The Ring1B 5′ UTR does not have cryptic promoter activity.

<p>To test cryptic promoter activity of the Ring1B 5′ UTR, the Ring1B and EMCV IRES dual luciferase constructs were cloned into a pcDNA3.1 vector without CMV promoter (A). The constructs correspond to the constructs depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g003" target="_blank">figure 3A</a>, with Renilla luciferase 5′ of the IRES and Firefly luciferase 3′ of the IRES. The y-axis shows the absolute counts of Firefly luciferase (cap-dependent) and Renilla luciferase (IRES-dependent) after transient transfection in COS7 cells. In the constructs without promoter, no mRNA is produced, therefore no translation occurs and no luciferase activity can be measured, indicating the Ring1B 5′ UTR does not contain a cryptic promoter. pEGFP vector was cotransfected in COS7 cells and showed equal fluorescence in every transfection. In a bicistronic construct with the EGFP ORF downstream of the Ring1B IRES we could clearly detect EGFP fluorescence with fluorescent microscopy (B, Ring1B IRES only) as well as EGFP protein on Western blot (C). The Ring1B IRES (IRES^R) seems to give a slightly higher expression than the EMCV IRES (IRES^E), but we did observe some variation in this in our different experiments. On average we would say the EGFP levels were equal. pEGFP served as a positive control, empty pcDNA3.1 as a negative control, and tubulin as a loading control.</p

Mouse and human Ring1B 5′ UTRs show a striking structural resemblance.

<p>Secondary structure prediction of the Ring1B IRES of human and mouse origin, showing a striking structure similarity (A). The first nucleotide is indicated with a black circle around it, the last triplet is the translational start. Loop numbers are indicated with filled black circles. In mutant IRES*1 and IRES*2 the indicated cytosines are replaced by adenines, creating mismatches. In mutant IRES*3 the indicated uracil is replaced by a cytosine, creating a matching nucleotide and therefore stabilizing the structure. Mutant IRES*4 lacks the entire second loop. Luciferase assays show the remaining activity of the mutant IRESes and the stabilized activity in case of the matching mutation (B). The same construct as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g003" target="_blank">figure 3a</a>, construct number 1, was used, but with the mutations introduced. The way of interpreting the graph is identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g003" target="_blank">figure 3b–f</a>. The mismatch point mutations almost completely abolish the IRES activity, whereas IRES*3 is, as expected, not affected by its stabilizing point mutation. Surprisingly, the IRES lacking the entire second loop still has some residual activity, indicating the relative importance of the remaining structure.</p

Ring1B has a remarkable genomic structure.

<p>The genomic structure of Ring1B is depicted in the upper pane, the approximate sizes of exons in the middle pane and introns in the lower pane, as well as the translational start and stop codons. Exon 7 greatly varies in length, sometimes measuring up to 3,5 kb (A). Apart from a 12 nucleotide stretch in the middle, which is only seen in human and pig Ring1B, IRESes of different mammals are virtually identical. The exact mouse sequence depicted, comprising the entire IRES until the translational start, is used in all our assays (B). A 5′ RLM-RACE was performed to determine the main transcriptional start (C). In lane 5 a 100 bp marker was loaded, with the most prominent band being 600 bp. In ES cells (lane 1), primary MEFs (lane 2) and 293 cells (lane 3) the most abundant fragment is approximately 380 bp long. This suggests a transcriptional start at approximately 154 bp 5′ of the 3′ splice site of the first exon, which was verified after sequencing the fragments. Lane 4 shows an internal control PCR, using the same 3′ Ring1B inner primer, but with a Ring1B 5′ primer instead of an adapter primer. The Ring1B 5′ primer is located downstream of the translational start, so this PCR serves as an internal control for RNA quality and primer specificity.</p

The Ring1B 5′ UTR harbours IRES activity in expression constructs.

<p>In this overview of the dual luciferase constructs used in the assays below, the CMV promoter, the Renilla (RLuc) and Firefly (FLuc) luciferase ORFs, the IRESes of EMCV (E.IRES) and Ring1B (R.IRES) and the SV40 polyadenylation signal are depicted (A). The diagrams show activity assays of the cellular IRESes of Ring1B (pLRL), PDGF (pLPL), VEGF (pLVL), c-myc (pLML) and the viral IRES of EMCV (pLEL). The assays were done in COS7 (B), 293 (C), U2OS (d), HACAT (E) and HeLa (F) cells. On the y-axis, the relative activity of the IRES is depicted by dividing the Firefly counts of the sample over the Firefly counts of the empty vector (pLL, which has no IRES between both luciferase ORFs). All samples are corrected for their relative Renilla counts, since Renilla luciferase translation is cap-dependent and generally a readout of transfection efficiency. All experiments were done in duplo and repeated several times. The assays show the varying activity of the IRESes in different cell types. Whereas the EMCV IRES is known to be highly active in most cell types, cellular IRESes are generally much less active. Nevertheless, the Ring1B IRES is as active as the EMCV IRES in COS7 and HeLa cells, and in every cell type the most active IRES among the cellular IRESes.</p

The Ring1B 5′ UTR is sensitive to 2A protease activity.

