source. Negative ionization mode, -4500V capillary voltage and 600°C source temperature were used. The optimized conditions were described as follows: declustering potential (DP), -18 V; entrance potential (EP), -4 V; collision cell exit potential (CXP), -15 V; collision energy (CE), -15 V; scan time, 200 msec. Multiple reaction monitoring (MRM) was used for the LC-MS/MS analysis. 2-HGwas detected by monitoring the quantification ion pairs of m/z 147 to 129.p values were calculated using the Student’s t-test. And p values< 0.05 were considered statistically significant.To assess the modification of nucleoside, triple quadrupole mass spectrometer was first applied to analyze 2’-deoxycytidine (dC), 5-methyl-2’-deoxycytidine (5mdC), 5-hydroxymethyl-2’-deoxycytidine (5hmdC), cytidine (C), 5-methylcytidine (5mC) and 5-hydroxymethylcytidine (5hmC). The nucleoside standards were separated by reverse phase ultra-performance liquid chromatography on a C18 column, with detection using AB SCIEX QTRAP 5500 LC/MS/MS in positive electrospray ionization mode. Multiple reaction monitoring (MRM) was used to monitor the nucleoside to base ion mass transitions: m/z 228 to 112 for dC; m/z 242 to 126 for 5mdC; m/z 258 to 142 for 5hmdC; m/z 244 to 112 for C; m/z 258 to 126 for 5mC; m/z 274 to 142 for 5hmC . Analyst−7) in HEK293 cells, whereas 5hmC in mRNA was present at an appreciable level (5hmC/C: ∼7 X 10−6)  . As show X 10−6) . To conf X 10−6) . The cat X 10−6) . And mas X 10−6) .To validate the role of IDH1/2 mutants in TET-promoted RNA oxidation, TET2 with/without IDH1/2 wts or mutants as noted were transfected into HEK293 or U2OS c−7) in human cells, whereas 5hmC of mRNA was present at an appreciable level (5hmC/C: ∼7 X 10−6). The enrichment of 5hmC in mRNA and the involvement of TETs in mRNA oxidation suggest that the roles of TETs are not restricted to the transcriptional regulation at the DNA level, but highly likely can be extended to the translational regulation at the mRNA level. Further studies are needed to define the site-specific localization of 5hmC in human mRNA, and the function of 5hmC modification in RNA biology. Due to the critical roles of TETs in various physical and pathological processes, the potential functions of TETs in RNA biology should also be evaluated.Although it is generally believed that TETs play roles in cellular processes through DNA oxidation, recent studies indicated that TET ortholog (CG43444) and TETs are also able to oxidate RNA 5mC to 5hmC , 17, 28.In addition, despite the recent exciting findings about TET ortholog (CG43444)-promoted RNA 5hmC , little However two limitations of this study should be noticed. First, we focused on the overexpression of TETs in this study, due to the abundance of 5hmC in RNA. Second, only HEK293 cells were applied to evaluate the role of IDH1/2 mutants in TET-promoted oxidation of RNA 5mC to 5hmC. Notwithstanding these two limitations, this study clearly demonstrates the enrichment of 5hmC in mRNA, and the ability of IDH1/2 mutants to inhibit the RNA oxidative activity of TETs.Collectively, we describe the mRNA enrichment of 5hmC in human cells, which suggests 5hmC might be involved in RNA biology including mRNA splicing, transporting, translation and degradation. Furthermore, we also demonstrate that IDH1/2 mutants can inhibit TET-promoted RNA 5hmC modification, which suggests an involvement of IDH1/2 mutations in tumorigenesis through the deregulation of RNA biology.S1 Fig(TIF)Click here for additional data file.S1 Methods and Materials(DOCX)Click here for additional data file.


In vivo studies of the MCMV orthologue, a 7.2 kb RNA, demonstrated that viruses that do not express the RNA fail to establish efficient persistent replication in the salivary glands of mice. To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail.HCMV encodes a stable 5 kb RNA of unknown function that is conserved across cytomegalovirus species. in vitro and in vivo assays.We performed multiple analyses to evaluate transcript expression kinetics, identify transcript termini and promoter elements. The half-lives of intron locus RNAs were quantified by measuring RNA levels following actinomycin D treatment in a qRT-PCR-based assay. We also constructed a series of recombinant viruses to evaluate protein coding potential in the locus and test the role of putative promoter elements. These recombinant viruses were tested in both in vivo. Low transcriptional output by the putative promoter element(s) is compensated by the long half-life of the 7.2 kb RNA of approximately 28.8 hours. Detailed analysis of viral spread prior to the establishment of persistence also showed that the intron is not likely required for efficient spread to the salivary gland, but rather enhances persistent replication in this tissue site.We show that the 7.2 kb RNA is expressed with late kinetics during productive infection of mouse fibroblasts. The termini of the precursor RNA that is processed to produce the intron were identified and we demonstrate that the m106 open reading frame, which resides on the spliced mRNA derived from precursor processing, can be translated during infection. Mapping the 5′ end of the primary transcript revealed minimal promoter elements located upstream that contribute to transcript expression. Analysis of recombinant viruses with deletions in the putative promoter elements, however, revealed these elements exert only minor effects on intron expression and viral persistence This data provides a comprehensive transcriptional analysis of the MCMV 7.2 kb intron locus. Our studies indicate that the 7.2 kb RNA is an extremely long-lived RNA, a feature which is likely to be important in its role promoting viral persistence in the salivary gland. Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily and a widespread human pathogen,2. HCMV in vivo, and contribute significantly to viral transmission. Healthy individuals may secrete virus in saliva, breast milk, and urine for long periods of time following primary infection[HCMV infection of healthy, immunocompetent individuals elicits a robust host immune response that effectively limits virus replication and pathogenesis. Despite this immune response, HCMV has adapted to long-term infection of humans by balancing persistent replication with clearance by the immune response. HCMV persistently replicates in glandular epithelial tissue and eventually establishes a life-long latent infection of the host. Glandular epithelial cells, such as those in the salivary gland, are key sites of HCMV persistent replication nfection,3. In ornfection-8. In adnfection,10. Few nfection-13. In anfection.During lytic replication, HCMV expresses a 5 kb lncRNA of unknown function -16. We sin vivo. MCMV expresses a 7.2 kb ortholog of the HCMV 5 kb RNA[in vivo. The specific molecular function of this conserved RNA is unknown, but we hypothesize that it mediates processes essential for the virus to evade immune surveillance and/or replicate efficiently at sites of viral persistence.Cytomegaloviruses exhibit strict species specificity and there is no animal model for HCMV infection. Murine cytomegalovirus (MCMV) infection of the mouse is widely used as an outstanding small-animal model of HCMV infection for several reasons. HCMV and MCMV share similar genomic sequence and organization and undergo similar replication cycles. Like HC5 kb RNA. We havein vivo, supporting our hypothesis that the 7.2 kb RNA functions to either evade the host response or maintain viral replication at sites of persistence.To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail. We confirmed that the intron locus RNAs are expressed with late gene kinetics. The 7.2 kb intron has an unusually long half-life, whereas the spliced mRNA that results from processing of the intron is metabolized with kinetics similar to most cellular mRNAs. Investigation of the promoter sequence that controls expression of the intron locus revealed a minimal promoter sequence contributing to the transcriptional output of the locus. Although there is no evidence for translation of the intron itself, we discovered that the spliced mRNA encodes a small protein that co-localizes with the RNA within the nuclei of infected cells. Importantly, we show that the RNA is not required for trafficking of virus to the salivary gland To determine the transcription kinetics of the 7.2 kb intron locus during productive MCMV infection, northern blot analysis was performed on total RNA prepared from cells treated with the translation inhibitor cycloheximide (CHX) or the DNA replication inhibitor phosphonoacetic acid (PAA) prior