Inverted repeat Alu elements in the human lincRNA-p21 adopt a conserved secondary structure that regulates RNA function

Abstract

LincRNA-p21 is a long intergenic non-coding RNA (lincRNA) involved in the p53-mediated stress response. We sequenced the human lincRNA-p21 (hLincRNA-p21) and found that it has a single exon that includes inverted repeat Alu elements (IRAlus). Sense and antisense Alu elements fold independently of one another into a secondary structure that is conserved in lincRNA-p21 among primates. Moreover, the structures formed by IRAlus are involved in the localization of hLincRNA-p21 in the nucleus, where hLincRNA-p21 colocalizes with paraspeckles. Our results underscore the importance of IRAlus structures for the function of hLincRNA-p21 during the stress response.

Figure 1.

The human lincRNA-21 is a single exon lncRNA that contains IRAlu elements. (A) Diagram of precursor (Pre-RNA) and mature mouse lincRNA-p21 (mLincRNA-p21) and of the previously reported partial sequence of human lincRNA-p21 (hLincRNA-p21) [adapted from Yoon et al. (9)]. The asterisk represents the p53 protein binding site. The shaded box represents a region of sequence homology between mLincRNA-p21 and hLincRNA-p21. (B) RT-PCR performed on total RNA from HEK293, Hela, H1299, MCF-10A, HCT-116 and MCF-7 cell lines and on primary dermal fibroblasts. Four different primer pairs were used to amplify portions of exon 1 (E1), the putative exon1-intron junction (US), the putative exon1–exon2 junction (Sp) and exon 2 (E2). H stands for housekeeping gene (tRNA-Lys for HeLa and ACTB for all other cell lines). MW is a DNA size ladder expressed in base pairs. (C) Comparative diagram of the full-length sequence isoforms of hLincRNA-p21 as obtained by 3′ RACE on HEK293 cells and the previously reported partial sequence of hLincRNA-p21. (D) Schematic representation of hLincRNA-p21 with associated UCSC Genome Browser tracks depicting ENCODE H3K4me3 and H3K27Ac ChIP-seq coverage as well as CSHL Long RNA-seq coverage (26). (E) Northern blot analysis of hLincRNA-p21 in nuclear RNA from HEK293 and HCT-116 cell lines and of total and nuclear RNA from primary dermal fibroblasts. A probe hybridizing with the 5′ end of hLincRNA-p21 was used. (F) Maximum CSF scores of hLincRNA-p21 isoforms and CDKN1A (p21) RNAs determined by analysis with PhyloCSF (27). (G) Quantification of hLincRNA-p21 copy number per cell determined in the HCT-116 cell line by qRT-PCR.

Figure 2.

Inverted repeat Alu elements (IRAlus) in hLincRNA-21 fold as independent structural domains in vitro. (A and B) Experimentally-derived secondary structures of sense (C) and antisense (D) Alu sequences of hLincRNA-p21. A schematic diagram of hLincRNA-p21 LIsoE2 isoform highlights the relative position of each structure in the RNA sequence (top). Colors of the circles around each nucleotide indicate SHAPE reactivity. The dotted lines represent putative tertiary contacts between the terminal loops of the three-way junctions. Data values are the mean of two biological replicates. Observed U to C transitions are indicated by colored arrowheads. The frequency values represent the fraction of observed transitions of n = 93 sequences from eight biological replicates for the sense Alu and n = 35 sequences from three biological replicates for the antisense Alu. Covariation of lincRNA-p21 in human and nine other primates is shown in boxes for selected regions (See also Supplementary Figure S3).

Figure 3.

The secondary structure of the inverted repeat Alu (IRAlu) elements is involved in hLincRNA-p21 nuclear localization. (A) Schematic representation of an Alu element indicating the strategy followed to disrupt the secondary structure of the RNA helices for each Alu element. (B) Relative subcellular distribution of various hLincRNA-p21 constructs quantified by qRT-PCR using nuclear and cytoplasmic RNA fractions. Wild-type (WT), disrupted IRAlu elements (dIR-Alu), disrupted sense Alu element (dS-Alu) and disrupted antisense Alu element (dA-Alu) constructs derived from the short isoform ending at site 1 (SIsoE1) and the long isoform ending at site 2 (LIsoE2) were used. A truncated 5E construct, retaining the first 920 nt of hLincRNA-p21 was also included. β-actin was used for normalization. Data represent the mean ± SEM of three biological replicates. The P-values obtained from unpaired t-test are <0.05 for all samples, assuming a level of significance α = 0.05. (C) Representative RNA-FISH maximum projection images depicting cellular distribution of WT and selected mutant hLincRNA-p21 constructs transfected in HEK293 cells. Cells were harvested 36 h after transfection and RNA-FISH was performed using a probe complementary to the MS2 hairpins (green). Nuclei were identified with NucRed staining (magenta). Schematic representations of the MS2-tailed constructs used for the heterologous expression of hLincRNA-p21 are also shown (upper right).

Figure 4.

The hLincRNA-p21 colocalizes with paraspeckles in the nucleus. (A) Quantification of endogenous hLincRNA-p21 by qRT-PCR in the nuclear and cytoplasmic fractions after the treatment with increasing concentrations of doxorubicin in different cell lines for 12 h. Data represent the mean ± SEM of two biological replicates. (B) The distribution of the fraction of NEAT1 spots that colocalize with hLincRNA-21 in doxorubicin-treated and untreated cells (n = 14 at 0 μM and n = 15 at 1 μM doxorubicin for MCF-7 and n = 18 at 0 μM and n = 18 at 1 μM doxorubicin for HCT-116). Mander's coefficients, indicating the degree of overlap, were obtained using Coloc2 (ImageJ) from RNA-FISH experiments performed in different cell lines. (C) Representative RNA-FISH single confocal plane images showing cellular distribution of endogenous hLincRNA-p21 and colocalization with NEAT1 in different cell lines at 1 μM doxorubicin. Overlapping foci between NEAT1 and hLincRNA-p21 are highlighted by arrows. Linescan analyses (right panel) were performed through the spots indicated by yellow arrows in the merge panel.