C. elegans. Here, we identify cytoplasmic signaling molecules required for UNC-6-mediated inhibition of filopodial protrusion involved in axon repulsion. We show that the Rac-like GTPases CED-10 and MIG-2, the Rac GTP exchange factor UNC-73/Trio, UNC-44/Ankyrin and UNC-33/CRMP act in inhibitory UNC-6 signaling. These molecules were required for the normal limitation of filopodial protrusion in developing growth cones and for inhibition of growth cone filopodial protrusion caused by activated MYR::UNC-40 and MYR::UNC-5 receptor signaling. Epistasis studies using activated CED-10 and MIG-2 indicated that UNC-44 and UNC-33 act downstream of the Rac-like GTPases in filopodial inhibition. UNC-73, UNC-33 and UNC-44 did not affect the accumulation of full-length UNC-5::GFP and UNC-40::GFP in growth cones, consistent with a model in which UNC-73, UNC-33 and UNC-44 influence cytoskeletal function during growth cone filopodial inhibition.UNC-6/Netrin is a conserved axon guidance cue that can mediate both attraction and repulsion. We previously discovered that attractive UNC-40/DCC receptor signaling stimulates growth cone filopodial protrusion and that repulsive UNC-40–UNC-5 heterodimers inhibit filopodial protrusion in  C. elegans, UNC-6, UNC-40 and UNC-5 mediate the dorsal-ventral circumferential migrations of growth cones and their axons . Loss of UNC-6 and UNC-5 resulted in VD growth cones that were larger and more protrusive , and activation of UNC-5 and UNC-40 using a myristoylated version of the UNC-40 cytoplasmic domain . UNC-40 was required for the excess protrusion in unc-5 mutants, indicating that, in repelled VD growth cones, UNC-6 and UNC-40 control both pro- and anti-protrusive activity. Furthermore, UNC-6 and UNC-5 were also required to bias protrusion asymmetrically to the dorsal side of the growth cone   . These dC. elegans, these pathways drive neuronal lamellipodial and filopodial protrusion. For example, the Rac-like GTPases CED-10 and MIG-2, CDC-42, UNC-34/Enabled, the Arp2/3 complex, the Rac-specific guanine nucleotide exchange factor (GEF) TIAM-1, and the actin-interacting protein UNC-115/abLIM stimulate neuronal protrusion and mediate attractive axon guidance in response to UNC-40   , which mn (CRMP) , 2010. uolecules . unc-44 f UNC-33 . Here, wunc-73, unc-44 and unc-33, consistent with these molecules acting downstream of UNC-6 receptor signaling.Previous studies found that UNC-73/Trio and MIG-2/RhoG affected the accumulation and distribution of the SAX-3/Robo and UNC-40 receptors in neurons , and thaunc-73(rh40) mutation eliminates the Rac GEF activity of UNC-73 but does not affect other activities (e.g. Rho GEF activity) (unc-73(rh40) mutants VD growth cones had significant increases in filopodial protrusiveness, exhibiting on average longer filopodia (e.g. 0.96 µm in wild type compared with 1.44 µm in unc-73(rh40); P<0.01) and a longer duration of the filopodia once formed (4.9 min in wild type compared with 8.2 min in unc-73(rh40); P<0.01) . Indeed, some filopodia endured throughout the length of the experiment (greater than 20 min). In some cases, the exceptionally long filopodia consolidated into neurites, resulting in a terminated axon with extensive branching. Indeed, unc-73 mutant adults exhibit extensive branching of the PDE neurons  . We foun neurons  and of t neurons , suggestranching .Fig. 2.C. elegans Rac GTPases. CED-10/Rac and MIG-2/RhoG redundantly control axon guidance and mediate neuronal protrusion downstream of UNC-40/DCC in neurons with axons attracted to UNC-6/Netrin , indicating that CED-10 and MIG-2 act redundantly in limiting filopodial length and duration in repelled VD growth cones.CED-10 is similar to the Rho GTPase Rac1  and MIG-6/Netrin . However neurons  ; unc-73(rh40) and ced-10(n1993); unc-73(rh40) displayed increased average growth cone filopodial length compared with wild type, the ced-10; mig-2 double mutant, and unc-73(rh40) (unc-73(rh40) is null for Rac GEF activity (ced-10(n1993) is not a null mutation and retains some function (mig-2(mu28); ced-10(n1993) mutants have wild-type ced-10(+) maternal contribution, so this double mutant does not represent a complete loss of ced-10 and mig-2 function, which might have a more severe filopodial phenotype than mig-2(mu28); ced-10(n1993).The Rac GEF domain of UNC-73/Trio acts as a GEF on CED-10/Rac and MIG-2/RhoG, but not s CDC-42 . To expl73(rh40) E. That uactivity  suggestsfunction , and migunc-33 and unc-44 mutants display branched and prematurely terminated axons, including the VDs  . unc-33 and unc-44 also displayed persistent filopodia that resolved into stable neurite-like structures , resulting in axon branching  . Additional alleles unc-33(e1197) and unc-44(e1193) showed the same effect (data not shown). Thus, UNC-73, UNC-33 and UNC-44 are required for inhibition of growth cone protrusion mediated by MYR::UNC-40. These results are in line with previous studies that identified unc-44 mutations in a screen for suppressors of UNC-5 axon repulsion activity  weakly but significantly suppressed the filopodial duration of myr::unc-40, and mig-2(mu28) weakly but significantly suppressed maximal length ; myr::unc-40 and unc-73(rh40); myr::unc-40 was significantly lower than that of unc-33(e204) and unc-73(rh40) alone, and the maximal length of unc-33; myr::unc-40 was significantly reduced compared with unc-33(e204) alone. This suggests that myr::unc-40 can slightly suppress unc-33 and unc-73, indicating that it might engage effectors in parallel to unc-33 and unc-73 to inhibit protrusion.While ced-10(G12V) and mig-2(G16V) expressed in the VD neurons by the unc-25 promoter . In the PDE axons that are attracted to UNC-6, activated CED-10(G12V) and MIG-2(G16V) result in excess protrusion , with a reduction in average filopodia duration  and length . This phenotype is the opposite of that observed in unc-73 and mig-2; ced-10 loss-of-function mutants, and resembled inhibition of growth cone protrusion caused by activated myr::unc-40  and mig-2(G16V) were introduced into an unc-73(rh40) loss-of-function background, the growth cones resembled those upon activated Rac GTPase expression alone, including significant reduction in filopodia duration and length . That activated CED-10 and MIG-2 were epistatic to unc-73 could mean that CED-10 and MIG-2 act downstream of UNC-73 in the same pathway, or that they act independently of UNC-73 in a parallel pathway, consistent with the double-mutant analysis in When unc-33 and unc-44 with ced-10(G12V) and mig-2(G16V) resembled unc-33 and unc-44 mutants (unc-33 and unc-44 were epistatic to activated Rac GTPases), with excessive growth cone filopodial protrusion as evidenced by increased filopodial length and duration (unc-33(e1197) and unc-44(e1193) showed the same effect (data not shown). These data indicate that UNC-33 and UNC-44 are required for Rac GTPases to inhibit growth cone protrusion and suggest that they act downstream of Rac GTPases in the process.By contrast, double mutants of duration . Additiounc-73 but not unc-33 and unc-44 (unc-73(rh40); mig-2(G16V) and unc-73(rh40); ced-10(G12V) mutants displayed reduced ectopic VD axon branching compared with unc-73(rh40) alone (mig-2(G16V) or ced-10(G12V) did not reduce ectopic axon branches of unc-33 and unc-44 mutants  alone , similar mutants , which iunc-25 promoter . This effect was not dependent upon functional UNC-6, but was dependent upon functional UNC-40, suggesting that a MYR::UNC-5–UNC-40 heterodimeric complex was involved (unc-73(rh40), unc-33 and unc-44 loss-of-function mutations suppressed the anti-protrusive activity of myr::unc-5   B, indicaunc-73, unc-33 and unc-44 mutants , unc-44(e1197) and unc-73 mutants (unc-33(e204) and unc-44(e362) alleles were also analyzed with similar results (data not shown).We used a full-length functional UNC-5::GFP-encoding transgene  to test  mutants . unc-33 (unc-73(rh40) double mutants with mig-2 and ced-10 display increased filopodial protrusion compared with both unc-73(rh40) and the ced-10(n1993); mig-2(mu28) double mutant. Activated ced-10 and mig-2 were epistatic to unc-73 loss of function , consistent with the known role of UNC-73/Trio as an upstream Rac regulator.Previous results suggested that UNC-6/Netrin and the receptor UNC-40/DCC can both stimulate and inhibit growth cone protrusion in the same repelled growth cone, which might result in directed