to MCMV infection. CHX pre-treatment of cells inhibits translation of immediate early (IE) genes blocking subsequent transcription of both early and late classes of viral genes. PAA treatment blocks DNA replication, on which expression of late (L) genes is dependent,2. TransIn high resolution northern blot analyses specific for the intron RNA, we routinely observed a doublet of bands near 7.2 kb: a major species at approximately 8.0 kb and a minor species migrating faster at 7.2 kb Figure A. These While the splice donor and acceptor sites used in the processing of the 7.2 kb RNA from the primary transcript were previously mapped, the termini of the primary transcript were not determined. To iden+ and A-). Taken together, our data suggest that a large precursor RNA is transcribed from the intron locus at late times of infection and processed to yield a single 7.2 kb stable intron and a spliced poly-adenylated mRNA consisting of two exons.A single 3′ end was identified at nucleotide position 153,872 by sequencing of 3′-RACE products [delSD:m106GFP infection, indicating that splicing of the mRNA is necessary for translation of m106  called m106. Positiom106GFP). This su Figure C. FurtheAlthough we previously demonstrated in fractionation studies that the HCMV 5 kb intron localizes to the nuclear compartment of infected cells, the specific sub-nuclear localization of the RNA was not examined. FluoresThe 7.2 kb RNA accumulates to high levels during infection as detected by northern blot analysis, suggesting it is unusually stable for an intron,19,26,27Little data is available regarding the sequence elements driving late transcriptional units of cytomegaloviruses. Therefore, the DNA sequence within the vicinity of the TSSs of the intron locus was examined for transcriptional regulatory sequences. A putative TATA box element was identified 24 nucleotides upstream of the transcriptional start site, as well as an additional TATA box located 127 nucleotides upstream Figure A. In orddelHP and MCMVdelSD)[delHP contains a 28 bp deletion spanning a predicted stem loop structure at the 3′end of the intron that is hypothesized to confer stability. Without this stem loop structure, processing of the primary transcript still occurs since the mRNA is detected by qRT-PCR and northern blot analysis, but accumulation of the intron is significantly reduced . The MCM Figure C. While  Figure C. In celin vivo, mice were inoculated with a subset of our panel of recombinant viruses and viral yields measured in the salivary gland at 14 days post-infection , highly basic, arginine-rich peptides. It is unknown if other UL106 orthologues are expressed during infection, but given the conservation of the genomic organization of the intron locus among CMVs, it is a distinct possibility to be explored.delSD in any organs at 14 dpi and genome copy number of the mutant virus was substantially reduced in liver and kidney. It is possible that the mechanisms that prevent establishment of intron-mutant virus persistence in the salivary gland may also promote accelerated clearance of that virus from liver and kidney. At this time, the adaptive immune response acts to limit viral replication and it is possible that the 7.2 kb intron is involved in modulating immune surveillance in some way. Some cellular lncRNAs are involved in transcriptional regulatory processes, therefore, a possible mechanism for evading the immune response could be to regulate cellular or viral genes that are involved in this host pathogen relationship[Production of the 7.2 kb RNA is required for the establishment of persistence in the salivary glands of mice. By analysis of multiple time points between the acute and persistent phases of infection in mice, we showed that recombinant virus lacking the intron appears to disseminate to the salivary gland as efficiently as wild-type MCMV. However, it is unable to maintain a highly productive replication program in the salivary glands as observed at 14 days post infection. In addition, we did not detect infectious MCMVtionship,45.This current study has provided a detailed transcriptional and functional analysis of the MCMV 7.2 kb RNA locus. Mapping the topography of the 7.2 kb RNA locus allows us to understand the genomic elements that not only comprise the locus but also control its expression. The unusual stability of the 7.2 kb RNA compensates for the weak transcriptional output by the putative promoter region observed and may also provide insight towards molecular function within the nucleus of infected cells. Uncovering the stability determinants of the 7.2 kb RNA will therefore allow us to understand the mechanisms that promote its retention within infected cells. Although a function has yet to be determined to the 7.2 kb RNA, this current analysis has provided the framework for investigating its function during viral persistence.2.10.1-mouse embryonic fibroblasts were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. Cells were maintained at 37°C with 5% COEscherichia coli strain GS1783 method or by using the FLP-recombinase method as previously described[50 assay on 10.1 fibroblasts.The BAC clone of the wild type Smith MCMV strain, pSM3fr, was used as the parent strain in this study,47. Recoescribed. Primer RACE products were cloned into pGEM-T-Easy (Promega) and sequenced. Reporter constructs were generated by PCR amplifying sequence upstream of the 7.2 kb RNA splice donor site. Primers used to generate the reporter constructs are indicated in Table 10 copies per well of each plasmid. Protein lysates were harvested 48 hours post transfection and assayed following the Duo-Glow Luciferase assay kit . Luciferase activity was normalized to renilla activity in each well and the data is expressed as the fold change of luciferase induction relative to the luciferase induction from the pGL3 promoter-less vector.Mouse fibroblasts were seeded in 24-well dishes and co-transfected with a pGL3 construct (see above) and phRL-TK using polyethylenimine (PEI) at a 6:1 ratio of PEI to plasmid DNA. The plasmids were cotransfected at a 1:1 ratio with 3 × 10To determine expression kinetics, 10.1 fibroblasts were pretreated with 100 μg/mL cyclohexamide or 200 μg/mL PAA 1 hour before MCMV infection. Total RNA was harvested from 10.1 fibroblasts at either 24 hours post infection (h p.i.) or 48 h p.i. with TRIzol LS (Life Technologies) according to the manufacturer’s protocol. RNA was resolved on either a 1.4% or 0.7% glyoxal gel for detection of the spliced mRNA or 7.2 kb RNA respectively. Northern blot analysis for intron locus RNAs was carried out as previously described using specific radio-labeled riboprobes.RNA half-life analysis was performed by infecting fibroblasts with wild-type MCMV at an MOI of 1.0. At 30 hours post infection, 4 ug/mL Actinomycin D was added to infected cells and RNA was harvested over a time course starting at time 0 and ending at 32 hours post treatment. Transcript levels were quantified by qRT-PCR at the different times points relative to RNA at time 0.Total RNA harvested from mock or WT MCMV infected 10.1 mouse fibroblasts at 48 hours post infection was analyzed by the First Choice RNA ligase-mediated rapid amplification of cDNA ends kit as recommended by the manufacturer . Amplification products were purified and TA cloned into pGEM-T-Easy  and sequenced . Quantitative PCR was performed using the LightCycler 480 Probes Master Mix (Roche) along with IDT hydrolysis probes specific for the intron locus RNAs and selected housekeeping genes Table . Ct valuCells were trypsinized, centrifuged, and collected in PBS. Cells were lysed in RIPA buffer  containing protease inhibitor cocktail (Roche). The cell lysate was briefly sonicated to facilitate nuclear protein release and insoluble debris was centrifuged. GFP tagged m106 protein was immunoblotted using a rabbit polyclonal antibody and detected with a fluorescently conjugated secondary antibody using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). HP1, heterochromatin associated protein 1, was detected similarly as a loading control (Santa Cruz).Fluorescently labeled RNA probes antisense to the 7.2 kb intron were generated using the FISH Tag kit Table  . 250 ng of DNA was analyzed by qPCR for each sample.All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado Denver. BALB/c mice were inoculated by intraperitoneal injection with 5×10The authors declare that they have no competing interests.TMS, L-AMV, CGA, and CAK participated in the design and execution of the study. TMS and CAK drafted the manuscript. All authors read and approved the final manuscript.


Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in   •Lin28a binding to a pre-miRNA is insufficient to trigger TuT4-mediated uridylation•The E3 ligase Trim25 binds to the conserved terminal loop of pre-let-7•Trim25 is an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation cis, thereby providing an additional level of specificity.Lin28a triggers TuT4-mediated pre-let-7 uridylation. Despite binding to other pre-microRNAs, only pre-let-7 is efficiently uridylated by TuT4. Choudhury et al. show that Trim25 is an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. These findings reveal that Trim25 can guide the function of canonical RNP complexes in  Next-generation studies on RNA binding proteins have supplied a constant stream of novel RNA-protein interactions . AdditioLin28a is an example of an RNA binding protein with a well-defined binding motif (GGAG) and roles in the control of microRNA (miRNA) maturation , mRNA trHere, we show that Lin28-mediated uridylation is dependent on the structure of the RNA and the sequence context of the Lin28a binding motif. Using RNA pull-down combined with high-throughput stable isotope labeling with amino acids in cell culture (SILAC) mass spectrometry, we discovered that the E3 ligase Trim25 is a factor that binds to pre-let-7. We found that Trim25 colocalizes and coimmunoprecipitates with Lin28a. We also found that Trim25 activates TuT4 and that drug inhibition of the E1 ubiquitin ligase reduces the levels of TuT4 and decreases pre-let-7 uridylation. Finally, using in vitro pre-miRNA processing and in vivo pre-miRNA quantification, we showed that Trim25 is an RNA-specific cofactor for Lin28a-mediated uridylation of pre-let-7. Altogether, our results uncover an important layer of RNA binding factors that can modify canonical ribonucleoprotein (RNP) complexes and control their molecular function.Previously, the GGAG motif was shown to be sufficient to induce Lin28a binding, but not uridylation . A fixedCo-overexpression of pri-let-7a-1 mutants with Lin28a in HeLa cells followed by northern blotting against let-7a confirmed the importance of the AU dinucleotide adjacent to the GGAG motif in the Lin28a-mediated inhibition of let-7a processing, as only the pri-let-7a-1@2 mutant was suppressed to the same extent as the wild-type pri-let-7a-1 . NotablyTo find the RNA-specific cofactor for Lin28a-mediated uridylation, we next used RNA pull-down combined with SILAC and high-throughput mass spectrometry A. Pre-leNext, we sought to establish whether Trim25 interacts with components of the pre-let-7/Lin28/TuT4 pathway. Using immunocytochemistry on mouse teratocarcinoma P19 cells, we found that Lin28a and Trim25 colocalize in vivo, supporting their functional link F. NotablTo determine whether Trim25 plays a role in Lin28a-mediated uridylation, we performed Trim25 RNAi in mouse teratocarcinoma P19 cells A. EfficiBecause Trim25 is an E3 ubiquitin ligase, we sought to determine whether interfering with the ubiquitination pathway would inhibit its downstream activity on the Lin28a/TuT4 pathway. After treating P19 cells with the E1 ubiquitin-activating enzyme inhibitor PYR-41  for 4 hrRNA binding proteins control gene expression programs in living cells by regulating RNA processing, modification, localization, translation, and turnover . They prThe RNA binding protein Lin28a is a pluripotency-promoting factor involved in downregulation of let-7 during early development and differentiation in metazoans . The majcis. Our model is that the sequence and structural features of the pre-let-7 CTL allow efficient Trim25 binding, which in turn stabilizes TuT4 leading to increased uridylation. In the future, it will be important to determine which type of modifications are responsible for this phenomenon and whether other members from the Trim family have similar RNA-specific functions. Because there are 75 Trim proteins annotated in the human genome, functional overlap, redundancy, and specification are very likely  were maintained in standard Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies), supplemented with 10% fetal bovine serum (Life Technologies). For SILAC mass spectrometry, undifferentiated cells were cultured in DMEM supplemented with “heavy” [13C]Arg/[13C]Lys isotopes, and differentiation was performed using DMEM supplemented with “light” [12C]Arg/[12Lys] isotopes . Where indicated, cells were treated with the E1 ubiquitin-activating enzyme inhibitor PYR-41  for 4 hr3 counts per minute [cpm]), approximately 20 pmol) were incubated in 30 μl reaction mixtures containing 50% (v/v) total P19 cell extract (approximately 10 μg × μl−1), 0.5 mM ATP, 20 mM creatine phosphate, and 3.2 mM MgCl2. The reactions were supplemented with 0.25 mM UTP and incubated at 37°C for 30 min followed by phenol-chloroform extraction, precipitation, and 8% (w/v) denaturing gel electrophoresis. The bands on the gel were visualized with a radiographic film or by exposure to a phosphorimaging screen and subsequent scanning on a FLA-5100 scanner (Fujifilm).Pre-miRNA transcripts were prepared by standard in vitro transcription with T7 RNA polymerase in the presence of [α-32P]-uridine-5'-triphosphate (UTP). Where indicated, pre-miRNA probes were 5′ end-labeled with [γ-32P]-ATP. The templates used to generate the transcripts were prepared by PCR amplification from larger fragments of the human genome using primers corresponding precisely to the ends of pre-miRNAs. The pre-let-7a-1@1, pre-let-7a-1@2, pre-let-7a-1@3, and pre-miRNA-363@1 templates were derived using mutagenesis of the wild-type pre-miRNAs. Generation of the template for pre-let-7a-1@16TL was described before . Gel-pur2, 10% (v/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 0.2 mM phenylmethanesulfonylfluoride). After the final wash, the proteins associated with the beads were analyzed by SDS-PAGE followed by in-gel digestion and mass spectrometry or western blotting.RNA pull-down and mass spectrometry were performed as previously described , with slTotal protein samples (100 μg per lane), isolated by sonication, were resolved by standard NuPAGE SDS-PAGE electrophoresis with MOPS running buffer (Life Technologies) and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 1:10 western blocking reagent (Roche) in Tris-buffered saline buffer with 0.1% Tween-20 (TBST). The following day, the membrane was incubated for 1 hr at room temperature with primary antibody solution in 1:20 western blocking reagent diluted in TBST: rabbit polyclonal anti-Lin28a (A177) , rabbit polyclonal anti-Trim25 , rabbit polyclonal anti-TuT4 , rabbit polyclonal anti-DHX9 , mouse-monoclonal anti-ubiquitin , and mouse-monoclonal anti-β-tubulin . After washing in TBST, the blots were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase and detected with SuperSignal West Pico detection reagent (Thermo Scientific). The membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (Chemicon) equilibrated in water, blocked in 1:10 western blocking solution in TBST, and reprobed as described above.−1, single-stranded DNA). A northern probe was prepared using the mirVana miR Probe Construction Kit (Life Technologies) and hybridized against the membrane for 2 hr at 40°C in 10 ml of hybridization buffer. Subsequently, the membrane was washed with wash buffer . The signal was registered with radiographic film or exposed to a phosphorimaging screen and scanned on a FLA-5100 scanner (Fujifilm).Northern blot analysis was performed as previously described . In briePools of siRNAs were obtained from Dharmacon in the format of three independent siRNAs targeting different regions of the mRNA coding for the protein of interest. Genomic fragments containing miRNAs were cloned into the pCG T7 plasmid and transiently expressed in HeLa cells. Four micrograms of siRNA was delivered in two transfection events separated by 48 hr using nucleofection technology (AMAXA), according to the manufacturer’s instructions. HeLa cells transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions.N.R.C. designed, performed, and analyzed the experiments and contributed to the writing of the manuscript. J.S.N. performed and analyzed the experiments. J.Z. and J.R. performed the mass spectrometry analysis. G.M. designed, performed, and analyzed the experiments, wrote the manuscript, and supervised the whole project.


