SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity

A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter

A CpG island (CGI) lies at the 5′ end of the Airn macro non-protein-coding (nc) RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs) occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start

Potencial alelopático de extratos aquosos foliares de aveia sobre azevém e amendoim-bravo Effect of extracts from the above-ground part of oat genotypes on ryegrass and wild poinsettia

O trabalho teve por objetivo avaliar o efeito alelopático de extratos aquosos de cinco cultivares de aveia branca (Avena sativa L.) e quatro de aveia preta (Avena strigosa Schreb), nas concentrações de 0, 25%, 50% e 100%, sobre a germinação e o desenvolvimento das plântulas de azevém (Lolium multiflorum Lam.) e amendoim-bravo (Euphorbia heterophylla L.). O delineamento utilizado foi o inteiramente casualizado distribuído em um trifatorial (genótipos de aveia x plantas infestantes x concentrações do extrato). Utilizaram-se placas de Petri contendo 25 sementes de plantas infestantes que foram umedecidas com extrato da parte aérea dos genótipos de aveia. As variáveis observadas foram: percentual de germinação, comprimento de radícula e de hipocótilo, avaliados aos 14 dias após a instalação do experimento. Os extratos aquosos das cultivares de aveia branca e preta inibiram o potencial alelopático sobre a germinação e o desenvolvimento das plantas daninhas testadas, e os genótipos de aveia branca 'IPR 126', 'UTF Iguaçu' e 'Fundacep FAPA 43' e de aveia preta 'UPFA 21 Moreninha' e 'UTG 9715' foram mais efetivos na inibição da germinação e no desenvolvimento do azevém e amendoim-bravo.<br>The objective of this study was to evaluate the allelopathic effect of aqueous extracts from five common oat (Avena sativa L.) and four black oat (Avena strigosa Schreb) cultivars, at concentrations of 0, 25, 50, and 100%, on the germination and development of ryegrass (Lolium multiflorum Lam.) and wild poinsettia (Euphorbia heterophylla L.) plantlets. A completely randomized design was adopted, distributed as a triple factorial arrangement (oat genotypes × weeds × concentrations). The experiment consisted of Petri dishes containing 25 weed seeds moistened with extract from the above-ground part of the oat genotypes. The following variables were observed: germination percentage, radicle length, and hypocotyl length, evaluated at 14 days after the experiment was installed. The aqueous extracts from common oat and black oat cultivars showed allelopathic potential on the germination and development of the weeds tested. Common oat genotypes 'IPR 126', 'UTF Iguaçu', and 'Fundacep FAPA 43', and black oat genotypes 'UPFA 21 Moreninha' and 'UTG 9715' were the most effective to inhibit ryegrass and wild poinsettia germination and development

The <i>Airn</i> CGI plays a major role in <i>Airn</i> transcription and function.

<p>(A) <i>Airn</i> expression by genome-tiling array (left axis) and strand-specific expression analysis by RNA-Seq (right axis) for differentiated <i>S12/+</i> and <i>S12/CGIΔ</i>-1A cells. Dashed arrows: sharp drop of <i>Airn</i> hybridisation signals in the <i>Airn</i>-specific region (single) and absence after 73 kb (doublet). Below: qPCR assays relative to <i>Airn</i>-TSS with colour code as (B,C). Striped box: overlapping START+RP11 assays. (B) qPCR of total+unspliced <i>Airn</i> in d0/d5/d14 differentiated <i>S12/+</i> and four <i>S12/CGIΔ</i> clones shows unspliced <i>Airn</i> is reduced by ∼40% at the 5′ end (RP11/154 bp), but when assayed downstream (<i>Airn</i>-middle/53 kb, <i>Airn</i>-end/99 kb) or at positions which include splice variants (START), is reduced by >70% in <i>S12/CGIΔ</i> cells. Shown are mean and standard deviation of three differentiation sets (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3A</a>). (C) <i>Airn</i> qPCR in <i>S12/+</i> and four <i>S12/CGIΔ</i> d14 clones shows that unspliced <i>Airn</i> is reduced by 79–83% at 0.57 kb and ∼85% at 7.3 kb, while spliced <i>Airn</i> reduced by >85%. Shown are mean and standard deviation of three differentiation sets (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3A</a>). (D) ChIP for Ser5P/Ser2P RNAPII in <i>S12/+</i>, <i>S12/TDRΔ</i>-1A and <i>S12/CGIΔ</i>-1A d11 cells shows unaffected <i>Airn</i> initiation and elongation (except at <i>Airn</i>-end) in <i>TDRΔ</i> and a sharp RNAPII decrease in the <i>CGIΔ</i> allele. The mean and standard deviation of three technical replicates is shown. Assay <i>Airn</i>-132 controls for background from the overlapping <i>Igf2r</i> transcript, which is 2-fold higher in <i>CGIΔ</i> that fails to repress the paternal <i>Igf2r</i> promoter. Map for qPCR assays as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a>. (E) DNA blot analysing methylation of the <i>Igf2r</i> promoter NotI site (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g004" target="_blank">Figure 4A</a>). *methylated fragment in d0 cells originating from feeder-cells. This blot shows that cells carrying a paternal <i>CGIΔ</i> allele contrary to wildtype cells do not gain the methylated 5 kb band on the paternal <i>Igf2r</i> promoter. White lines: indicate the order of samples run on the same gel was changed electronically. (F) qPCR quantifying allelic expression shows absence of <i>Igf2r</i> imprinted expression (Mat∶Pat ratio is close to 1), in four <i>CGIΔ</i> (<i>S12/CGIΔ</i>) cell lines compared to wildtype (<i>S12/+</i>). Three differentiation sets are shown separately due to variability in Mat∶Pat ratios in wildtype controls for each set. Bars represent the mean, error bars the standard deviation of 3 technical replicates (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g004" target="_blank">Figure 4C</a>).</p