protrusion and migration away from UNC-6/Netrin . Our res73(rh40) . HoweverC. elegans CRMP-like molecule, is required for the inhibition of growth cone filopodial protrusion caused by the UNC-6/Netrin receptors UNC-40 and UNC-5. In cultured mammalian neurons, CRMP4 (DPYSL3) knockdown results in longer filopodial protrusions and more axon branches on myelin-derived substrates  are required for semaphorin-3A-mediated growth cone collapse through a receptor complex that includes Plexin-A and Neuropilin-1 . Here, wbstrates , consistunc-33 and unc-44 mutations were epistatic to activated MIG-2/RhoG and CED-10/Rac, as the double-mutant growth cones displayed excess filopodial protrusions similar to unc-33 and unc-44 mutants alone. This suggests that UNC-33/CRMP and UNC-44/Ankyrin are required for the effects of activated Rac GTPases and that they act downstream of them to mediate filopodial inhibition. CRMPs interact with both the actin and microtubule cytoskeletons. CRMP4 interacts with F-actin in vitro , unc-33 and unc-44, suggesting that these molecules are not involved in guidance receptor localization to the growth cone in this context. Therefore, our results are consistent with a model in which the Rac-like GTPases MIG-2 and CED-10, UNC-73 and UNC-33 directly regulate cytoskeletal organization downstream of MYR::UNC-40–UNC-5 receptors to inhibit protrusion ; I: unc-73(rh40); II: juIs76 [Punc-25::gfp]; IV: unc-5(e53 and e152), unc-33(e204 and e1197), unc-44, ced-10(n1993). Chromosomal locations not determined: lqIs128 [Punc-25::myr::unc-40::gfp], lqIs242 [Punc-25::myr::unc-5::gfp], lqIs204 [Punc-25::ced-10(G12V)] and lqIs182 [Punc-25::mig-2(G16V)]. Extrachromosomal arrays were attained by injection into the germline, and then integrated into the genome via standard techniques  and Punc-25::mig-2(G16V) were analyzed with similar effect, and one was chosen for integration and further analysis. The mig-2(mu28); ced-10(n1993M+) strain was balanced with the nT1 balancer.Experiments were performed at 20°C using standard chniques . Mutatiochniques . Multiplmyr::unc-5::gfp transgene included the coding region, containing both exons and introns, for the C-terminal 557 residues of the UNC-5A isoform (GenBank accession AAB23867), consisting of all of the cytoplasmic domain but not the transmembrane domain or the extracellular domain. This sequence was placed downstream of the unc-25 promoter and fused in frame to the myristoylation sequence MGSSKS at the N-terminus as previously described . In unc-73(rh40); unc-5::gfp, which were subviable and sterile. unc-73(rh40); unc-5::gfp growth cones were not quantified, but those observed showed no gross change in UNC-5::GFP growth cone localization.Images of VD growth cones with MYR::UNC-40::GFP and full-length UNC-40::GFP and UNC-5::GFP were taken as described above. Using ImageJ, the perimeters of the growth cones were traced, and the average pixel intensity in the defined growth cone area was reported. At least ten growth cones for each genotype were analyzed, except for


Using data from the 2011 Behavioral Risk Factor Surveillance System (BRFSS), we examined households in 13 states  in which the respondent or another adult household member experienced increased confusion or memory loss (ICML) in the preceding 12 months. A total of 12.6% of households reported at least 1 adult who experienced ICML, and in 5.4% of households all adults experienced ICML. Based on these results, an estimated 4 million households in these 13 states have a member with ICML, potentially affecting more than 10 million people. This study can inform public health communication campaigns aimed at increasing awareness of the signs and symptoms of cognitive decline and augment community planning efforts so that the needs of households in which 1 or more adults has cognitive decline are considered. In 2011, the Behavioral Risk Factor Surveillance System (BRFSS) added questions about increased confusion or memory loss (ICML) experienced during the previous 12 months by adult(s) in the household. Thus, unique information about ICML at the household level is available. Our objective was to describe the number and characteristics of households that have members with ICML using available household-level variables . Because memory problems can affect medication adherence and can cause safety concerns, we also described characteristics of households in which all adults (aged ≥18) experienced ICML.http://www.cdc.gov/brfss/annual_data/annual_2011.htm. In BRFSS, 1 adult is randomly selected to represent each household. Our study focused on the household, defined as a unit consisting of all people occupying a residence intended for use as a separate living quarter.The BRFSS consists of annual state-based telephone surveys of randomly selected noninstitutionalized adults on health practices and risk behaviors. Details of the 2011 BRFSS and its response rates are available at Responses to 2 questions were used to determine the number of adults in the household who experienced ICML: 1) “During the past 12 months, have you experienced confusion or memory loss that is happening more often or is getting worse?,” and 2) “How many adults 18 years or older in your household  experienced confusion or memory loss that is happening more often or is getting worse during the past 12 months?”Data were included from 13 states that had available household weights and included ICML questions on their 2011 surveys .Cellular telephone and split-sample surveys were excluded because household weights were not available. Data on the reported number of people in a household with ICML were compared with data collected as part of the initial BRFSS selection process on the number of people in the household. Inconsistent reports were found in less than 0.3% of total households and were removed. Additionally, data on respondents with missing values for ICML were excluded.Households  were classified according to whether any person in the household experienced ICML and whether all persons in the household experienced ICML. Analyses were performed from January through May 2014 using Stata version 11 (StataCorp LP) and adjusted using household weights. We used the weighted estimates from the BRFSS along with data from the 2011 American Community Survey to estimate the number of households with members experiencing ICML and the number of people in those households.A total of 12.6%  of households reported at least 1 adult with ICML, and 5.4% of households reported that all of the adults had ICML . In 6.9%Among households with at least 2 adults, 13.4%  had at least 1 adult with ICML. In 4.7%  of households with at least 2 adults, only the respondent had ICML; in 5.5% , the respondent reported that at least 1 other adult had ICML; and in 3.1% , the respondent and at least 1 other adult had ICML.The percentage of households in which at least 1 adult had ICML increased with the number of adults in the household, ranging from 11.2% in households of 1 adult to 18.5% in households of 3 or more adults . In housOur study is the first to report on ICML in households and provides a glimpse of the reach of ICML directly or indirectly into households. Other research on ICML in individuals and associations with functional difficulties and chronic conditions, using data from 21 states, is being reported in this issue by Anderson and colleagues –5. CogniOf all chronic conditions, brain-related conditions impose the greatest risk to the psychological well-being of other family members . BecauseOur findings are subject to limitations. Data were collected from 13 states and cannot be construed as national averages. Data are self-reported and subject to bias, particularly when respondents report on “other” adult(s). The survey design is cross-sectional, so causality cannot be determined. The ICML measure is not clinically validated and may not correspond to specific diagnoses. Finally, using household weights restricted analyses to household-level variables. Additional questions for households would enhance future research.These findings highlight the magnitude of the problem of cognitive decline and can help inform public health programs and policies. For example, increasing awareness about recognition of signs and symptoms of cognitive decline in self or others can allow household members to seek medical advice and plan for future needs.


Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of  Xist residing within exon 1. However, the function of exon 7 of Xist RNA, which is the second longest exon, remains poorly understood. Our objective was to clarify the role of this exon in X-inactivation through the use of Xist truncation mutant female ES cells. Here, we provide evidence that Xist exon 7 is required for the stable localization of Xist RNA and X-linked gene silencing on the inactive X-chromosome.To balance gene expression from X-chromosomes between males and females, one of the two X-chromosomes is inactivated in female mammals. X-chromosome inactivation is a chromosome-wide epigenetic gene silencing mechanism regulated by long non-coding Xist RNA. Mouse Xist RNA is commonly organized into 7 exons, with the extensively studied and known important domains of  In eukaryotes, an overwhelming majority of genomes are transcribed as non-protein-coding RNAs (ncRNAs) ,2. One oXic) are known to interact to initiate X-inactivation in one of the two X-chromosomes in females [Xic of the future inactive X-chromosome (Xi) at the onset of X-inactivation and has a pivotal role in initiating X-inactivation in cis [XIST/Xist [Xist deletion from the Xi induces the partial de-repression of the X-linked genes [Xist in murine hematopoietic stem cells after the establishment of X-inactivation leads to a genome-wide aberration in gene expression, especially in the expression of X-linked genes, and an induction of highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome in a female-specific manner [Xist in the maintenance phase of X-inactivation to prevent cancer transformation and progression. Therefore, proper regulation of Xist is critical in both the initiation and maintenance phases for cell survival, cellular differentiation and development, and the prevention of cancer pathogenesis in mammalian species.X inactive-specific transcript (Xist) RNA is one such lncRNA, which regulates chromatin organization and transcriptional gene silencing in one of the two X-chromosomes to equalize the X-linked gene dosage between males and females . In the  females ,12. Xistn in cis . Highly n in cis  and recrn in cis ,16. Xistn in cis . There iIST/Xist ,19; howeed genes ,20. Indec manner . This fiXist transgene has identified the functional domain of Xist RNA for gene silencing, and broad redundant region for Xist RNA localization on the Xi and formation of macrochromatin bodies (MCB) associated with histone variant macroH2A1 [Xist-specific repeat motifs contained within exon 1 and conserved in various eutherian mammalian species are involved in the localization of Xist RNA on the Xi [Xist gene body through repeat C of Xist RNA and YY1 binding sites near repeat F in Xist, respectively; it is believed to anchor the Xist RNA to the Xist gene on the Xi as a nucleation center for Xist RNA spreading [Xist repeat C with peptide nucleic acids (PNAs) or locked nucleic acids (LNAs). This fact contrasts with findings that a lack of repeat C does not affect the localization of Xist RNA on the Xi of the transgene assay [Xist RNA has multiple functional domains and directly or indirectly interacts with various proteins such as transcription factors, chromatin modifying enzymes and scaffold proteins . ComprehacroH2A1 . This apacroH2A1 –31. Seven the Xi –34. Tranne assay ,32,33.Heterogeneous nuclear ribonucleoprotein U  has an essential role for Xist RNA localization on the Xi . InteresXist exon 7 in X-inactivation, we generated Xist exon 7-truncated mutant ES cells by inserting tandem polyadenylation (tpA) signals. In this paper, we report our novel findings regarding the interplay between the Xist exon 7 and hnRNP U for stable localization of Xist RNA on the Xi and X-linked gene silencing.While most Xist RNA functional domains for RNA localization and gene silencing are mapped within exon 1 of Xist RNA, the function of exon 7 in Xist RNA is unknown in spite of the presence of a well-conserved repeat E among the eutherian mammals . To elucXist coupled with splice acceptor (SA)-internal ribosomal entry site (Ires)-hygromycin (Hyg)-pA for Tsix truncation to induce non-random X-inactivation of the mutant X. This resulted in the simultaneous truncation of both Xist and Tsix (XistdelEx7TsixTST6) (Mus musculus 129SvJ (129) X-chromosome and one Mus castaneous (Cast) X, which enabled us to perform allele-specific analysis based on single nucleotide polymorphisms (SNPs) between these mouse strains [Tsix truncation mutant by inserting SA-Ires-Hyg-pA at Xist intron 6, resulting in TsixTST6 female ES cells  in control TsixTST6 and two XistdelEx7/TsixTST6 mutant cells, the Tsix transcript was efficiently truncated downstream  of the pA insertion site. These mutant ES cells allow us to address the effect of the Xist mutation on X-inactivation because the Tsix mutation caused a non-random inactivation of the mutant 129 X-chromosome [To investigate the role of exon 7 in mouse Xist RNA, we inserted two tandem polyadenylation signals (2xtpA) into the end of exon 6 of sixTST6) . We used strains ,48. As aES cells . Allele-mutation . While tromosome .Xist exon 7  by allele-specific RT-PCR analysis using a polymorphic restriction enzyme digestion with wild-type, control TsixTST6 and XistdelEx7TsixTST6 mutant cells  [Xist truncation mutant deletes approximately 40% of full length Xist RNA (7.5 kb out of 17.8 kb), we investigated whether an Xist truncation mutation lacking exon 7 affects the stability of the RNA by measuring the half-lives of 5-ethynyl uridine (EU) pulse-labeled Xist RNAs  of the Xist RNA lacks a large region of exon 7 in a long isoform (L-isoform) except for a 1.1 kb repeat E and 0.5 kb region of its 3´ end by alternative splicing, the S-isoform Xist RNA is similar to the mutant XistdelEx7 RNA lacking the entire exon 7. We examined whether the L- and S-isoforms Xist RNA have a different stability. The L-isoforms of the Xist RNA exhibited a slightly longer half-life in wildtype 16.7 and cells TsixTST6 mutant  . Furtherist RNAs . The hal we used . Similar6 mutant , suggestXist truncation mutation influences the ES cell differentiation (TST6 and XistdelEx7TsixTST6 mutant cells were similarly decreased as the embryoid body (EB) differentiation progressed. Moreover, the outgrowth of the XistdelEx7TsixTST6 mutant cells was similar to that of the control TsixTST6 mutant cells at day 8 upon differentiation, with a slightly slower growth  to observe the accumulation of Xist RNA and a hallmark of facultative heterochromatin marker, H3K27me3, on the Xi upon differentiation. In the TsixTST6 mutant cells, the number of robust (strong) Xist RNA cloud- and H3K27me3-positive cells gradually increased upon differentiation. This is greater than that in the wild-type 16.7 ES cells at each time point, likely due to faster Xist induction by the Tsix mutation in TsixTST6 mutant cells [TST6 mutant cells, the immuno-RNA FISH results in the XistdelEx7TsixTST6 mutant cells indicated a significantly reduced number of Xist RNA cloud- and H3K27me3-positive cells, despite the comparable levels of Xist expression between the mutant and control TsixTST6 cells  Xist signal. While the TsixTST6 mutant cells exhibited strong Xist clouds associated with H3K27me3 staining in approximately 65~70% of the cells on both day 8 and day 12, the co-localization of strong Xist clouds and H3K27me3 in the XistdelEx7TsixTST6 mutant cells was less than 20% and 14% on days 8 and 12, respectively. The difference in terms of the percentages of the Xist cloud-positive nuclei between the TsixTST6 and XistdelEx7TsixTST6 mutant cells became more significant as differentiation progressed (delEx7TsixTST6 mutant cells on days 8 and 12 were significantly decreased from 45% to 24% (clone #1) and from 37% to 19% (clone #2), respectively. In contrast, the TsixTST6 cells maintained a high percentage of Xist cloud-positive cells (over 75% Xist cloud-positive) during the transitional period from day 8 to day 12. Our results suggest that exon 7 of the Xist RNA is essential for stable Xist RNA localization, especially in the maintenance of Xist RNA localization and associated H3K27me3 modifications on the Xi.Having shown that neither the control Tsixnt cells . In contlls Figs  and 2. Iogressed . NotablydelEx7TsixTST6 mutant clones exhibited impaired localization of the Xist RNA and H3K27me3 on the Xi. This observation prompted us to investigate whether the compromised Xist RNA and H3K27me3 localization on the Xi affects the expression status of X-linked genes on the Xi during EB differentiation using two different approaches , respectively. In contrast to the control cells, the mutant 129 allele-specific RT-qPCR for Mecp2 and Pgk1 in the XistdelEx7TsixTST6 mutant clones showed that the partial silencing of Mecp2  and Pgk1  occurred on day 8 instead of on day 0. The partial silencing of Mecp2 and Pgk1 in the XistdelEx7TsixTST6 mutant cells was then followed by the reactivation of Mecp2  and Pgk1  on day 12 upon differentiation, although the initial phase of silencing in Mecp2 and Pgk1 on day 4 was similar between the TsixTST6 and XistdelEx7TsixTST6 mutant cells. Additionally, to confirm the compromised Mecp2 and