For larger squares, we need statistical approaches for estimating the number. For this purpose, we formulated the problem as a combinatorial optimization problem and applied the Multicanonical Monte Carlo method (MMC), which has been developed in the field of computational statistical physics. Among all the possible arrangements of the numbers 1; 2, …, n2 in an n × n square, the probability of finding a magic square decreases faster than the exponential of n. We estimated the number of magic squares for n ≤ 30. The number of magic squares for n = 30 was estimated to be 6.56(29) × 102056 and the corresponding probability is as small as 10−212. Thus the MMC is effective for counting very rare configurations.How rare are magic squares? So far, the exact number of magic squares of order  Besides the classic magic squares, there are many variations, and some rigorous results have been found for them. But not much is known about classic magic squares [Making magic squares is a popular form of mathematical recreation. It is also used in classrooms as an elementary mathematical exercise. The classic (or ordinary) magic square of order There are some algorithms for making magic squares of any size. They, however, provide some special classes of magic squares, which gives rise to the question: Among all the possible arrangements of numbers in a square of a given size, how many of them form magic squares? Putting the question in another form: Is there any chance of making a magic square by putting numbers randomly in a square? It may be surprising to know that the exact number of possible magic squares is so far only known up to order 5. There is currently no hope of exact enumeration for a larger system. In this paper, we apply a Monte Carlo method to this problem, and estimate the number of the magic squares of each size up to order 30.n. Let Nn denote the total number of magic squares. Since possible configurations increase as n2!, counting the magic squares rapidly becomes more difficult. Currently, only the following three values of Nn are known exactly: N3 = 1, N4 = 880, and N5 = 275,305,224 [To state the problem explicitly, we consider classic magic squares order N6 and N7. Their results are N6 = 1.7745(16) × 1019 and N7 = 3.760(52) × 1034, where the digits in parentheses indicate the statistical error of the lowest digits. Trump proposed a more efficient method, called Monte Carlo Backtracking, and estimated Nn for n ≤ 20. [For larger squares, we need to employ statistical approaches for estimating the number of magic squares. There have been two studies in this direction. Pinn and Wieczerkowski applied the exchange Monte Carlo method (EMC)  to this EMC belongs to a family of extended ensemble Monte Carlo methods . Extenden. In order to apply the MMC, we define the energyE(C) of a configuration of numbers C as follows:Si, Sj, and Sk are the sums of the numbers for the ith column, that for the jth row, and that for the kth diagonal. E(C) is zero if and only if C is a magic square, and it takes a positive value otherwise. Thus, the lowest energy (E = 0) configurations provide magic squares. In other words, finding problem of magic squares are formulated as a combinatorial optimization problem. The number of optimal configurations are very large in this case, and we estimate the number using the MMC.Let us consider magic squares of order T as the steady-state distribution of the Markov process, the MMC aims to give a flat energy distribution over the entire energy range. This flatness enables us to estimate the number of configurations of any energy. For that purpose, a predetermined weight function W(E) is used in the MMC instead of the canonical weight eE/T− used in the Metropolis method. W(E) is prepared so that the energy histogram H(E) obtained by Monte Carlo sampling is sufficiently flat. Since H(E) is proportional to the product of W(E) and the number of states g(E) having energy E, we can then estimate relative values of g(E) from W(E) and H(E) asThe MMC was proposed by Berg and Neuhaus in the field of statistical physics to overcome slow convergence of Metropolis-type Markov chain Monte Carlo methods when applied to the sampling of low temperature states of complex systems . In contE = ɛ in randomly arranged configurations of numbers from 1 to n2 is estimated byPn is given by Pn = P(E = 0). Since the total number of configurations is n2!, we can also estimate the total number of magic squares Nn by Pn × n2!/8. It should be noted that, in principle, MMC gives statistically unbiased estimates for the number of magic squares.The appearance probability of energy W(E), we carried out preliminary runs using the Wang–Landau method [W(E) is updated at each Monte Carlo trial until it finally gives a sufficiently flat histogram H(E). We then fixed W(E) and carried out long measurement runs using the entropic sampling method [n, and took averages of Nn and Nn was then estimated as three times the standard error:t is the number of the measurement runs.To determine u method , in whicg method , which in2−1 with n2 were used as an elementary process of the Monte Carlo trial, following Ref. [Only the sequential transposition of adjacent numbers, 1 with 2, then 2 with 3, ⋯, ing Ref. .n. For the largest system with n = 30, for example, we made 40 independent measurement runs of 1.1 × 1012 Monte Carlo trials each. Flatness of the histogram was confirmed by a long independent run that was four times longer than the measurement run. The number of configurations in each bin of the histogram falls within the range from 0.93 to 1.01 of the mean value, which we decided as sufficiently flat.The number of measurement runs and the length of each run are different for each n dependence of Pn. Our estimates of Pn and Nn up to n = 30 are listed in Nn for n = 3,4 and 5 and the previous estimates of Nn by Trump for 6 ≤ n ≤ 20 are also shown for comparison. We obtained N30 = 6.56(29) × 102056. In contrast to this extremely large value, its appearance probability, P30, is smaller than 10−212. Thus the magic squares are numerous but rare.n = 30 is much larger than that which has been calculated previously. The estimates of N3, N4, and N5 agree with the exact values within the statistical error, and the estimates up to n = 17 are consistent with Trump’s values within the statistical error. However, there are appreciable discrepancies between the present results and those of Trump for n = 18 and 19; our values are larger for these sizes. In fact, Trump himself pointed out that the true values for N18 and N19 might be smaller than his estimates based on his own extrapolation formula. We thus think that our estimates are reliable. He also gave estimates of Nn for n > 20 obtained by the abovementioned extrapolation formula. Compared to the present estimates, his extrapolation results have two-digits accuracy up to n = 30.The largest size of Pn decreases rapidly with n. In other words, magic squares become rarer rapidly as n increases. This raises the question: how fast does their appearance probability decrease? It clearly decreases faster than the exponential function exp(−an) with constant a. On the other hand, since the number of possible configurations is n2!, Pn should decrease slower than exp(−n2logn). Considering these facts, we first tried to fit logPn for n ≥ 10 by the second-order polynomial. But the reduced χ2 was larger than 6000 and thus the fitting function was inappropriate. Next we tried functions including logn. The fitting function (An + B)logn + Cn + D with the constants A, B, C and D gave A = −4.99 ± 0.07 with the reduced χ2 = 2.55 and the fitting function (En + F)log(n + G) + H with the constants E, F, G and H gave E = −4.880 ± 0.008 with the reduced χ2 = 2.42. Fitted curves using these two functions are shown in n. Other functions we tried gave larger values of reduced χ2. The