Ribosomal RNA 2'-O-methylation dynamics impact cell fate decisions.

Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2'-O-methylation (2'-O-me) of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here, we examine changes in rRNA 2'-O-me during mouse brain development and tri-lineage differentiation of human embryonic stem cells (hESCs). We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site impacting neuronal differentiation. Modulation of its methylation levels affects ribosome association of the fragile X mental retardation protein (FMRP) and is accompanied by an altered translation of WNT pathway-related mRNAs. Together, these data identify ribosome heterogeneity through rRNA 2'-O-me during early development and differentiation and suggest a direct role for ribosomes in regulating translation during cell fate acquisition.

Regulation of translation by site-specific ribosomal RNA methylation.

Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms and can arise from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2'-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signaling pathways, such as MYC oncogene expression. Ablation of one prominent methylation resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2'-O-methylation can give rise to ribosomes with specialized function. This suggests a broader mechanism where the specific regulation of rRNA modification patterns fine tunes translation.

Profiling of 2'-O-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.

A dual program for translation regulation in cellular proliferation and differentiation.

A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found that genes serving cell-autonomous functions and genes involved in multicellularity obey distinct codon usage. Proliferation-induced and differentiation-induced tRNAs often carry anticodons that correspond to the codons enriched among the cell-autonomous and the multicellularity genes, respectively. Because mRNAs of cell-autonomous genes are induced in proliferation and cancer in particular, the concomitant induction of their codon-enriched tRNAs suggests coordination between transcription and translation. Histone modifications indeed change similarly in the vicinity of cell-autonomous genes and their corresponding tRNAs, and in multicellularity genes and their tRNAs, suggesting the existence of transcriptional programs coordinating tRNA supply and demand. Hence, we describe the existence of two distinct translation programs that operate during proliferation and differentiation.

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 lpo and collaborates with activated Wnt signaling in inducing intestinal neoplasia in female mice.

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 Hox 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 miR-10a knock out mouse. Here we show that, in the Apc(min) mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10(+/+) and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a 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 Lpo 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.

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.