Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized1–4; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR–mRNA interaction occurs through a 25-nucleotide ‘TINCR box’ motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR–STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

MicroRNAs (miRNAs) are short, highly conserved non-coding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene’s protein expression in the presence and absence of regulation by miRNA. We find that while the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target messenger RNA (mRNA) below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results demonstrate that miRNAs can act both as a switch and as a fine-tuner of gene expression.

Short RNA expression in several distinct stages of T-lymphocyte development was comprehensively profiled. The total number of microRNAs (miRNAs) expressed per cell at different stages of development varies over nearly an order of magnitude in parallel with changes in total cellular RNA content, suggesting that global miRNA levels are coregulated with the translational capacity of the cell. However, individual miRNAs were dynamically regulated during T-cell development, with at least one miRNA or miRNA family overrepresented at each developmental stage. miRNA regulation in this developmental pathway is characterized by analog rather than switch-like behavior, with temporal enrichments at distinct stages of development observed against a background of constant, basal expression of the miRNA. Enrichments of these miRNAs are temporally correlated with depletions of the transcript levels of targets containing seed matches to the specific miRNAs, and may have specific functional consequences. miR-181a, which is specifically enriched at the CD4 + CD8 + (DP) stage of thymocyte development, can repress the expression of Bcl-2, CD69, and the T-cell receptor, all of which are coordinately involved in positive selection.[Keywords: CD69; development; genomics; microRNAs; microarray; thymocyte] Supplemental material is available at http://www.genesdev.org.

Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.

MicroRNAs (miRNAs) are 19-22nt non-coding RNAs that post-transcriptionally regulate mRNA targets. To identify endogenous miRNA binding sites, we performed photo-crosslinking immunoprecipitation using Ago2 antibodies, followed by deep-sequencing of RNAs (CLIP-seq) in mouse embryonic stem cells (mESCs). We also performed CLIP-seq in Dicer−/− mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA-dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ∼68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences crosslinked to Ago2 in the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.

Summary C. elegans 21U-RNAs are equivalent to the piRNAs discovered in other metazoans and have important roles in gametogenesis and transposon control. The biogenesis and molecular function of 21U-RNAs and piRNAs are poorly understood. Here, we demonstrate that transcription of each 21U-RNA is regulated separately through a conserved upstream DNA motif. We use genomic analysis to show that this motif is associated with low nucleosome occupancy, a characteristic of many promoters that drive expression of protein-coding genes, and that RNA polymerase II is localized to this nucleosome-depleted region. We establish that the most conserved 8-mer sequence in the upstream region of 21U-RNAs, CTGTTTCA, is absolutely required for their individual expression. Furthermore, we demonstrate that the 8-mer is specifically recognized by Forkhead family (FKH) transcription factors and that 21U-RNA expression is diminished in several FKH mutants. Our results suggest that thousands of small non-coding transcription units are regulated by FKH proteins.

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.

Long noncoding RNAs (lncRNAs) are important regulators of cell fate, yet little is known about mechanisms controlling lncRNA expression. Here we show that transcription is quantitatively different for lncRNAs and mRNAs—as revealed by deficiency of Dicer (Dcr), a key RNase that generates microRNAs (miRNAs). Dcr loss in mouse embryonic stem cells led unexpectedly to decreased levels of hundreds of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA transcriptional initiation and elongation. Computational and genetic epistasis analyses demonstrated that Dcr activation of the oncogenic transcription factor cMyc is partly responsible for lncRNA expression. A quantitative metric of mRNA-lncRNA decoupling revealed that Dcr and cMyc differentially regulate lncRNAs versus mRNAs in diverse cell types and in vivo. Thus, numerous lncRNAs may be modulated as a class in development and disease, notably where Dcr and cMyc act.