reduced χ2 for both functions, however, are still large, and the fittings are not fully satisfactory. We consider the reason is that n = 10 is not yet at the asymptotic region. In fact, when we tried to fit the same functions to data only for larger sizes, n ≥ 20, we obtained the reduced χ2 = 1.30 and 1.37, respectively. Although the errors of the parameters are large, A = −4.6 ± 0.5 and E = −4.88 ± 0.06, reduced χ2 for both functions are rather satisfactory.As seen in Pn/exp{(An + B)logn + Cn + D} and Pn/exp{(En + F)log(n + G) + H}. Both functions seem to express Pn equally well. In any case, since the slope of logPn varies slowly, it is difficult to determine the appropriate functional form from the present results. We need Pn for much larger systems to provide a solid conclusion. Even so, we may conjecture that the logPn decreases asymptotically as an log n with a ≃ 5.E(C). The MMC directly gives the number of the optimal configurations from the histogram of the lowest energy configurations. The present work demonstrates that the MMC is a powerful tool for counting rare configurations of combinatorial problems. We can estimate the appearance probabilities of the optimal configurations as small as 10−212.In this paper, we applied the Multicanonical Monte Carlo method to a combinatorial optimization problem by defining appropriate an energy function


RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between the two cell lines. Sequence analysis revealed that AU-rich elements (AREs) are highly enriched in the 3′UTR of these Wig-1-bound mRNAs. Network enrichment analysis showed that Wig-1 preferentially binds mRNAs involved in cell cycle regulation. Moreover, we identified a 2D Wig-1 binding motif in HIF1A mRNA. Our findings confirm that Wig-1 is an ARE-BP that regulates cell cycle-related processes and provide a novel view of how Wig-1 may bind mRNA through a putative structural motif. We also significantly extend the repertoire of Wig-1 target mRNAs. Since Wig-1 is a transcriptional target of the tumor suppressor p53, these results have implications for our understanding of p53-dependent stress responses and tumor suppression. Wig-1, also known as Zmat3 and PAG608, is an RNA-binding protein (RBP) that can bind and regulate expression of multiple mRNAs. Wig-1 contains three highly conserved C2H2-type zinc-finger domains that mediate RNA-protein interactions . The effWe have previously shown that Wig-1 binds to mRNAs through AU-rich element motifs (AREs) –7. AREs In this study we have performed a systematic analysis of Wig-1-associated mRNAs. Through RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells, we found 286 Wig-1-bound mRNAs that are common in the two cell lines. Network enrichment analysis revealed that Wig-1 target mRNAs are highly connected to Cell Cycle regulation pathways. Furthermore, integration of the data from the RIP-Seq in HCT116 with our previously published gene expression data from the same cells upon Wig-1 knockdown  resultedTo isolate Wig-1-bound mRNAs, we performed RIP assays followed by high-throughput sequencing in HCT116 and Saos-2 cells expressing Flag-tagged Wig-1 (W), as well as Saos-2 cells expressing Flag-tagged Wig-1 with a point mutation in the first zinc-finger (Wmut), which is therefore unable to bind to RNA  Figure . We perf2FC) > 1). We plotted the mean counts of each sequenced transcript in the input sample versus the RIP sample in HCT116 and Saos-2 cells and observed that Wig-1-bound RNAs  gave a total of 2335 and 354 RNA targets in HCT116 and Saos-2 cells, respectively Figure  and S3, Moreover, we integrated the data from our previous study including a microarray analysis in HCT116 cells after Wig-1 knockdown by siRNA , and comp-value < 0.05 by Fisher Exact test). As for MF, a total of 90 and 30 genes from our list corresponded to proteins with catalytic or transferase activity respectively. Among the BP categories, “cellular metabolic process” , “protein localization” (9%) and “cell cycle” (5%) were enriched. In terms of CC, components of the membrane-bounded organelle (59%), endoplasmic reticulum (14%), and mitochondrion (11%) are over-represented in our list of Wig-1-bound mRNAs (To determine the functions of the Wig-1-associated RNAs we grouped them into molecular function (MF), biological process (BP) and cellular compartment (CC) by Gene Ontology (GO) term using the gene set enrichment analysis of the DAVID bioinformatics resource tool . Two hunnd mRNAs .p-values, while the complete list is included in To further characterize the list of Wig-1 associated transcripts we performed network enrichment analysis (NEA) and identified as many as 300 pathways as enriched at significance level NEA false discovery rate (FDR) < 0.01 . We deciWe also performed NEA on the list of genes whose mRNA was bound and regulated by Wig-1 in HCT116 cells . Table 22FC value ranging from 1.3 to 2.6, these targets were neither among the most enriched targets or the bottom group just above our cutoff of log2FC > 1 (corresponding to a twofold enrichment) and 3) presence of AREs in their 3′UTRs as determined by RSAT .In order to validate Wig-1-bound mRNAs, we selected nine Wig-1-associated mRNAs enriched in both HCT116 and Saos-2 RIP-Seq experiments: MAD2L1 (Mad2 Mitotic Arrest Deficient-Like 1), MTHFD2 (Methylenetetrahydrofolate Dehydrogenase (NAPD+ Dependent) 2), CCNG1 (Cyclin G1), EIF4E (Eukaryotic Translation Initiation Factor 4E), CHEK1 (Checkpoint Kinase 1), RMI1 (RecQ Mediated Genome Instability 1), HIF1A , AMD1 (Adenosylmethionine Decarboxylase 1) and CAV1 (Caveolin 1) Table . The selWe performed RIP and assessed the levels of these mRNA targets by qRT-PCR. We also included two negative controls for the validation: the tumor protein p53 inducible protein 3, TP53I3, and the tryptophanyl-tRNA synthetase, WARS, both of which were detected but not enriched after Wig-1 RIP in both HCT116 and Saos-2 cells. To verify whether these targets are also regulated by Wig-1, we knocked down Wig-1 using siRNA (siW1). Our data showed that Wig-1 knockdown leads to decrease levels of MTHFD2, EIF4E, RMI1, and CAV1, while we observed an increase in the levels of HIF1A mRNA. The remaining targets showed little or no regulation, including the negative control WARS and TP53I3 Figure . Intereshttp://www.rsat.eu/) [p-value = 0.018, two-sided proportionality test function in R), while the canonical pentamer (AUUUA) is present at least once in 87% of the Wig-1-bound mRNAs and in 54% of the unbound control group . Moreover, 43% of the unbound control group does not contain any pentamer, while the corresponding number for the Wig-1-bound group is 5%. Another sub-group of AREs is classified as stretches of uridines (Us). Using the same program, we found that 7% of the Wig-1-bound and 2% of the unbound control mRNAs contain a U-stretch  . We conclude that the ARE motifs are significantly enriched in the 3′UTR of the Wig-1-associated RNAs, thus supporting previous results demonstrating that Wig-1 is an ARE-BP.Next, we set out to investigate if the mRNAs bound by Wig-1 share any particular sequence elements. The interaction of Wig-1 and its target mRNAs has been shown to be mediated predominantly by ARE motifs  locatedsat.eu/) . As negae-value < 10−5). In contrast, the opposite analysis and comparison of 3′UTRs from the unbound mRNAs to the bound mRNAs revealed that 3′UTRs from unbound mRNAs are rich in G and C  and among the top motives  when compared to the unbound set. As expected, we note that both the Wig-1-bound and unbound sets were enriched for the cleavage and poly-A signal (AAUAAA) when compared to the random control  in terms of sequence content and secondary structure. These sequences range in length between 425 and 1994 nts, have an average GC content of 32.6%, which is lower than the average GC content of human 3′UTR sequences , and a To further