Pgk1 expression from the 129 Xi relative to that of the wild-type Cast Xa in the XistdelEx7TsixTST6 mutant cells, allele-specific RT-PCR analysis based on a polymorphic restriction enzyme digestion was performed  analysis was carried out using Neuro2a cells as previously reported  on day 12 upon differentiation   . Approxills Figs  and 5E. lls Figs  or to inT6 cells . These dXist exon 7 is necessary for the localization of Xist RNA during X-inactivation. Our results show that the aberrant localization of mutant Xist RNA lacking exon 7 on the Xi leads to the loss of the Xist cloud and the H3K27me3 modification on the Xi and impairs X-linked gene silencing due to the impaired binding affinity of Xist RNA to hnRNP U which bridges the Xist RNA and the Xi.In this study, we provided novel evidence that Xist mutations on chromosome silencing and coating, tetracycline (tet)-inducible mutant Xist RNA constructs containing exon 7 specific deletions were generated and introduced into the Hprt locus of male ES cells [Xist truncation mutant at the endogenous locus exhibited a severe phenotype in terms of X-inactivation  displayed dispersed localization of XIST RNA on the targeted locus, yet could still induce adjacent EGFP silencing [XIST, exon 6, might be more crucial for XIST RNA localization on the Xi than exon 1 in human cells.Interestingly, the tet-inducible ilencing . Furtherilencing . Whereasilencing ,33,51. TXist gene showed that hnRNP U broadly bound not only to exon 1 of Xist RNA but also to exon 7  for homologous recombination in bacteria was inserted into the BstBI/SacI of pBS-2xF3-Zeo, yielding pBS-2xF3-R85. The SpeI (Klenow-filled)-NotI SA-Ires-Hyg-tpA of pBS-SA-Ires-Hyg-tpA and the left arm  were inserted into the HindIII (Klenow-filled)-XhoI of pBS-2xF3-Zeo-R85. tpA was replaced by an Eco53kI-NotI beta-actin pA (bpA) fragment of pGEM11-DTAL. For the Xist/Tsix truncation, the SA-tpA cassette was cloned into pBS-SKII(-). SA was replaced with an additional tpA fragment which was amplified with pA-BamXmaI-F and pA-BglII-R, yielding pBS-2xtpA-F. The left arm of the Tsix-truncation targeting construct for bacterial recombination was replaced with the left arm for Xist/Tsix-truncation (adaptor with XST-F7 and XST-R7) and EcoRV-NotI 2xtpA cassette from pBS-2xtpA-F. Selection cassettes with left and right homology arms for bacterial recombination were released by SalI-Bsu36I digestion and inserted into pGEM-Xex2-7-DTAR at position chrX: 103,468,781 and chrX: 103,468,781–103,468,821 in GRCm38/mm10 (UCSC genome browser) for the Tsix- and Xist/Tsix-truncation constructs, respectively. The Zeo selection marker was finally removed from the targeting constructs for recombination in bacteria by arabinose-inducible Flpe recombinase in the SW105 E. coli. The Tsix- and Xist/Tsix-truncation vector was linearized by XhoI digestion for homologous recombination in the ES cells.The targeting vectors for the bacteria . A 9.3 kP1 clone  was clonP1 clone , yieldinP1 clone  was subcP1 clone  was clon6 cells and 40 μg of the linearized targeting vector was used for electroporation using the BioRad GenePulser . 250 μg/ml hygromycin was added at 24 hours after transfection, and hygromycin-resistant colonies were picked on 8–9 days after electroporation. Colonies were screened by genomic PCR using 7.2-F and Hyg-F or SA-R and 7.2In-R primer pairs ; Nanog ; Oct3/4 ; Xist exon 1–3 (X1) for 129 allele ; Xist exon 7 (X2) for 129 allele ; Tsix intron 3–4 (T1) for 129  and Cast  alleles; Tsix exon 4 (T2) for 129 (Tsix129-E4-F and Tsix129-E4-R) and Cast  alleles; Mecp2 for 129 allele ; Pgk1 for 129 allele . The ΔΔCt method was employed to analyze the relative changes in the gene expression levels from the real-time qPCR experiments, and Gapdh was used to normalize the data.The total RNA was isolated from undifferentiated ES and differentiating cells on days 0, 4, 8 and 12 upon differentiation by NucleoSpin RNA II (Clontech). A 2.5 μg of total RNA was converted into cDNA using Maxima H Minus reverse transcriptase (Thermo Scientific) according to the manufacturer's instructions. Real-time PCR was performed on Step One Plus real-time PCR System (Life Technologies) using the Fast SYBR Green Master Mix (Life Technologies). The primers and annealing temperature were: 32P-end-labeled oligonucleotide probe. The primers, probes and restriction enzymes were: Xist . Typhoon phosphorimager  was used to obtain quantitative measures. The data were quantified using GelEval  or ImageQuant .Allele-specific RT-PCR was performed as described . Briefly-R; XSP1 ; ScrFI);-R; NS65 ; DdeI); Xist exon1-3 (X1), the S-isoform Xist specific primer set (XiI7SRT-F and XiI7SRT-R) or the L-isoform Xist specific primer set (XiI7LRT-F and XiI7LRT-R).The half-life assay for Xist RNA was performed with Click-iT Nascent RNA Capture Kit (Life Technologies) following the manufacturer’s instructions. Briefly, undifferentiated ES cells or EB were seeded to gelatinized 6-cm dishes and cultured with ES medium containing 0.1 mM 5-ethynyl uridine (EU) for 22 hrs. The next day, the EU-containing medium was replaced with EU-free medium, and cells were harvested at 0, 40, 80, 120, 180 and 240 min after changing the medium. 5 μg of total RNA extracted as above was biotinylated via the Click-iT reaction, precipitated by ethanol precipitation and dissolved in 50 μl of water. 1 μg of the biotinylated RNA was bound to Dynabeads MyOne Streptavidin T1 magnetic beads (Life Technologies) for 30 minutes at room temperature, and washed with 5x Click-iT reaction buffer 1 and 5x Click-iT reaction buffer 2. The biotinylated RNA-bound beads were resuspended with 12 μl of 5x Click-iT reaction buffer 2 and used as a template for RT-qPCR. RT-qPCR was performed as described above, with the 129 allele-specific primer set for Immuno-FISH was performed as described  with somImmunofluorescence was carried out as described previously  using hu2 at 254 nm. The cells were lysed in SDS buffer  and incubated on ice for 10 min. Samples were sheared for 6 cycles of 30 sec ON at high power/30 sec OFF with the Bioruptor (Diagnode). Sonicated lysates were diluted 10-fold with dilution buffer . The soluble fraction was obtained by centrifuging at 15,000 rpm for 15 min at 4°C. One-tenth of the lysates for immunoprecipitation was kept as the “10% of input”. Anti-Flag M2 beads (Sigma), or protein G beads (Millipore) conjugated with 3 μg of control IgG (Jackson or Millipore) or anti-hnRNP-U  were added to the cell lysates and rotated at 4°C for 2 h. The beads were washed twice with high salt buffer  and three times with low salt buffer . The washed beads were treated with proteinase K at 37°C for 1 h, and the RNA was extracted with Trizol LS Reagent (Life Technologies) according to the manufacturer’s instructions. Purified RNA treated with DNase I was dissolved in 20 μl nuclease free water, and cDNA was synthesized using 2 μl of the RNA samples. Quantitative PCR was performed using the primers listed in UV-Crosslinking RIP was performed following a previously described procedure  with som6 ES cells were plated on 6-well plates with feeder cells 8 hours before transfection. Cells were transfected with FuGENE HD (Roche) according to the manufacturer’s instructions, with 1 μg pX459FE-hnRNPU vector and 1 μg single-stranded oligodeoxynucleotide (hnRNPU-FLAGHA-KI) for the homology directed repair (HDR)-mediated FLAG-HA knock-in. 16 hour after transfection, 2 μg/ml of puromycin was added to the ES media and ES cells were cultured for 24 hours. After puromycin selection, cells were trypsinized and 1/5 of cells were plated onto a 10 cm dish with feeder cells without any selection drug. Colonies were picked on 8 days after plating.pSpCas9(BB)-2A-Puro   set on “HIGH” for 4 cycles of 30 sec ON and 30 sec OFF. Equal amounts of total protein (20 μg per lane) were applied and run on a 4–15% SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore). Membranes were blocked by 5% skim milk in TBST  at room temperature for 1 h, and incubated using primary antibodies against FLAG M2 , hnRNP U  and alpha Tubulin DM1A  overnight at 4°C. After incubation with the secondary antibody conjugated to horseradish peroxidase (HRP)  at room temperature for 1 h, immune complexes were visualized using an enhanced chemiluminescence (ECL) detection system (Thermo Scientific) or Immobilon Western chemiluminescent HRP substrate (Millipore), and quantitated by AlphaView (Alpha Innotech).S1 FigXist/Tsix locus. Arrows indicate the position and direction of the primers used. (B) Genomic PCR analysis to confirm FLAG-HA knock-in.(A) Map of the mouse (EPS)Click here for additional data file.S2 FigXist with the 3´ end of the XistdelEx7 mutant RNA. (B) RT-qPCR was performed using the S-isoform-specific (XiI7SRT-F and XiI7SRT-R) and L-isoform-specific primer sets (XiI7LRT-F and XiI7LRT-R). The mean ± SD values from two independent experiments are shown.(A) A map shows alternative splicing of (EPS)Click here for additional data file.S3 Fig(A) RT-qPCR analysis for the expression of pluripotent markers (Nanog and Oct3/4). Each Nanog and Oct3/4 expression level was normalized to that of differentiation day 0 (set to 1) and Gapdh, which was used as an internal control. The mean ± SD bar for three independent experiments is shown. (B) Representative phase contrast images of the differentiated EB cells on differentiation at day 8.