A potential role of extended simple sequence repeats in competing endogenous RNA crosstalk

Abstract

MicroRNA (miRNA)-mediated crosstalk between coding and non-coding RNAs of various types is known as the competing endogenous RNA (ceRNA) concept. Here, we propose that there is a specific variant of the ceRNA language that takes advantage of simple sequence repeat (SSR) wording. We applied bioinformatics tools to identify human transcripts that may be regarded as repeat-associated ceRNAs (raceRNAs). Multiple protein-coding transcripts, transcribed pseudogenes, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) showing this potential were identified, and numerous miRNAs were predicted to bind to SSRs. We propose that simple repeats expanded in various hereditary neurological diseases may act as sponges for miRNAs containing complementary repeats that would affect raceRNA crosstalk. Based on the representation of specific SSRs in transcripts, expression data for SSR-binding miRNAs and expression profiling data from patients, we determined that raceRNA crosstalk is most likely to be perturbed in the case of myotonic dystrophy type 1 (DM1) and type 2 (DM2).

Keywords: ceRNA hypothesis; miRNA cooperativity; miRNA sponge; microsatellite repeats; myotonic dystrophy; non-coding RNAs; repeat expansion diseases.

Figure 1.

Characterization of SSRs found in human coding and non-coding transcripts. (A) Representation rate of all triplet repeats in the human RNAome compared with their occurrence in the human genome. Tracts of at least 5 consecutive triplet repeats were analyzed in the genome, protein-coding transcripts, lncRNAs, pseudogenes and circRNAs. Positive and negative values represent the fold over- and under-representation, respectively. Repeats capable of forming stable RNA structures are marked with an asterisk. (B) Number of non-coding transcripts containing at least 5 consecutive disease-relevant SSRs. No RNAs were found for AUUCU, GGCCUG and GGGGCC repeat tracts. (C) Distribution of the lengths of tracts containing at least 5 consecutive disease-relevant triplet repeats in the genome and RNAome, * − P-value < 0.05; ** − P-value < 0.01, *** − P-value < 0.001. Boxes and whiskers represent the minimum and maximum values.

Figure 2.

Identification of miRNAs binding to SSR tracts. (A) Number of miRNAs predicted to bind to disease-relevant SSRs showing 7-nt and 6-nt complementarity to SSRs within their seed regions. (B) Secondary structure representations of complexes between CUG repeats and miR-15a-5p and between CAG repeats and miR-370-3p. SSR tracts are highlighted in yellow; miRNA seed regions are highlighted in red. (C) Distribution of the number of predicted MREs for miRNAs interacting with SSRs in the 3ʹ UTRs of protein-coding transcripts and in ncRNAs (lncRNAs, pseudogenes and circRNAs). Each point represents a single RNA; only RNAs with a minimum of 10 MREs are shown. (D) The density of predicted MREs was calculated as the number of MREs per 1 kb of the analyzed RNAs with multiple (minimum of 10) MREs and is depicted as the mean value with the SEM (symbols with bars).

Figure 3.

SSRs mediate raceRNA crosstalk. (A) Expression levels of CUG- and CCUG repeat-binding miRNAs in muscle (upper graphs) and expression of CAG and UGGAA repeat-binding miRNAs in the brain (lower graphs). Expressed miRNAs are depicted with gray diamond-shaped symbols; miRNAs binding to repeats are shown in red. MiRNAs showing 7-nt matching within the seed region are underscored. (B) Modeling of miRNA-mediated crosstalk between CUG repeats and a group of CUG-binding miRNAs (with a 6-nt AGCAGC seed region (hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-195-5p, hsa-miR-424-5p, hsa-miR-497-5p, hsa-miR-503-5p, hsa-miR-646 and hsa-miR-6838-5p)) in human myoblasts. The graph depicts the effects on miRNA site occupancy upon DMPK transcript expression (normal (~2 MREs), with premutation (~5 MREs), in adult-onset DM1 (60 MREs) or congenital DM1 (240 MREs)). (C) Modeling of miRNA crosstalk between CCUG repeats and a group of CCUG-binding miRNAs (with a 6-nt AGGCAG seed region (hsa-miR-34b-5p, hsa-miR-449c-5p, hsa-miR-940, hsa-miR-1910-3p, hsa-miR-2682-5p, hsa-miR-6808-5p, hsa-miR-6893-5p and hsa-miR-6511a-5p)) in human myoblasts. The graph depicts the effects on miRNA site occupancy upon of CNBP transcript expression (normal (3 MREs) or present in DM2 (800 MREs)). (D) The graph depicts the percentage of mRNAs bearing putative conserved MREs for conserved CUG-binding miRNAs in their 3ʹ UTRs in all human mRNAs and mRNAs downregulated or upregulated in DM1 patients (Supplementary Table III), *** − P-value < 0.001. (E) The graph depicts the percentage of mRNAs bearing putative conserved MREs for conserved CCUG-binding miRNAs in their 3ʹ UTRs in all human mRNAs and mRNAs downregulated or upregulated in DM2 patients (Supplementary Table III), ** − P-value < 0.01. (F) The graph depicts the percentage of mRNAs bearing putative conserved MREs for all conserved miRNAs in their 3ʹ UTRs in in all human mRNAs, mRNAs downregulated or upregulated in DM1 patients and mRNAs downregulated or upregulated in DM2 patients, *** − P-value < 0.001.

Figure 4.

The concept of miRNA-mediated crosstalk between coding and non-coding transcripts using SSR tracts. Different types of RNAs containing SSR tracts (protein-coding RNAs and ncRNAs, including pseudogenes, lncRNAs and circRNAs) can function as ceRNAs. SSR-binding miRNAs play a central role in the crosstalk between various cellular RNAs. Aberrant expansion of SSRs may lead to disruption of this network.