characterize the nine bound 3′UTR sequences we predicted their complete 2D sequence structures using RNAfold software . We assein vitro pull-down assays using biotinylated RNA probes containing the whole candidate 3′UTR or the candidate 3′UTR with the consensus 2D motif deleted  for further validation, along with two additional mRNAs that were detected in the RNA-Seq but not enriched in the Wig-1 RIP sample as negative controls (TP53I3 and WARS). The validation confirmed the RIP-Seq data for all the targets in HCT116 cell line. For Saos-2 we confirmed six targets out of nine as compared to negative control: MAD2L1, MTHFD2, RMI1, EIF4E, CHEK1 and AMD1. For CCNG1, HIF1A and CAV1 the enrichment compared to Wig-1 mutant control was not statistically significant. This validation rates of 100% in HCT116 cells and 67% in Saos-2 cells compare well to results reported by others –31. The Wig-1 knockdown by siRNA led to decreased levels of MTHFD2 , EIF4E,The fact that these five genes  are all very relevant in tumor biology further emphasizes a role of Wig-1 in the regulation of cell cycle and cell proliferation, as well as tumor onset, progression and metastasis. For example, MTHFD2 is a mitochondrial enzyme fundamental in one-carbon metabolism, a metabolic system recently implicated in rapid cancer cell proliferation . Indeed,Caveolin-1 (CAV1) is a membrane protein involved in signal transduction, as well as in numerous other cellular processes including cell cycle, vesicular transport, cholesterol homeostasis and cell migration . During The eukaryotic translation initiation factor 4E (eIF4E) is a key player in translational control. Silencing of eIF4E slows down proliferation, and causes arrest of the cell cycle in the G0/G1 phase, as well as increased apoptosis in many cancer types –43. An aRMI1 is an essential member of the RecQ-topoisomerase III complex that has a role in DNA replication and the replication stress response, and is involved in suppressing sister chromatid exchange and tumorigenesis . Thus, iLastly, HIF-1 alpha (HIF1A), a transcription factor that regulates key genes involved in the glycolysis pathway , is upreIn line with previous reports on Wig-1 bound mRNAs, our current data demonstrate that Wig-1 can regulate targets both positively and negatively, and affect both pro- and anti-growth factors. This is in agreement with what has been reported for other ARE-BPs such as AUF1 and HuR –56. Nonein vivo UV crosslinking in order to create covalent and irreversible bonds between RNA and protein. The bound RNA is then fragmented, resulting in recovery and sequencing of only the portion of the RNA that is bound by the protein, which allows for exact determination of the RNA sequence bound by the protein. The crosslinking also enables precipitation of transient interactions. However, because CLIP-Seq allows identification of also transient and potentially unspecific interactions, while RIP-Seq generally identifies stronger and more stable interacting RNA partners that remain bound without crosslinking through purification and washes, we chose to use RIP-Seq rather than CLIP-Seq, favoring the identification of potentially stronger interactions with Wig-1 at the cost of less information on the exact motives bound by Wig-1. Analyzing these strong Wig-1 bound candidates for common sequence elements that may explain their interaction with Wig-1, we show that Wig-1 preferentially binds to mRNAs that contain AREs in their 3′UTR and 95% of the mRNAs bound by Wig-1 contain at least one AUUUA pentamer. Moreover, de novo motif enrichment analysis revealed that apart from classical AU-rich elements, Wig-1 also favors the binding of motifs that are generally rich in A and Us compared to an unbound control groups. Since protein/RNA interaction may occur over a wide range of affinities, depending on the involvement of both primary and secondary structure elements, we analyzed predicted secondary structures generated from the whole 3′UTR sequences from Wig-1-bound RNAs using the LocARNA software. Our data show that shared consensus secondary structures (the consensus 2D motif) for Wig-1-interacting RNAs have a very low GC content (11%). Interestingly, this consensus 2D motif is localized, in the majority of the cases (eight out of nine), in close proximity to an AU-rich element in the primary sequence of the 3′UTR or – in the ninth case - in the secondary folded structure on the 3′UTR. Moreover, we could experimentally validate that the consensus 2D motif on HIF1A 3′UTR is critical for Wig-1 binding. However, the relevance of the in silico predicted structural elements for binding to other Wig-1 targets need to be tested carefully. As shown in this study, the same 2D consensus element was not required for binding of Wig-1 to MTHFD2 3′UTR. It is not fully understood to what degree RNA secondary structures, as opposed to sequence motifs such as AREs, are important for Wig-1 binding to RNA. It is conceivable that Wig-1 RNA binding is affected by the recognition of a secondary RNA structure, while the effect of Wig-1 on mRNA stability could be conferred by a specific RNA sequence motif.The RNA-immunoprecipitation method used here results in the pull-down of the entire target transcripts, not allowing the identification of the specific RNA sequence required for the RNA-protein interaction. Alternatively, the global RNA interaction partners of a protein can be assayed by CLIP-seq (crosslinking immuno-precipitation and sequencing), which utilizes In conclusion, our study provides a novel and comprehensive view of the RNA-binding properties of Wig-1 and defines more precisely the Wig-1-RNA interaction network. Our data confirm that Wig-1 is an ARE-BP involved in regulation of cell cycle progression and cell proliferation. We also show that Wig-1 is primarily a destabilizing RNA-binding protein. We significantly expand the list of known Wig-1 targets, and provide novel insights into preferred Wig-1 RNA binding motifs. Importantly, we identify a putative 2D motif for Wig-1 mRNA binding to HIF1A mRNA. Since Wig-1 is a target of the p53 transcription factor, our results should provide a better understanding of p53-mediated tumor suppression through its target Wig-1, extending the frontiers of gene expression control from the transcriptional to the post-transcriptional level.2 at 37°C. Cell were transfected with 10 nM all star negative control (siC) or Wig-1_1 (siW1) siRNAs using HiPerFect (Qiagen) according to manufacturers protocol. Transient plasmid transfections were performed using PEI reagent according to standard protocol.HCT116 and Saos-2 cells were grown in IMDM supplemented with 10% fetal bovine serum  or fetal bovine serum with reduced tetracycline  for the tetracycline-regulated Wig-1-expressing Saos-2 cells , 2 mM L-HCT116 or Saos-2 cells were harvested 24 h after transfection or 48 h after induction with 1 μg/ml doxycycline, respectively. RIP and qRT-PCR was performed as described in . Brieflyin vitro transcription using Sp6 polymerase  in the presence of biotin labeling mix containing biotinylated UTPs (Roche) according to the manufacturer's protocol. 