(EPS)Click here for additional data file.S4 FigImmunofluorescence of hnRNP U (magenta) and histone macroH2A (green) in the control and hnRNP U knockdown HEK293T cells. The arrowhead indicates a hnRNP U knockdown cell.(EPS)Click here for additional data file.S5 FigXist exons 1 to 3. The expression value was normalized to that of TsixTST6 at day 0  and Gapdh was used as an internal control. The graph shows the mean ± SD bar from three independent experiments. (B) 129 allele-specific RT-qPCR analysis of the X-linked Pgk1 gene upon differentiation, normalized to undifferentiated cells (set to 1), with Gapdh used as an internal control. The mean ± SD bar from three independent experiments is shown. P values were calculated using an unpaired t test . (C) Top: Immuno-FISH for Xist RNA (green) and H3K27me3 (red) in FLAG-HA targeted knock-in ES cells after differentiation (day 12). Nuclei were counterstained with DAPI. Scale bar, 10 μm. Bottom: frequency of the Xist cloud- and H3K27me3-positive cells on day 12 upon differentiation. More than 300 nuclei in each ES cell line at each time point from three independent experiments were counted and classified based on the Xist RNA and H3K27me3 signal.(A) 129 allele-specific RT-qPCR analysis of Xist expression in the FLAG-HA targeted knock-in ES cells using primers that extend through (AI)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds. Trypanosoma brucei—an extracellular parasite that multiplies in the blood and the lymphatic and cerebrospinal fluids. Some progress in the development of new trypanocidal compounds has recently been made by reformulating existing drugs while at least two other compounds are currently being tested in clinical trials  = -0.9xΔn [Absorbance ed as in . The numed as in ,32. The 2/ΔH0)Δn .CD measurements of pre-annealed gRNA/pre-mRNA hybrid RNAs were carried out at 27°C in 10mM Na cacodylate, pH6.8 and 65mM NaCl. Spectra were recorded from 205–315nm at an RNA concentration of 12μM. Neomycin B was added from a concentrated stock solution in 10mM Na cacodylate, pH6.8 and 75mM NaCl to yield final concentrations between 0–0.6mM. Samples were equilibrated for 5min before measuring. Resulting spectra were Savitzky-Golay smoothed  and corr© 2.0 (Pentafolium-Soft). Neomycin B molecules were docked into the modeled RNAs using Autodock Vina 1.1.2 [www.pymol.org). Autodock Tools [Guide RNA/pre-mRNA hybrid RNAs were interactively modeled using ERNA3Dna 1.1.2 . File cona 1.1.2 . All docck Tools  was usedck Tools  for geneck Tools . Surfacein vitro RNA editing assays [50) inhibited at a concentration of 30±8μM and the IC50-value for inhibiting U-deletion editing is 31±2μM. All data are summarized in 50-values of 10±1μM and 13±2μM (U-deletion/U-insertion) followed by neamin (31/30μM), tobramycin (47/34μM) and sisomycin (36/60μM). The macrolides erythromycin, azithromycin and spiramycin and the aminocyclitols apramycin, streptomycin and hygromycin B showed no effect (IC50≥100μM).Based on the published data on the inhibition of translation, pre-mRNA splicing and other RNA-driven reactions  we testeg assays ,28. BothImportantly, the inhibitory effect shows pleiotropic characteristics: next to inhibiting the formation of the fully edited pre-mRNA other steps of the processing cycle are affected as well. In the case of the U-deletion, the exoUase trimming reaction at the 3’-end of the 5’-pre-mRNA fragment is inhibited, although to a lesser degree. Furthermore, religation of the “faithfully” edited reaction product is more sensitive to the aminosugars in comparison to the formation of the unedited ligation side-product. However, while the formation of edited products in both cases is completely stalled at concentrations ≥100μM, the inhibition of the exoUase is not. Lastly, we also analyzed the endonuclease cleavage step of the editing cycle. mRNA cleavage is similarly inhibited in a dose dependent manner and is completely abolished at ≥30μM neomycin B .d) of 1.1μM for the U-insertion gRNA/pre-mRNA hybrid and of 1.0μM for the U-deletion RNA  from which the fraction of RNA molecules having 1, 2 or 3 binding sites occupied was determined  binding experiments. At neomycin B concentrations up to 10-fold above the determined Kentified . Similardetected .m-values were derived from 1st-derivative plots and identified melting midpoints at temperatures ranging from 47°C to 65°C. In the presence of 1.3μM neomycin B (1.3-fold above Kd) the individual melting transitions shift to higher temperatures with ΔTm’s between 11–13°C. This indicates a strong stabilization of the helical elements in both RNAs equivalent to ΔΔG-values of-7 to-23kJ/mol. All binding reactions show saturation characteristics, which allowed the determination of the number of neomycin B binding sites per RNA  of the RNA/aminosugar interaction. For that we performed UV-melting experiments of gRNA/pre-mRNAs hybrid RNAs in the presence of defined concentrations of neomycin B. Representative melting curves for the U-insertion and U-deletion RNAs are shown in  per RNA . For the+-ion concentrations (5–250mM) in the absence and presence of neomycin B. According to polyelectrolyte theory the melting temperature (Tm) varies logarithmically with the salt concentration and the slope of the linear relationship is proportional to the difference in bound counter ions (Δn) in the folded and unfolded RNA. m = f(log cNa+) plots are characterized by positive slopes (∂Tm/∂logcNa+) indicating a release of counter ions upon RNA melting. Conversely, in the presence of 13μM neomycin B (13-fold over Kd) the slopes are negative demonstrating an uptake of Na+-ions during melting. This implies that the temperature-induced strand separation of the individual RNA helices causes the bound aminoglycoside molecules to dissociate thereby freeing negative charges, which are neutralized by an uptake of Na+-ions. The calculated Δn-values vary from 1.8 to 2.6 in the absence of neomycin B and from-1.1 to-1.7 for the neomycin B-bound RNAs  the conformational adaptability of the sugar molecules and (ii) their “overall” electrostatically-driven RNA binding mode ,23. In ound RNAs . DependiTo analyze whether the aminosugar-induced thermodynamic stabilization of editing substrate RNAs is a result of structural changes in the gRNA/pre-mRNA hybrid molecules we performed a conformational analysis using circular dichroism (CD) spectroscopy. Representative spectra are shown in © 2.0 using standard A-form geometries for the helical elements and maximal stacking of the ss-nucleotides at the editing junction [In order to gain a structural understanding of the aminoglycosid-mediated inhibition of editing we decided to model the two gRNA/pre-mRNA hybrid RNAs based on the above-described experimental data and to predict possible neomycin B binding sites by ligand docking. The two RNAs were interactively modeled with the help of ERNA3Djunction . Neomycijunction ,43,44. NThe RNA editing reaction within the mitochondria of African trypanosomes represents an ideal drug target for two reasons: First, editing is a required biochemical pathway for the survival of the parasite and second, the reaction cycle involves a multi-enzyme machinery (the editosome) and a class of small, non-coding RNAs (gRNAs) that only exist in the parasite. Although some editosome-interacting compounds have been identified , no inhi3+ groups contribute to a single neomycin B-gRNA/pre-mRNA interaction. Within the context of the editing reaction this suggests that the editosome/substrate RNA interaction relies on a defined number of negative charges, which when neutralized cause the processing reaction to stall. This is further supported by the fact that in vitro RNA editing is performed at low monovalent cation concentrations (≤30mM) because the reaction is known to be sensitve to high salt conditions.Here we demonstrate that the aminosugars neamin, neomycin B, tobramycin and sisomycin inhibit RNA editing at micromolar concentrations. These values are in a similar range to the inhibitory concentrations for other RNA-driven biochemical processes such as the neomycin B-induced inhibition of the hammerhead ribozyme , the inhAsp, locks the RNA in a non-functional conformation [Furthermore, the data suggest that the processing reaction requires a defined structural flexibility of the gRNA/pre-mRNA hybrid RNAs. Aminosugar binding induces a stabilization or “stiffening” of the substrate RNAs, which leads to inhibition. This scenario is supported by the observation that editosomes are known to tolerate dynamic RNA rearrangements at editing sites simply because the primary sequence of the pre- and partially edited mRNAs is constantly changed as a result of the numerous U-insertions and deletions. An inability to re-organize the structure of the gRNA/pre-mRNA hybrid molecules might therefore stall the reaction. This is supported by the fact that editosomes have been shown to execute RNA chaperone activity to resolve folded RNA motifs  in additormation  and the ormation .st-hydration shell of RNAs have been shown to add to the plasticity of RNA/ligand interactions [in vitro RNA editing is inhibited at crowded solution conditions [Importantly, the inhibitory effect is not a result of a direct structural interference of the aminosugar molecules at the editing site. This is supported by our CD and modeling/docking data. Instead, it’s a consequence of multiple binding events distal to the RNA editing site, which is transduced to the catalytic domain. A likely canditate for this mediator function might be the water structure of the substrate gRNA/pre-mRNA hybrid RNAs. Electrostatically confined water molecules in the 1ractions ,60,23. Tnditions . Synthetnditions ,62.The described results can be used to define a set of criteria to search for improved RNA-interacting editing inhibitors for instance by structure-guided approaches as in the case of HIV-1 RNA  or by viS1 Fig2- or OH-substituents that distinguish the different aminosugars are listed next to their names. Sisomicin and geneticin, both 4,6–2-DOS-linked aminoglycosides, share a common scaffold consisting of rings II and III.Roman numbers annotate the different ring structures, arabian numbers label individual C-atoms. Ring II represents the core 2-DOS scaffold. Grey lines are used to delimit the individual aminoglycosides. The various NH(TIF)Click here for additional data file.S2 FigTop: pre-cleaved U-insertion gRNA/pre-mRNA hybrid RNA. Bottom: pre-cleaved U-deletion gRNA/pre-mRNA. gRNA-“guiding” nucleotides are in grey. U-nucleotides to be deleted are in black.(TIF)Click here for additional data file.S3 Fig32P) gRNA/pre-mRNA substrate RNAs were incubated with 20S editosomes in the presence of increasing concentrations of neamin (1.6μM-1.7mM). The formation of edited products is plotted as a function of the neamin concentration to derive half-maximal inhibitory concentrations (IC50). Errors are standard deviations (s.d.).Radioactively labelled (5’-(TIF)Click here for additional data file.S4 Fig32P).The depicted radioactively labelled pre-mRNA/gRNA substrate RNA was incubated with 20S editosomes in the presence of increasing concentrations of neomycin B . Endonucleolytic cleavage at the editing site (arrow) generates a 5’-mRNA cleavage fragment that was electrophoretically separated and densitometrically quantified (bar graph). M: mock treated sample. *: position of the radioactive label ((TIF)Click here for additional data file.S5 Fig50-value for the inhibition of the editing reaction. Fifty percent of the free RNA are complexed at ~0.8μM (dahed grey line), which agrees with the macroscopic Kd of the neo/RNA interaction.The graph shows the result of a numeric simulation of the concentration-dependent probability of forming neo/RNA complexes with one (yellow), two (orange) or three (red) neomycin B molecules per substrate RNA. Free RNA is shown in black. Half-maximal occupancy of all three binding sites is achieved at ~12μM (dashed grey line) in agreement with the derived IC(TIF)Click here for additional data file.S6 Figd, 10xKd, 100xKd). Measurements were performed as in [d of the RNA/neomycin B complex no binding was observed. Only at a ≥100-fold excess a weak interaction is visible.Surface plasmon resonance-based time trace of surface-immobilized 20S editosomes (20S ed) incubated with increasing concentrations of neomycin B (1xKed as in . Even at(TIF)Click here for additional data file.S7 FigT. brucei apocytochrome b (Cyb) mRNA (blue trace) and further incubated with neomycin B  (red traces). Measurements were performed as in [Surface plasmon resonance-based time trace of surface-immobilized 20S editosomes (20S ed) complexed with unedited ed as in . Even at(TIF)Click here for additional data file.S1 Table3’-end amino modifications in two of the oligoribonucleotides were introduced to prevent self ligation.(TIF)Click here for additional data file.


How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for  Furthus (HDV) –7. Howevus (HDV) . This vius (HDV) –13. The us (HDV)  or as cous (HDV) –15. Morenstrated . Very innstrated  and as pnstrated . Howevernstrated ,12, a laWhat might be the advantage of RNA being in a circular form rather than in the traditional linear one? The most obvious reason is certainly stability. Since exonucleases are ubiquitously spread, circRNAs are advantageous in terms of being protected against degradation. The covalently closed ring structure may be not only favorable for endogenous RNAs, but should also be beneficial to the application of RNAs for example as antisense-RNAs, aptamers, ribozymes or siRNAs –23.In vivo, circRNAs usually result from splicing events, either as exonic circRNA from circularization of exons or as intronic circRNA, such as, for example, circular tRNA and rRNA introns produced from archaeal splicing. Circular RNAs appear in viruses and viroids and as viroid-like satellite RNAs. In vitro, RNA circularization involves the intramolecular formation of a 3′, 5′-phosphodiester bond, requiring close proximity of the 3′- and 5′-terminus of the linear precursor. Here we review pathways of circRNA formation in vivo and strategies for RNA circularization in vitro. Furthermore, we provide a collection of protocols to be used for the purpose of RNA circularization (Supplementary data).What are the strategies for production of circRNAs? In eukaryotic cells the spliceosome acts to remove introns from primary transcripts in a two-step mechanism. In the first step, the 2′-OH group of a defined adenosine within the intron (branch point adenosine) attacks the 5′-splice site, generating a free 3′-OH group at the 5′-exon and the lariat intermediate. The second step involves nucleophilic attack of the generated 3′-OH group onto the 3′-splice site, producing the final products: an excised lariat intron and a linear RNA composed of the two combined exons Figure .In addition, exonic circRNAs may result from spliceosomal action. They were first observed in 1991 . Since tin vivo, although direct backsplicing is favored as the more frequently used pathway , thus generating the circularized intron and the 5′-exon  as an external nucleophile that initiates splicing by nucleophilic attack onto the 5′-splice site and thereby becomes attached to the 5′-end of the intron. Upon second transesterification, exons are ligated, and a linear catalytic intron is released (reviewed in ). Interein vivo .Tetrahymena intron, full-length intron circles were reported to be minor and barely detectable in vivo , hairpin ribozyme (HPR), HDV ribozyme) perform this reaction. Intramolecular ligation appears to be catalyzed mainly by host enzymes, although in vitro self-ligation was also observed  together with morpholino derivatives such as 2-(N-morpholino)-ethane sulfonic acid (MES) for linking two DNA strands carrying terminal 5′-hydroxyl and 3′-phosphate groups  carbodiimide (EDC) were used to support phosphodiester bond formation; however, BrCN appears to be the more widely used reagent ,63. Chemet al. reported an approach for solid phase synthesis of circular RNAs of 2–21 nucleotides length  (1 (T4 RNA ligase 1 was used for circularization in a number of applications with short and long RNAs as substrates (for exemplary protocols see Supplementary data (Protocols S7  and S8 ). An imp (CEV-A) . FurtherEV-A) (1 . The autEV-A) (1 . In the EV-A) (1  (ProtocoEV-A)   (1 . AnotherEV-A) (1 . BeaudryEV-A) (1 . In anotEV-A) -pp-(5′)G intermediate. Subsequently, the 3′, 5′-phosphodiester bond is formed by nucleophilic attack of the 5′-terminal hydroxyl group onto the activated 3′-phosphate of the intermediate  of linear forms of the HPR containing 2′,3′-cyclic phosphate and 5′-OH termini  viruses. Thus, evolution itself demonstrates the natural importance of circular RNAs. CircRNAs appear as intermediates and/or final product of RNA processing pathways and their occurrence appears to be ultimately linked to function ,121–122.in vivo, there has been growing interest in strategies for RNA circularization in vitro. Suitable models are needed for studying the structural and functional features of circular RNA occurring in vivo. Furthermore, applications in molecular biology, diagnostics or medicine will profit from the higher stability of circRNAs compared to their linear counterparts.Encouraged by the discovery of more and more circular RNA species in vivo; however, it does not allow for insertion of modified bases and/or sugar-phosphate backbones. In vitro production of circRNAs can satisfy this aim: modified nucleotides can be incorporated in RNA fragments by chemical synthesis, followed by chemical or enzymatic ligation/circularization. In general, the choice of the method should be based on three key points: the size of the circRNA, its nature  and the site of production (in vitro or in vivo). Taken together, there are circularization strategies for all kinds of RNAs, and one may anticipate that the toolbox is still growing.Dependent on the size and nature of the circular RNA to be prepared, the appropriate circularization method may be chosen. For example, the PIE method or group II intron inverse splicing can generate circRNAs Supplementary Data are available at NAR Online.


