Profiling of 2'-<i>O</i>-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity

Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2′-O-Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2′-O-Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2′-O-Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings

Identification of expressed and conserved human noncoding RNAs

The past decade has shown mammalian genomes to be pervasively transcribed and identified thousands of noncoding (nc) transcripts. It is currently unclear to what extent these transcripts are of functional importance, as experimental functional evidence exists for only a small fraction. Here, we characterize the expression and evolutionary conservation properties of 12,115 known and novel nc transcripts, including structural RNAs, long nc RNAs (lncRNAs), antisense RNAs, EvoFold predictions, ultraconserved elements, and expressed nc regions. Expression levels are evaluated across 12 human tissues using a custom-designed microarray, supplemented with RNAseq. Conservation levels are evaluated at both the base level and at the syntenic level. We combine these measures with epigenetic mark annotations to identify subsets of novel nc transcripts that show characteristics similar to known functional ncRNAs. Few novel nc transcripts show both high expression and conservation levels. However, overall, we observe a positive correlation between expression and both conservation and epigenetic annotations, suggesting that a subset of the expressed transcripts are under purifying selection and likely functional. The identified subsets of expressed and conserved novel nc transcripts may form the basis for further functional characterization

Loss of miR-10a Activates <i>Lpo</i> and Collaborates with Activated Wnt Signaling in Inducing Intestinal Neoplasia in Female Mice

<div><p>miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is particularly interesting considering its conserved location in the <i>Hox</i> cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a <i>miR-10a</i> knock out mouse. Here we show that, in the <i>Apc^min</i> mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10^+/+ and male controls. We further found that <i>Lpo</i> is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using <i>in vitro</i> assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of <i>Lpo</i>, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of <i>miR-10a</i> knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that <i>Lpo</i> upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.</p></div

<i>Lpo</i> is transcriptionally upregulated in the intestines of <i>miR-10a</i> deficient female mice.

<p>(A) <i>Lpo</i> mRNA is ∼29-fold upregulated in intestines of <i>miR-10a</i> KO compared to WT mice as shown by qRT-PCR. <i>Lpo</i> mRNA levels are normalized to <i>Actb</i> and values ± SD are shown relative to the first WT sample. (B) Western-blot from same tissue samples as in (A) confirming upregulation on protein level. Vinculin was used as loading control. As evident from Lpo and Vinculin control as well as ponceau staining (now shown), sample 4 did not contain any protein for unknown reason. (C) Representative immunohistochemistry staining of Lpo in <i>miR-10a</i> WT and KO intestine. Scale bar 100 µm. (D) Scoring of Lpo expression level estimated by distribution and staining intensity in Lpo stained intestines of WT and miR-10a^−/− mice. Scoring is divided into low, medium or high expression. Consistent with qRT-PCR and Western blotting analysis a significant difference (<i>p</i>≤0.006, Pearson chi-square test with exact probability) in Lpo expression is observed between the different genotypes.</p

Transcription factor <i>KLF4</i> is regulated by miR-10a and can regulate the <i>LPO</i> promoter <i>in vitro</i>.

<p>HCT-116 cells were transfected with a miR-10a duplex or control for 72 h. (A) Relative mRNA levels of <i>KLF4</i> were measured by qRT-PCR and <i>ACTB</i> was used for normalization. Data are shown as mean ± S.D. of three replicates relative to the control and are representative of three independent experiments. * p<0.05 using a two-tailed <i>t</i>-test. (B) Protein levels in miR-10a or control transfected cells were assessed by Western-blot using antibodies against KLF4. GAPDH was used as loading control. (C) Western Blot showing the over expression from the pcDNA3.1-KLF4 vector. GAPDH was used as loading control. (D) Luciferase reporter assay in HCT-116 cells (24 h) with pGL4-luc2 holding part of the <i>LPO</i> promoter (1 kb upstream TSS) or the pGL4-luc2 empty vector co-transfected with a vector over-expressing KLF4 or a control vector (pcDNA3.1+). Data are shown as mean ± S.D. of three replicates relative to the pcDNA3.1 transfected control and are representative of eleven independent experiments. **** p<0.0001 using a two-tailed <i>t</i>-test. (E) HCT-116 cells were transfected with KLF4 siRNA for 48 h or 72 h. Relative mRNA levels of <i>LPO</i> were measured by qRT-PCR and <i>ACTB</i> was used for normalization. Data are shown as mean ± S.D. of three replicates relative to the control and are representative of five independent experiments.</p

Klf4 is upregulated in <i>miR-10a</i> KO intestines.

<p>(A) mRNA levels of <i>Klf4</i> were measured by qRT-PCR, <i>Actb, Ubc, Hprt</i> and <i>36b4</i> were used for normalization. Data are shown as mean ± S.D. of <i>miR-10a</i> KO (n = 16) and WT (n = 13) samples relative to an average of the controls. * p<0.05 using a Mann-Whitney test. (B) Representative immunohistochemistry staining of Klf4 in <i>miR-10a</i> WT (n = 5) and KO (n = 8) intestine. Scale bar 100 µm (C) VisiomorphDP software scoring of Klf4 expression level estimated by distribution and staining intensity in Klf4 stained intestines of WT and miR-10a KO mice. * p = 0.019, students t-test.</p

The miR-10 microRNA precursor family

The miR-10 microRNA precursor family encodes a group of short non-coding RNAs involved in gene regulation. The miR-10 family is highly conserved and has sparked the interest of many research groups because of the genomic localization in the vicinity of, coexpression with and regulation of the Hox gene developmental regulators. Here, we review the current knowledge of the evolution, physiological function and involvement in cancer of this family of microRNAs