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

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus from rRNAs and ribosomal proteins, exported as premature complexes, and processed in final maturation steps in the cytoplasm. Ltv1 is a conserved 40S ribosome biogenesis factor that interacts with pre-40S complexes in vivo and is proposed to function in yeast in nuclear export. Cells lacking LTV1 grow slowly and are significantly impaired in mature 40S subunit production. Here we show that mutation or deletion of a putative nuclear export sequence in LTV1 is strongly dominant negative, but the protein does not accumulate in the nucleus, as expected for a mutation affecting export. In fact, most of the mutant protein is cytoplasmic and associated with pre-40S subunits. Cells expressing mutant Ltv1 have a 40S biogenesis defect, accumulate 20S rRNA in the cytoplasm as detected by FISH, and retain the late-acting biogenesis factor Tsr1 in the cytoplasm. Finally, overexpression of mutant Ltv1 is associated with nuclear retention of 40S subunit marker proteins, RpS2–GFP and RpS3–GFP. We suggest that the proximal consequence of these LTV1 mutations is inhibition of the cytoplasmic maturation of 40S subunits and that nuclear retention of pre-40S subunits is a downstream consequence of the failure to release and recycle critical factors back to the nucleus

Ltv1 Is Required for Efficient Nuclear Export of the Ribosomal Small Subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Δltv1 mutants, as judged by the altered distribution of the 5′-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell—endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell

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

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

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

The methylation-free state of the paternal ICE depends on the CGI.

<p>(A) DNA blot assaying methylation of the <i>Airn</i> promoter MluI site as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a>, in undifferentiated ES cells carrying a paternal <i>CGIΔ</i> or wildtype (<i>+</i>) allele. The 5.0 kb band identified by probe MEi (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map) indicates a gain of methylation on the <i>CGIΔ</i> paternal allele. This band is weaker in cells with lower passage numbers that still retain the selection cassette (<i>S12/CGIΔ+cas</i>-1,-2) compared to cells that have been in culture for 8 more passages (<i>S12/CGIΔ</i>-1A,-1B,-2A,-2B) with a deleted selection cassette. The lower panel confirms this by showing a matching loss of the unmethylated 1.1 kb fragment specific to the paternal allele in cells with a higher passage number. Both panels were from the same blot and the intervening area lacking any hybridisation signal removed. (B) DNA blot as in (A) assaying <i>Airn</i> promoter MluI methylation during ES cell differentiation showing that the level of paternal methylation on the <i>CGIΔ</i> allele in undifferentiated ES (d0) cells (5 kb band) does not change in differentiated d5 and d14 cells. Probe MEi is a 1 kb EcoRI-MluI fragment shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map. (C) Bisulfite sequencing of two undifferentiated <i>S12/CGIΔ</i> ES cell clones using primers spanning the deletion that specifically amplify the paternal <i>CGIΔ</i> allele, confirms the strong gain of DNA methylation, but also shows that some alleles are more methylated than others (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5B</a>).</p

TDR absence has a minor effect on paternal <i>Igf2r</i> repression.

<p>(A) Genomic DNA digested with EcoRI (E) or EcoRI+methyl-sensitive NotI (E/N) hybridised with probe EEi. wt:wildtype, met:methylated, unmet:unmethylated, <i>Thp</i>:deletes the entire <i>Igf2r</i> cluster. Quantification of the methylated/unmethylated hybridisation signal shown below for d14, shows equal gain of DNA methylation on the paternal <i>Igf2r</i> promoter in <i>S12/+</i> and <i>S12/TDRΔ</i> cells. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s004" target="_blank">Figure S4A</a> shows two further differentiation sets. (B) RT-PCR followed by digestion of a paternal-specific PstI site to assay allelic <i>Igf2r</i> expression in ES cells carrying a paternal wild type (<i>S12/+</i>) or mutated (<i>S12/TDRΔ</i>) allele in four targeted clones. Two further differentiation sets are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s004" target="_blank">Figure S4B</a>. -: minus RT, u: undigested, P: PstI digested, Mat: maternal, Pat: paternal. Impaired paternal <i>Igf2r</i> repression indicated by the clear presence of two paternal bands at d5 and faint presence at d14 (*) was seen in all four <i>S12/TDRΔ</i> cell lines. (C) Allele-specific qPCR quantifying <i>Igf2r</i> expression using the same SNP as in (B). The mean maternal∶paternal <i>Igf2r</i> expression ratio and standard deviation of three differentiation sets is displayed. As undifferentiated ES cells show biallelic <i>Igf2r</i> expression the ratio was set to 1 in <i>S12/+</i> d0 cells. Also the <i>S12/TDRΔ</i> cells show biallelic expression in undifferentiated ES cells, as the ratio mat/pat is around 1. During differentiation, the ratio in <i>S12/+</i> cells increases twofold more compared to <i>S12/TDRΔ</i> cells, indicating a compromised although not statistically significantly impaired imprinted expression of <i>Igf2r</i> in <i>S12/TDRΔ</i> cells. <i>S12/+</i> and <i>S12/TDRΔ</i> were compared using an unpaired t-test. (D) qPCR of total <i>Igf2r</i> steady-state levels in 12.5–13.5 dpc mouse embryos shows a minor loss of paternal <i>Igf2r</i> repression. Embryos from 3 litters carrying wildtype (<i>+/+</i>, <i>Thp/+</i>) or <i>TDRΔ</i> (<i>+/TDRΔ</i>, <i>Thp/TDRΔ</i>) paternal alleles were assayed and compared using an unpaired t-test. The <i>Thp</i> allele carries a deletion of the entire <i>Igf2r</i> cluster thus only the paternal allele is present. Details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3C</a>.</p