7 nM of probes were then incubated with approximately 500 μg of cell lysate of HCT116 cells transiently transfected with pCMVtag2bhWig-1 and used for the pulldown as described in [The pCRII-FAS-3′UTR, pCRII-HIF1A-3′UTR, pCRII-HIF1A-3′UTR-delta2D, pCRII-MTHFD2–3′UTR and pCRII-MTHFD2–3′UTR-delta2D vectors were linearized with Not1 or XhoI and used as templates for RNA libraries for sequencing were prepared using TruSeq RNA Sample Prep  (non-strand-specific) according to the manufacturer's instructions with the following changes. The protocols were automated using an MBS 1200 pipetting station  and all purification steps and gel-cuts were replaced by the magnetic bead clean-up methods as previously described . The cluhttp://www.huber.embl.de/users/anders/HTSeq) was used to calculate gene-level read counts. These read counts were used as input to limma (Linear Models for Microarray Data) [2FC) > 1) and an adjusted p-value less than 0.05 for the difference (IP.W-IP.C)-(input.W-input.C) or (IP.W-IP.C)-(input.W-input.C) and (IP.W-IP.Wmut)-(input.W-input.Wmut) for HCT116 and Saos-2 cell line experiments respectively. C: control (empty vector); W: Flag-Wig-1; Wmut: Flag-Wig-1-point mutant; IP: immunoprecipitated.We used TopHat  (v. 1.3.ay Data)  to perfoWe used DAVID  to perform gene ontology (GO) enrichment analysis . Wig-1-aFor the network enrichment analysis (NEA), we probabilistically estimated putative functional relations between gene sets as described in . The advThe global interaction network was based on the one from FunCoup , a databThe occurrences of the ARE motifs in the 3′UTR sequences of the Wig-1 associated mRNAs were determined using RSAT , a regulPrediction of 2D sequence structure was performed using the program RNAfold (default parameter settings) contained in the ViennaRNA Package 2 . Shared http://emboss.bioinformatics.nl/cgi-bin/emboss/geecee).GC content was calculated using the software geecee (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2840.RIP-Seq data are available in the ArrayExpress database (


Pseudomonas aeruginosa the KH-S1 domains of PNPase are required for the type III secretion system (T3SS) and bacterial virulence. Transcriptome analysis revealed a pleiotropic role of PNPase in gene regulation. Particularly, the RNA level of exsA was decreased in the ΔKH-S1 mutant, which was responsible for the reduced T3SS expression. Meanwhile, the pilus biosynthesis genes were down regulated and the type VI secretion system (T6SS) genes were up regulated in the ΔKH-S1 mutant, which were caused by increased levels of small RNAs, RsmY, and RsmZ. Further studies revealed that deletion of the KH-S1 domains did not affect the transcription of RsmY/Z, but increased their stabilities. An in vivo pull-down and in vitro electrophoretic mobility shift assay (EMSA) demonstrated a direct interaction between RsmY/Z and the KH-S1 fragment. Overall, this study reveals the roles of PNPase in the regulation of virulence factors and stabilities of small RNAs in P. aeruginosa.Post-transcriptional regulation enables bacteria to quickly response to environmental stresses. Polynucleotide phosphorylase (PNPase), which contains an N-terminal catalytic core and C-terminal RNA binding KH-S1 domains, is involved in RNA processing. Here we demonstrate that in  PNPase is a phosphate-dependent 3–5′ exonuclease with homologs identified in bacteria and eukaryotes , reciprocally regulate acute and chronic infection associated virulence factors. RsmY/Z directly bind to RsmA and suppress its function  of 1.0. The bacterial cells were collected and resuspended in phosphate-buffered saline (PBS). Female BALB/c mice (6–8 weeks old) were anesthetized with 0.1 ml of 7.5% chloral hydrate injected intraperitoneally. Then the mice were inoculated intranasally with 2 × 107 CFU bacteria. Twelve hours post infection, the mice were sacrificed. The lungs were isolated and homogenized in 1% proteose peptone. The bacterial loads were determined by serial dilutions and plating. Statistical analysis was performed with the GraphPad Prism software.All animal experiments complied with Nankai University and Chinese national guidelines regarding the use of animals in research. The protocol was approved by the institutional animal care and use committee of the college of life sciences of Nankai University with permit number: NK-04-2012. Overnight bacterial culture was diluted in fresh LB and grown to an optical density at 600 nm (OD5 HeLa cells were seeded into each well of a 24-well plate and cultured in Dulbecco's modified Eagle medium (DMEM) with 10% (vol/vol) heat-inactivated fetal bovine serum (hiFBS), penicillin G (100U/ml), and streptomycin (100 μg/ml) at 37°C with 5% CO2 for 18 h. The medium was replaced with antibiotic free DMEM with 10% hiFBS 1 h before bacterial infection. Overnight bacterial culture was diluted 50-fold in fresh LB and grown to an OD600 of 1.0. Bacteria were washed twice and resuspended in PBS. HeLa cells were infected with indicated strains at a multiplicity of infection (MOI) of 20. After 3 h, the medium in each well was removed. Cells remaining attached were washed twice with PBS and stained with 0.1% crystal violet for 15 min. Then, each well was washed twice with water. For quantification, the stained crystal violet was dissolved in 95% ethanol and measured at the wavelength of 590 nm.Bacterial cytotoxicity was determined by measuring detachment of mammalian cells after bacterial infection as described previously . The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and probed with a rabbit polyclonal antibody against ExoS or a mouse monoclonal antibody against FLAG (Sigma). Signals were detected with the ECL-plus kit (Millipore).proC was used as an internal control.Bacteria were grown in LB medium to indicated growth phases. Total RNA was isolated with an RNA prep Pure cell/Bacteria Kit (Tiangen Biotec). cDNA was synthesized with a PrimeScript Reverse Transcriptase and random primers (Takara). The cDNA was mixed with indicated primers and FastStart Essential DNA Green Master (Roche). The pyrroline-5-carboxylate reductase coding gene www.pseudomonas.com). The RSEM software (V 1.2.15) was used to align the input reads against the reference gene with Bowtie2 and expression values were calculated using the FPKM (fragments per kilobase of transcript per million reads) method. The software edger (V3.4.2) (Bioconductor) was used to calculate p-values.The Transcriptome sequencing and analysis were performed by GENEWIZ . Briefly, total RNA of each sample was quantified and qualified by an Agilent 2100 Bioanalyzer (Agilent Technologies). One microgram total RNA with RIN value above seven was used for library preparation. Large ribosomal RNA was depleted from bacterial total RNA using RiboMinus Bacteria Module (Invitrogen) and the rRNA-depleted mRNA was then fragmented, and primed with random primers. Pair-end index libraries were constructed according to the manufacturer's protocol . The RNA expression analysis was based on the annotations of PAO1  with recombinant RNase inhibitor  and lysed by sonication. After centrifugation, the supernatant was incubated with 30 μl Ni-NTA agarose beads (Qiagen) at 4°C for 1 h. Then the beads were washed 5 times with 1 ml lysis buffer, and incubated with 30 μl elution buffer  for 10 min. After centrifugation, protein and RNAs in the supernatant were collected and subjected to RNA purification with an RNA prep Pure cell/Bacteria Kit (Tiangen Biotec). The purified RNA was analyzed by real time PCR. The gfp RNA was used as the reference to determine the relative level of each sRNA.The C-terminus His-tagged full length PNPase or KH-S1 fragment was constructed in pMMB67EH and transferred into wild type PAK constitutively expressing a 2, 10% glycerol, 1 U recombinant RNase inhibitor (Takara)] for 30 min at room temperature. Fifteen microliters of each sample was loaded onto a non-denaturing 7% polyacrylamide gel. Electrophoresis was performed at 100 V for 150 min with 0.5 × TBE buffer (Tris-borate-ethylenediaminetetraacetic acid) at 4°C. The RNA bands were observed by staining with ethidium bromide (Takara) for 15 min.The sRNA transcripts were synthesized from PCR products using the Riboprobe System-T7 (Promega) according to the manufacturer's instructions. The primers used in PCR are shown in supporting information Table . The RNAP. aeruginosa genome, the PNPase coding gene pnp is 2106 base pairs (bp) long. Our previous study demonstrated that transposon (Tn) insertion at the 1655 bp of the pnp gene in a wild type strain PAK resulted in reduced ExoS secretion  on wild type PAK and the ΔKH-S1 mutant. The two strains were grown to log phase and total RNAs were purified. Compared to wild type PAK, expression of 309 genes were altered, with 182 genes down regulated, and 127 genes up regulated in the ΔKH-S1 mutant Table . ConsistexsA mRNA in the ΔKH-S1 mutant  RpsL levels were similar between PAK and the ΔKH-S1 mutant with or without rifampicin treatment, suggesting a similar degradation rate , suggesting a similar role of PNPase in P. aeruginosa.RNA metabolism and processing are essential for bacterial survival and response to environmental stresses. In Y. pseudotuberculosis and Y. pestis, it was shown that the Δpnp strain was less virulent ; National Basic Research Program of China  and Science and Technology Committee of Tianjin .