Motivation: RNA-Seq is a method for profiling transcription using high-throughput sequencing and is an important component of many research projects that wish to study transcript isoforms, condition specific expression and transcriptional structure. The methods, tools and technologies used to perform RNA-Seq analysis continue to change, creating a bioinformatics challenge for researchers who wish to exploit these data. Resources that bring together genomic data, analysis tools, educational material and computational infrastructure can minimize the overhead required of life science researchers.Results: RNA-Rocket is a free service that provides access to RNA-Seq and ChIP-Seq analysis tools for studying infectious diseases. The site makes available thousands of pre-indexed genomes, their annotations and the ability to stream results to the bioinformatics resources VectorBase, EuPathDB and PATRIC. The site also provides a combination of experimental data and metadata, examples of pre-computed analysis, step-by-step guides and a user interface designed to enable both novice and experienced users of RNA-Seq data.Availability and implementation: RNA-Rocket is available at rnaseq.pathogenportal.org. Source code for this project can be found at github.com/cidvbi/PathogenPortal.Contact:anwarren@vt.eduSupplementary information:Supplementary materials are available at Bioinformatics online. Transcriptomic analysis using high-throughput sequencing continues to increase in popularity due to low sequencing costs, its sensitivity, reproducibility and its ability to sample the entire transcriptome  is the all-bacteria Bioinformatics Resource Center (patricbrc.org) , Cryptosporidium (cryptosporidiosis) and kinetoplastida (i.e. Trypanosoma brucei and Leishmania species.) (Anopheles species), tsetse flies, ticks, lice, kissing bugs and sandflies.The RNA-Rocket takes advantage of many different open-source projects to enable users to upload and analyze their own data. We use the Galaxy system to consolidate and provide the tools and services necessary to process high-throughput sequencing data. The use of Galaxy has many benefits: showing provenance information for data creation, including the tools and parameters used to process data; support for batch analysis for multiple samples; providing a mechanism for results sharing across research groups and publishing for external references such as presentations or publications and its integration of tools and projects in the larger bioinformatics community.Before users can run analysis on the RNA-Rocket site they must first upload their data in FASTQ format. Using standard Galaxy interfaces RNA-Rocket supports upload via URL, FTP, HTTP and direct transfer via the European Nucleotide Archive  or TopHaThe site also provides the ability to conduct differential expression analysis using Cuffdiff, part of the Cufflinks suite and the ability to visualize data generated using CummeRbund , based on the concept that the user is interested in. Clicking on a concept diagram component gives information about the corresponding processing steps that fulfill the component . To assihttp://www.bioinformatics.babraham.ac.uk/projects/fastqc) for determining the quality profile of sample reads, as well as Sickle (https://github.com/najoshi/sickle) and Trimmomatic  to ‘collapse’ multiple read pairs that have identical coordinates for the first and second read into a single representative pair.RNA-Rocket also provides a quality control option for removing PCR bias that can occur as a result of library preparation . For paiIn addition to RNA-Seq analysis, RNA-Rocket also supports ChIP-Seq (Chromatin Immunoprecipitation-Sequencing) analysis by providing access to the peak calling program MACS  initiative, research projects competitively enabled through NIAID’s BRC program designed to drive innovation at the BRC sites based on the needs of the research community. Each dataset is provided as both ‘before’ and ‘after’ project spaces so that users can import the project into their own user space, run their own analysis and view pre-existing results. See the By combining experimental concepts with file-based requirements, the user interface aims to guide life science researchers through the process of RNA-Seq data analysis while making them aware of quality control caveats. After results have been computed at the RNA-Rocket site they can be streamed back to the respective BRC depending on the reference organism selected for analysis. This provides users with the ability to process and analyze their RNA-Seq data remotely without having to download potentially large files to their own computer.RNA-Rocket is a free service that can be used by life-science researchers to process and analyze their RNA-Seq data. The site maintains up-to-date genome and annotation data through NIAD’s Bioinformatic Resource Centers. By leveraging BRC data and the Galaxy system, the RNA-Rocket project can provide up-to-date tools and capability despite the rapidly changing landscape of RNA-Seq analysis.


Life devoted to The Origins and Early Evolution of RNA, 17 papers explore a remarkably broad range of topics surrounding this hypothesis. I would not go so far as to say the hypothesis is experiencing a mid-life crisis. However it is clear that it has generated a spectrum of viewpoints, from ardent devotees to outright skeptics. I perceive all these vantage points as giving us a richer appreciation of the chemical origins of life.The RNA World is now some four billion years behind us, but only recently turned 50 as a human hypothesis. As early as 1962 Alex Rich suggested that RNA might have a phenotype in addition to its informational role [i.e., RNA enzymes, and thus operating on reversible reactions) generate such products. Martin et al. also briefly consider the role of 2´,3´-cyclic phosphates in their paper, which is an in-depth survey of the problems and potentials of RNA ligase, nucleotide synthase, and RNA replicase ribozymes [The Issue was initiated by a cogent argument from Scott and colleagues that the 2´,3´-cyclic phosphate version of nucleotides should be considered a viable prebiotic source of monomers for the abiotic polymerization into RNA [et al. revisit the role of formamide as both a reactant and a solvent for activated nucleotide synthesis that obviates much of the water problem [The key problem of how RNA nucleotides, if present prebiotically, could polymerize into oligomers in the face of an uphill battle against hydrolysis in an aqueous medium, was taken up by two papers in the Special Issue. Hashizume  details  problem . Of note problem ,7.The transition from “short-mers” to “longer-mers” was also investigated by Mungi and Rajamani  and KanaOnce RNAs were long enough, the RNA World hypothesis generally turns to how the evolutionary process could speed up. Kun and Szathmáry, utilizing a recent influx of experimental data on RNA function, explore the nature of RNA fitness landscapes . A very et al. [et al. [e.g., [Another “next step” in RNA World discussions is the origin and evolution of the genetic code. This problem was pondered long ago by Jukes , Woese , and othet al.  take thiet al.  takes th [et al.  also tak [et al.  also con. [e.g., , and woret al. [Once the RNA World had a foothold, a next logical question is, how did life bring in DNA? In today’s biology, DNA synthesis requires a very challenging enzymatic reaction catalyzed by the ribonucleotide reductase (RNR) protein enzymes, which use RNA as a substrate. Given the complexity, yet apparent antiquity, of this reaction, the evolutionary provenance of these enzymes is of extreme importance. Lundin et al.  provide i.e., RNA per se). The same stance is taken by van der Gulik and Speijer, who argue forcefully on catalytic grounds that an RNA world without amino acids could never have existed [et al. [The last group of papers in the Issue deal with the highly probable case where the RNA World was not strictly consisting of RNA, and that peptides or other molecules either initiated life or co-evolved with RNA. The so-called “RNP World” – for ribonuleoprotein world – posits that RNA could not have had enough catalytic prowess without the help of peptide cofactors. This is a rather attractive hypothesis and various authors have varying degrees of opinions on its strength. The “Strong RNP World”  is advo existed . Smith e [et al. , perhaps [et al.  makes pe [et al. .In sum, all of these papers serve to richen the discussion of the RNA World, in all of its various forms. Although definitive confirmation of any of these ideas may require a time machine, I sense that we are at the precipice of a unified theory that accommodates a wide spectrum of RNA-related observations.