TDRs are required for the regulation of ICE DNA methylation.

<p>(A) Genomic DNA digested with EcoRI (E) or EcoRI+methyl-sensitive MluI (E/M) and hybridised with probe <i>Airn</i>T. Cells carrying a paternal <i>TDRΔ</i> (<i>S12/TDRΔ</i>) or wildtype (<i>S12/+</i>) allele have a 6.2 kb band from the maternally-methylated plus 4.3 kb or 5.0 kb from the paternally-unmethylated allele. <i>S12/TDRΔ</i> cells additionally show at d0, a faint 5.5 kb band representing a paternal partially methylated allele (*) that is lost by d14 in differentiated cells. (B) Bisulfite sequencing of undifferentiated <i>S12/TDRΔ</i>-1A ES cells. Each row represents one allele, single columns: one CpG, double columns: two neighbouring CpGs. White/black circles: unmethylated/methylated CpGs. Arrows: position of MluI site and <i>TDRΔ</i> deletion. Primers DMR2-4F and -9R specifically amplify the paternal allele in <i>S12/TDRΔ</i> ES cells (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s005" target="_blank">Figure S5B</a>). Bisulfite sequencing confirms a low level of DNA methylation on the paternal <i>TDRΔ</i> allele at d0 only. (C) Percent methylation levels for two <i>S12/TDRΔ</i> and control <i>R2Δ/+</i> ES cells. The <i>R2</i>Δ deletion removes the maternal ICE, thus only the wildtype paternal ICE is detected. Each point represents one sequenced clone and shows that low-level DNA methylation is a general feature of the paternal ICE in d0 ES cells and not a consequence of the TDR deletion. (D) DNA blot analysing the MluI methylation status as in (A) for 12.5–13.5 dpc embryos carrying a maternal <i>TDRΔ</i> (<i>TDRΔ/+, TDRΔ/Thp</i>) or wildtype (<i>+/+, +/Thp</i>) allele. The 5.0 kb methylated band is faint or absent in <i>TDRΔ/+</i> embryos, but present in <i>+/+</i> embryos (6.2 kb), showing that maternal inheritance of the <i>TDRΔ</i> allele leads to loss of DNA methylation. (E) Bisulfite sequencing of embryonic genomic DNA (details as in B) shows that maternal transmission of the TDR deletion leads to a major loss in DNA methylation. For <i>TDRΔ/Thp</i> only the maternal allele, for <i>+/+</i> both parental alleles were analysed. (F) Percent methylation level for <i>TDRΔ/Thp</i> and <i>+/+</i> embryos as in (C).</p

CpG islands lie upstream and downstream of the transcription start site.