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev.The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.Our results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2.The online version of this article (doi:10.1186/s12977-014-0115-7) contains supplementary material, which is available to authorized users. The Rev protein of lentiviruses mediates nuclear export of singly spliced and unspliced viral RNA transcripts. HIV-1 Rev binds its target RNA, the Rev responsive element (RRE), as a monomer, then multimerizes along the RRE RNA before being shuttled to the cytoplasm through association with the Crm1 host export factor. Several well-characterized motifs mediate known functions of HIV-1 Rev: a nuclear localization signal (NLS), which overlaps an RNA-binding arginine-rich motif (ARM); a nuclear export signal; and a pair of oligomerization domains that flank the ARM  -6. BiochNMR studies of HIV-1 Rev revealed that the C-terminal half of the protein is intrinsically disordered ; howeverin vitro .EIAV Rev R1 [GenBank:AAG53100] was used as the reference amino acid sequence for generating full-length EIAV Rev165 structural models. R1 was isolated from a pony experimentally infected with EIAV of EIAV . Additio of EIAV ,43; and  of EIAV , the fulgenerate comparative models” option was used for PROTINFO. For the ITASSER server, in addition to default settings, Rev was modeled using an HIV Rev crystal structure (PDB:3lph) [specify template without an alignment” mode; and ii) the “specify template with alignment” mode. Pairwise alignment of R1 and HIV-1 Rev 3lph:A amino acid sequences was generated with the T-Coffee webserver [The QUARK, ITASSER, LOMETS, and PROTINFO protein structure prediction servers were used for automated modeling of Rev and are described in ,37,45,46DB:3lph)  as the sebserver . All modThe QMEAN ,48 and PPairwise protein alignments were performed with the T-Coffee webserver , using ta’ and ‘d’. For the PARCOIL server, a 28-residue window width and a p-score cut-off of 0.05 were used. Default settings were used for CCHMMPROF. The DrawCoil 1.0 server [Coiled-coil motif prediction was performed using the COILS ,52, PAIR0 server  was usedTwo hundred distinct EIAV Rev amino acid sequences from the US, Ireland, and China, were retrieved from the NCBI GenBank protein database  was modeled with ITASSER. The ClusPro 2.0 docking server was used to generate dimeric structures, using default parameters -63.E. coli strain Rosetta Gami in NZY media as described previously [MBP-Rev fusion proteins were cloned and expressed in eviously . FollowiPurified MBP-Rev protein samples were added to 6X Blue native sample loading buffer  supplemented with 0.2% SDS. Samples were analyzed by electrophoresis in 8% Blue native polyacrylamide gels with 50 mM Bis-Tris pH 7.0 anode buffer and 50 mM Tricine, 15 mM Bis-Tris pH 7.0, 0.002% Coomassie brilliant blue G-250 cathode buffer.4 cpm of 32P-labeled EIAV RRE RNA in binding buffer  for 20 min at room temperature. Following incubation, samples were UV-irradiated with 3×105 μJ at 254 nm for 7 min, followed by treatment with 0.1 mg/ml RNase A at 37°C for 2 min. Samples were boiled in an equal volume of SDS for 5 min and separated in 12% SDS-PAGE in Tris-glycine buffer. Gels were fixed in 50% methanol-10% acetic acid, dried, and exposed to phosphorimager screens overnight. UV cross-linked complexes were detected using a PersonalFX scanner and Quality One software .UV-crosslinking RNA binding assays were described previously . Briefly


Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3’ untranslated regions (3’UTRs) in testes.By deep sequencing of the 3’ end region of poly(A) + transcripts, we report widespread shortening of 3’UTR through APA during the first wave of spermatogenesis in mouse, with 3’UTR size being the shortest in spermatids. Using genes without APA as a control, we show that shortening of 3’UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear highly potent during spermatogenesis. We additionally found widespread regulation of APA events in introns and exons that can affect the coding sequence of transcripts and global activation of antisense transcripts upstream of the transcription start site, suggesting modulation of splicing and initiation of transcription during spermatogenesis. Importantly, genes that display significant 3’UTR shortening tend to have functions critical for further sperm maturation, and testis-specific genes display greater 3’UTR shortening than ubiquitously expressed ones, indicating functional relevance of APA to spermatogenesis. Interestingly, genes with shortened 3’UTRs tend to have higher RNA polymerase II and H3K4me3 levels in spermatids as compared to spermatocytes, features previously known to be associated with open chromatin state.Our data suggest that open chromatin may create a favorable cis environment for 3’ end processing, leading to global shortening of 3’UTR during spermatogenesis. mRNAs with shortened 3’UTRs are relatively stable thanks to evasion of powerful mRNA degradation mechanisms acting on 3’UTR elements. Stable mRNAs generated in spermatids may be important for protein production at later stages of sperm maturation, when transcription is globally halted.The online version of this article (doi:10.1186/s12915-016-0229-6) contains supplementary material, which is available to authorized users. Pre-mRNA cleavage/polyadenylation (C/P) is a 3’ end processing mechanism employed in eukaryotes for expression of almost all protein-coding transcripts and long non-coding RNAs by RNA polymerase II (RNAPII) , 2. The Most mammalian genes express alternative cleavage and polyadenylation (APA) isoforms –12. APA A sizable fraction of mammalian genes display APA in introns and exons, which affects coding sequences (CDS) , 25. TheSpermatogenesis is the developmental process to generate mature sperms from spermatogonia  . Two wavWe and others have previously found that pAs usage in testis is rather unique as compared to other tissues , 16, 39.To examine how APA is regulated during spermatogenesis, we extracted RNA from mouse testes at different time points after birth, namely 1 week (w), 2w, 3w, 4w, 5w and 6w. These time points correspond to key phases in the first wave of spermatogenesis  isoform and distal pA (Dis-pA) isoform, respectively , our recently developed method to statistically identify regulated APA events using a bootstrapping approach , and gloCep57 is shown in Fig. There are several cell types in the testis, including spermatogonia, spermatocytes, spermatids, mature sperms and Sertoli cells. Since 2w testes are highly enriched with spermatocytes and 4w testes with spermatids, our result suggests significant 3’UTR shortening during maturation of spermatocytes into spermatids. To confirm cell specificity of APA, we analyzed a strand-specific RNA-seq data set with different cell types purified from the testis . BecauseP <2 × 10−16, Kolmogorov–Smirnov, or K–S, test). To corroborate this result, we analyzed a RNA-seq data set, for which RNAs from testes were sequenced by a strand-specific method .