ATP-binding cassette (ABC) proteins were first recognized for their role in multidrug resistance (MDR) in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR). In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is necessary to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps), multidrug-resistance-associated proteins (MRPs 1-5) and breast cancer resistance associated protein (BCRP). In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of the detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants that can act as chemosensitizers, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in (1) regulation and functioning of ABC proteins; (2) cooperation with phase I and II biotransformation enzymes; and (3) ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clearly suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish to underlay the mechanism to consider their use as biomarkers in environmental monitoring. The maintenance of the overall environmental health is quite dependent on aquatic ecosystems. Species overexploitation, pollution from urban, industrial and agricultural areas, as well as habitat loss and alteration have been contributing to the decline of aquatic environmental health. Toxic heavy metals resultant from industrial processes, persistent organic pollutants (POPs) from agricultural and industrial sources and polycyclic aromatic hydrocarbons (PAHs) resultant from oil spills are among the sources of major concern regarding water pollution, due to their known toxic effects on the aquatic biota  the regulation and functioning of ABC transporters; (2) the cooperation with phase I and II biotransformation enzymes; and (3) the ecotoxicological relevance of emergent pollutants with ability to modulate ABC transporters expression and activity. Recent studies have raised the possibility of a coordinated action between these groups of proteins, resulting in a powerful and effective mechanism of cellular detoxification.The ABC genes represent one of the largest family of transmembrane transporter proteins encoded in the human genome , the latter being composed by 6–10 membrane spanning α-helices that confer the substrate specificity Figure . EukaryoABCB1 gene, was the first ABC transporter to gain importance and later to be related to cellular detoxification. From this point, several other members of the ABC transporter efflux family, other than ABCB, have been identified as capable of interacting with endo- or xenobiotic compounds, including members of the ABCC and ABCG subfamilies . In humans, Pgp is encoded by two different isoforms of the gene (MDR1 and MDR3); the class 1 has been implicated in drug resistance whereas the function of class 3 isoform is still unknown , follows the same predicted structure as ABCB1 Figure , and stuAfter the discovery of ABCB1, the study on cancer cells displaying MDR phenotype not associated with ABCB1 expression, led to the discovery of ABCC1, the founding member of the ABCC subfamily   , northern-blotting and activity assays (through the measurement of efflux or accumulation of fluorescent model substrates).Identification of ABC transporters and characterization of the MXR mechanism has been achieved by the use of several detection methods, including quantitative or semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR or RT-PCR) and Most studies directed to the determination of organ distribution pattern of ABC transporters in aquatic organisms have been directed to Pgp by the use of the mammalian anti-Pgp monoclonal antibody (mAb) C219. This mAb which recognizes an epitope common to all known Pgps (in human MDR1 and MDR3) and also to Spgp (ABCB11)  and xenobiotics  were shown to inhibit Pgp activity in fishes, resulting in an increase of the bioaccumulation of toxic compounds like 2-aminoanthracene, hydrocarbon-rich Diesel-2 oil, and/or model Pgp substrates like rhodamine B and rhodamine 123 , while ABCCs and ABCG2 transport phase I and II metabolites, therefore acting in phase III of cellular detoxification Bard,  Figure . CompareUnderstanding the underlying facts behind the mechanisms of cellular detoxification has implications both for human and environmental health. The medical relevance of these transporters is evidenced by their MDR abilities and, under this point of view there is a fundamental need to know how to properly block the action of these transporters to assure the targeting efficacy of chemotherapeutic drugs. In addition, the study of ABC efflux transporters is also very important from an environmental point of view, since they are believed to be an integral part of the cellular detoxification system. For environmentalists, the goal is to understand how these efflux transporters keep toxicants out of the cells, and to ensure that they operate optimally in order to protect the cells from environmental contaminants. The effects of ABC transporters on the toxicity of environmental contaminants is still poorly understood, thus efforts must be made to provide insights into the interactions of contaminants in fish species. In particular, interactions with classical and emergent compounds and the environmental relevance of fish ABC transporters is yet to be thoroughly addressed. The search for chemosensitizers among emergent compounds is relevant since some contaminants considered to be weak or moderately toxic at concentrations found in the aquatic environment can be potent inhibitors of ABC transporters and modulate toxicity of other xenobiotics and led to unpredicted effects to exposed species. Therefore, and in particular with emergent compounds, it is important to evaluate interaction of a large array of contaminants with ABC transporters when assessing toxicity of xenobiotics to the aquatic environment. One difficulty to fully address the role of ABC transporters in the detoxification process and the interactions with xenobiotics with the different classes of contaminants is that the information available on fish species is dispersed in different studies with different goals. So in the future, research must be directed to the study of regulation of ABC transporters and to unravel the importance of these efflux proteins to the overall maintenance of the homeostasis of fish species chronically exposed to foreign chemicals.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.


EV71 (enterovirus 71) RNA contains an internal ribosomal entry site (IRES) that directs cap-independent initiation of translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs) from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2) as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication. Enterovirus 71 (EV71), a member of the family Picornaviridae, poses a persistent global public health problem. EV71 infections usually cause hand-foot-and-mouth disease (HFMD) or herpangina, yet EV71 has also been implicated as the etiological agent in several large-scale outbreaks of severe neurological disorders in children worldwide –6. SeverEV71 is a positive-stranded RNA virus –17.Stem-loop II (SL-II) within the EV71 5’ UTR is essential for EV71 viral translation. Our previous studies showed that hnRNP A1 and AUF1 associate with this region ,15. WhilSF268 (human glioblastoma) , RD . Upon confluence, cell extracts were prepared, and pull-down assays of complexes between cellular proteins and biotinylated RNA were performed as described . ProteinTwo micrograms of plasmid expressing control short hairpin (sh)RNA or target shRNA were mixed in 100 μl serum-free MEM and combined with 10 μl SuperFect reagent (Qiagen); this mixture was incubated at room temperature for 10 min before addition to cell cultures. Knockdown efficiency was monitored by Western blotting. Short hairpin plasmids: Control, pSilencer-U6-hygro/shCTRL; shAUF1, pSilencer-U6-hygro/shAUF1 ; shAgo2-Western blot was carried out as described . PrimarySF268 cells were infected with EV71 at an MOI = 2. Cells were harvested at various time points, and total RNA was extracted from cells with an RNeasy minikit (Qiagen). Viral RNA was determined by real time RT-PCR as described .SF268 cells were seeded in 12-well plates in antibiotic-free RPMI and transfected with RLuc-EV71-5’UTR-FLuc RNA as described . IRES acTo assess whether Ago2 and HuR associate with EV71 viral RNA in infected cells, RNP immunoprecipitation and Northern blotting were performed. SF268 cells were infected with EV71 at an MOI = 40 and cell lysates were prepared 8 h post-infection. RNA-protein complexes were immunoprecipitated with non-immune rabbit serum or antiserum specific to Ago2 or HuR. RNA was isolated from the immune precipitates and analyzed by Northern blotting with a radiolabeled probe specific to EV71 RNA. Immunoprecipitation with Ago2 or HuR antibodies co-precipitated EV71 RNA while non-immune serum did not . No RNA The EV71 5’ UTR contains a 5’ cloverleaf-like structure important for viral RNA synthesis and an internal ribosomal entry site (IRES) that is critical for viral mRNA translation. To determine the region(s) of the 5’UTR with which Ago2 and HuR interact, different stem-loops of the EV71 5’UTR were synthesized in vitro with biotin-UTP. These regions are shown in To determine whether Ago2 and HuR affect EV71 replication, levels of Ago2, HuR, or both proteins together were reduced by transfection of SF268 cells with the respective plasmids expressing shRNAs against Ago2, HuR, or a control shRNA. Western blot analysis indicated that Ago2 and HuR were reduced >80% when compared to serial dilutions of lysate from cells expressing the control shRNA, shCTRL . TranslaEffects of Ago2 and HuR knockdown on viral RNA synthesis, translation, and titer were examined next. To determine the effect of Ago2 and HuR knockdown on viral RNA synthesis, cells expressing shAgo2, shHuR, or shAgo2+shHuR were infected with EV71 at an MOI = 2 and were cultured with 2 μg/ml actinomycin D. Viral positive-strand RNA levels were examined by real time RT-PCR 24 h post infection. Knockdown of either Ago2 or HuR, or both proteins, had no effect on viral RNA levels . WesternOur previous work  and the As such, the effects of knocking down expression of two proteins on RNA binding by the third protein were examined. The Western blots show efficient, dual knockdowns upon transfection of SF268 cells with plasmids expressing two target shRNAs  to SL-II . InfecteRNA-induced silencing mediated by RISC is an important defense against viral infections –28. Our In addition to stabilizing subsets of cellular mRNAs to which it binds, HuR affects virus gene expression as well. For example, it binds to the HCV 3’UTR, though the significance of this binding is not clear . During AUF1, HuR, and Ago2 normally control host mRNA degradation and/or translation. Interestingly these three ITAFs associate with SL-II and vsRNA1 promotes their association with SL-II. The molecular details underlying the interactions between these ITAFs and vsRNA1 are not known. It is tempting to speculate that vsRNA1 may act to remodel local RNA structure within SL-II to favor or disfavor RNA binding among AUF1, Ago2, and HuR. Alternatively, vsRNA1 may recruit other host factors to SL-II to affect translation. For example, a miR-122–Ago2 complex binds the 5’UTR of HCV viral RNA to enhance RNA stability and translation –37. In a