<p>Western blot showing cleaved eIF4GI isoforms upon transfection of viral protease 2A (lane 1) in 293 cells, whereas eIF4GI remains intact in the absence of 2A (lane 2) (A). Molecular weight markers are indicated. This Western blot was done on the same lysates we used in one of the experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g004" target="_blank">figure 4B</a>. Results are representative of three independent experiments. The graphs depict the absolute counts of Renilla and Firefly luciferase activity (B) and the ratio of the Firefly luciferase counts over the Renilla luciferase counts (C). Ring1B IRES-dependent Firefly luciferase is clearly affected by the protease to a similar extend as the cap-dependent Renilla luciferase, showing no difference in their relative activity and therefore no difference in the ratio. As a control, in the presence of 2A protease the EMCV IRES-dependent Firefly luciferase is equally active whereas the cap-dependent Renilla luciferase activity is reduced over two-fold, resulting in an over two-fold increase in the ratio. This experiment clearly shows the remarkable apparent cap-dependency of the Ring1B IRES. pIND-2A, the protease-expressing plasmid, was co-transfected at 5 µg per transfection, without muristerone-A induction. Experiments were performed four times. Transfection efficiences were checked to be comparable between samples by cotransfection of 50 ng EGFP-expressing plasmid.</p

Expression of Ring1B is influenced by the 5′ UTR.

<p>In this overview of the Ring1B IRES validation constructs used in our assays, the position of the CMV promoter, the flag and myc tags, the Ring1B ORF, the 140 bp stable hairpin structure (hp) blocking translation, the Ring1B IRES, the SV40 polyadenylation signal and the MSCV-Puro LTRs are depicted (A). Western blots indicate the different expression levels of our Ring1B constructs (B). Numbers correspond to the constructs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g002" target="_blank">figure 2A</a>. All constructs were based on pcDNA3.1 and expression analyses were done with transient transfections in 293 cells, except construct #4, which was based on the retroviral vector MSCV-Puro and was infected in primary MEFs. 50 ng of EGFP-expressing plasmid was cotransfected to be able to judge by fluorescent microscopy whether transfection was comparably efficient. One can clearly appreciate the increased exogenous Ring1B protein levels relative to tubulin when the 5′ UTR is present, as well as the decreased protein levels when the stem-loop-stem hairpin is cloned in front of the 5′ UTR, and the absence of exogenous Ring1B protein when the CMV promoter was removed from the vector. Ring1B RNA levels (both endogenous and transfected measured together) in the same cell populations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g002" target="_blank">figure 2B</a> were very much alike, ruling out that the differences in protein levels could be accounted for by differences in transcription efficiency (C). RNA levels in this graph were typical for several independent experiments and normalized against beta-actin; normalization against Hprt gave the same result. Ring1B (1) and Ring1B (2) indicate different primer sets. Bar numbers correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002322#pone-0002322-g002" target="_blank">figure 2A</a>.</p

Chromatin Landscapes of Retroviral and Transposon Integration Profiles

<div><p>The ability of retroviruses and transposons to insert their genetic material into host DNA makes them widely used tools in molecular biology, cancer research and gene therapy. However, these systems have biases that may strongly affect research outcomes. To address this issue, we generated very large datasets consisting of to unselected integrations in the mouse genome for the Sleeping Beauty (SB) and piggyBac (PB) transposons, and the Mouse Mammary Tumor Virus (MMTV). We analyzed (epi)genomic features to generate bias maps at both local and genome-wide scales. MMTV showed a remarkably uniform distribution of integrations across the genome. More distinct preferences were observed for the two transposons, with PB showing remarkable resemblance to bias profiles of the Murine Leukemia Virus. Furthermore, we present a model where target site selection is directed at multiple scales. At a large scale, target site selection is similar across systems, and defined by domain-oriented features, namely expression of proximal genes, proximity to CpG islands and to genic features, chromatin compaction and replication timing. Notable differences between the systems are mainly observed at smaller scales, and are directed by a diverse range of features. To study the effect of these biases on integration sites occupied under selective pressure, we turned to insertional mutagenesis (IM) screens. In IM screens, putative cancer genes are identified by finding frequently targeted genomic regions, or Common Integration Sites (CISs). Within three recently completed IM screens, we identified 7%–33% putative false positive CISs, which are likely not the result of the oncogenic selection process. Moreover, results indicate that PB, compared to SB, is more suited to tag oncogenes.</p></div

Genome and chromatin profiling data used in this paper.

<p>Genome and chromatin profiling data used in this paper.</p

Unselected integration profiles and CIS designation.

<p>A) The bias of unselected integrations relative to CIS integrations, on a scale from blue (more CIS integrations) to red (more unselected integrations). B) log2 ratio of activating CISs and repressing CISs. A CIS is activating if it is not within a gene, or within a gene and 90% homogeneous with regard to orientation relative to that gene. Otherwise it is repressive. C) Bias of unselected integrations for CIS regions in a (i) genome-wide background, (ii) genic background (+/−100 kb), and (iii) intergenic background (whole genome except genes +/−100 kb), as measured by the log2 ratio of observed (unselected integrations) and expected (matched controls). D) CIS integration counts vs. unselected integration counts. CISs are annotated with the nearest TSS. Note that a single gene can be associated with multiple CISs. Spurious CISs were determined by a one-sided binomial test to determine if the CIS contained more CIS integrations than unselected integrations (, FDR-corrected).</p

Integration datasets used in this paper.

<p>Integration datasets used in this paper.</p