<p>(A) ES cell imprinting model. In undifferentiated ES cells <i>Igf2r</i> is expressed from both chromosomes and <i>Airn</i> is not expressed <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540-Latos1" target="_blank">[31]</a>. The ICE, which contains the <i>Airn</i> promoter, is methylated on the maternal allele (black hexagon) and unmethylated on the paternal allele (white hexagon). The <i>Igf2r</i> promoter is unmethylated on both parental alleles (white oval). During differentiation, <i>Airn</i> (wavy line) is expressed from the unmethylated paternal allele. <i>Igf2r</i> is upregulated up to 20-fold on the maternal allele, but its upregulation on the paternal allele is blocked by <i>Airn</i> expression <i>in cis</i>. The repressed paternal <i>Igf2r</i> promoter gains DNA methylation late in differentiation (black oval). (B) <i>Airn</i> 5′ region. T1,T2,T3: multiple closely-spaced <i>Airn</i> TSSs. 53 bp splice donor: shared by all <i>Airn</i> splice variants. MluI: MluI restriction site. Grey shading: CGI. Grey triangles labelled TDRs: tandem direct repeats. <i>CGIΔ</i>/<i>TDRΔ</i>: 1129/692 bp deletions generated here. <i>AirnPΔ</i>: 959 bp deletion defining <i>Airn</i> promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540-Stricker1" target="_blank">[32]</a> that lies upstream of the CGI. <i>R2Δ</i>: 3656 bp deletion defining the imprint control element or ICE <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540-Wutz1" target="_blank">[10]</a>. Thicker grey line on <i>R2Δ</i>: maternally methylated region. Possible roles of the CGI and the TDRs on the two parental alleles are listed below. (C) Transcription start sites (TSSs) of mouse RefSeq genes are plotted relative to their CpG island (CGI) and 50% of the CGI length upstream and downstream (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#s4" target="_blank">Materials and Methods</a>). Dotted line: TSS of <i>Airn</i>, <i>Kcnq1ot1</i> and <i>Igf2r</i>. The <i>Airn</i> TSS lies upstream to the CGI in contrast to the majority of TSSs that lie inside of the CGI.</p

Tandem direct repeats play a role in <i>Airn</i> processivity.

<p><i>Airn</i> expression by genome tiling array in day 5 differentiated ES cells carrying a paternal wildtype (<i>S12/+</i>) or mutated (<i>S12/TDRΔ</i>-2A and <i>+/TDRΔ</i>) allele. Note the maternal allele is always written on the left side (Mat/Pat). x-axis: basepairs, y-axis: averaged relative signal intensities with standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#s4" target="_blank">Materials and Methods</a>). Single and double dashed arrows: position after which consistent differences between wildtype and two <i>TDRΔ</i> cell lines are seen. Grey arrow: <i>Airn</i> hybridisation signals are lost after 90 kb in two <i>TDRΔ</i> cell lines. Below: <i>Airn</i> (wavy arrow) and <i>Airn</i> splice variants (black boxes: exons). Grey font: <i>Airn</i> qPCR assays with their distance from <i>Airn</i>-TSS. RP11, RP6, RP21, RP5, RP4 were combined with FP1+TQ-AS. This analysis shows that <i>Airn</i> in <i>TDRΔ</i> cells is reduced after 68 kb and lost after 90 kb.</p

Tandem direct repeats regulate the length of <i>Airn</i>.

<p>(A) qPCR of total (spliced+unspliced) <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a> map for location of qPCR assays). Relative <i>Airn</i> levels were set to 100% in <i>S12/+</i> cells at d14. Bars and error bars: mean and standard deviation of three differentiation sets. <i>S12/+</i> and <i>S12/TDRΔ</i> were compared using an unpaired t-test (*P = 0.1–0.5, **P = 0.001–0.01, ***P<0.001). The data show that <i>Airn</i> steady-state levels are unchanged up to 53 kb but are greatly reduced and lost at the 3′ end. (B) qPCR of spliced <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells. Details as in (A). These data show that the TDR deletion does not affect <i>Airn</i> splicing suppression but leads to a shortening at the 3′ end. (C) qPCR of unspliced <i>Airn</i> in 12.5–13.5 dpc mouse embryos confirms the significant loss of <i>Airn</i> steady-state levels at the 3′ end as seen in differentiated ES cells (A,B). Embryos from 3 litters were assayed carrying wildtype (<i>+/+</i>, <i>Thp/+</i>) or <i>TDRΔ</i> (<i>+</i>/<i>TDRΔ</i>, <i>Thp/TDRΔ</i>) paternal alleles. The <i>Thp</i> allele carries a deletion of the entire <i>Igf2r</i> cluster thus only the paternal allele is present. Samples of the same genotype were averaged and the horizontal lines and error bars show mean and standard deviation. Values for individual embryos are plotted as single data points. The number of samples is given below the genotype (n). Relative <i>Airn</i> levels were set to 100% for <i>+/+</i>, all others are displayed relative to it. Samples were compared to <i>+/+</i> using an unpaired t-test. Details as (A).</p