Control of somatic tissue differentiation by the long non-coding RNA TINCR

Abstract

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; 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.

Figure 1. TINCR is induced during epidermal differentiation

a, Mean-centred, hierarchical clustering of 258 annotated non-coding RNAs altered (>twofold change) in undifferentiated cells (day 0) and during days of calcium-induced differentiation in vitro. b, Schematic of TINCR genomic locus on chromosome 19. Day 0, 3 and 6 of keratinocyte (KC) differentiation; blue rectangles represent exons. c, Relative TINCR abundance in fragments per kilobase of exon model per million mapped fragments (FPKM). d, TINCR qRT–PCR. Error bars are s.d., n = 4. e, Northern blot analysis, with TINCR the single band seen in differentiation; bp, base pairs.

Figure 2. TINCR regulates epidermal differentiation genes involved in barrier formation

a, Loss of differentiation proteins in TINCR-depleted organotypic human epidermis by independent TINCR siRNAs (siTINCRA and siTINCRB) versus scrambled control (siControl); nuclei stained blue (Hoechst 33342). Scale bars, 50 μm. b, mRNA expression in TINCR-deficient tissue versus control; duplicate biological replicates for duplicate independent TINCR siRNAs. Error bars are s.d., n = 6. c, GO terms significantly enriched in the TINCR-depleted gene subset. d, mRNA expression of lipid barrier synthesis genes in TINCR-depleted tissue. Error bars denote s.d., n = 4. e, Loss of protein-rich keratohyalin granules (arrows in control) in TINCR-deficient organotypic human epidermis. St, stratum. Scale bars, 10 μm. f, Loss of normal lipid-containing lamellar bodies (arrows in control, top image) in TINCR-depleted tissue (bottom image) (n = 3). Scale bars, 100 nm.

Figure 3. TINCR interacts with differentiation mRNAs and STAU1 protein

a, Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b, Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c, STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST) represent lncRNA controls. d, Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.

Figure 4. Differentiation regulation by TINCR RNA and STAU1 protein

a, Diminished expression of TINCR-regulated genes in STAU1-depleted organotypic tissue in independent biological replicates for duplicate independent STAU1 siRNA treatments. Error bars are s.d., n = 3. b, Overlap of 672 genes regulated by TINCR and STAU1 c, Location of the TINCR box motif in TINCR as well as selected TINCR-associated differentiation mRNAs. RIA, RNA interactome analysis. d, TINCR motif base-pairing between the TINCR transcript and PGLYRP3 differentiation gene mRNA. e, Biotinylated TINCR RNA with or without STAU1 protein pulls down the full-length 1,077-bp PGLYRP3 mRNA at higher efficiency than TINCR box-depleted PGLYRP3 (PGLYRP3Δ96 bp). Error bars denote s.d. and are too small to be visible for the PGLYRP3Δ96 samples; n = 3.