miRNA Combinatorics and its Role in Cell State Control-A Probabilistic Approach.

A hallmark of cancer evolution is that the tumor may change its cell identity and improve its survival and fitness. Drastic change in microRNA (miRNA) composition and quantities accompany such dynamic processes. Cancer samples are composed of cells' mixtures of varying stages of cancerous progress. Therefore, cell-specific molecular profiling represents cellular averaging. In this study, we consider the degree to which altering miRNAs composition shifts cell behavior. We used COMICS, an iterative framework that simulates the stochastic events of miRNA-mRNA pairing, using a probabilistic approach. COMICS simulates the likelihood that cells change their transcriptome following many iterations (100 k). Results of COMICS from the human cell line (HeLa) confirmed that most genes are resistant to miRNA regulation. However, COMICS results suggest that the composition of the abundant miRNAs dictates the nature of the cells (across three cell lines) regardless of its actual mRNA steady-state. In silico perturbations of cell lines (i.e., by overexpressing miRNAs) allowed to classify genes according to their sensitivity and resilience to any combination of miRNA perturbations. Our results expose an overlooked quantitative dimension for a set of genes and miRNA regulation in living cells. The immediate implication is that even relatively modest overexpression of specific miRNAs may shift cell identity and impact cancer evolution.

Spliceosome-Associated microRNAs Signify Breast Cancer Cells and Portray Potential Novel Nuclear Targets.

MicroRNAs (miRNAs) act as negative regulators of gene expression in the cytoplasm. Previous studies have identified the presence of miRNAs in the nucleus. Here we study human breast cancer-derived cell-lines (MCF-7 and MDA-MB-231) and a non-tumorigenic cell-line (MCF-10A) and compare their miRNA sequences at the spliceosome fraction (SF). We report that the levels of miRNAs found in the spliceosome, their identity, and pre-miRNA segmental composition are cell-line specific. One such miRNA is miR-7704 whose genomic position overlaps HAGLR, a cancer-related lncRNA. We detected an inverse expression of miR-7704 and HAGLR in the tested cell lines. Specifically, inhibition of miR-7704 caused an increase in HAGLR expression. Furthermore, elevated levels of miR-7704 slightly altered the cell-cycle in MDA-MB-231. Altogether, we show that SF-miR-7704 acts as a tumor-suppressor gene with HAGLR being its nuclear target. The relative levels of miRNAs found in the spliceosome fractions (e.g., miR-100, miR-30a, and let-7 family) in non-tumorigenic relative to cancer-derived cell-lines was monitored. We found that the expression trend of the abundant miRNAs in SF was different from that reported in the literature and from the observation of large cohorts of breast cancer patients, suggesting that many SF-miRNAs act on targets that are different from the cytoplasmic ones. Altogether, we report on the potential of SF-miRNAs as an unexplored route for cancerous cell state.

A cell-based probabilistic approach unveils the concerted action of miRNAs.

Mature microRNAs (miRNAs) regulate most human genes through direct base-pairing with mRNAs. We investigate the underlying principles of miRNA regulation in living cells. To this end, we overexpressed miRNAs in different cell types and measured the mRNA decay rate under a paradigm of a transcriptional arrest. Based on an exhaustive matrix of mRNA-miRNA binding probabilities, and parameters extracted from our experiments, we developed a computational framework that captures the cooperative action of miRNAs in living cells. The framework, called COMICS, simulates the stochastic binding events between miRNAs and mRNAs in cells. The input of COMICS is cell-specific profiles of mRNAs and miRNAs, and the outcome is the retention level of each mRNA at the end of 100,000 iterations. The results of COMICS from thousands of miRNA manipulations reveal gene sets that exhibit coordinated behavior with respect to all miRNAs (total of 248 families). We identified a small set of genes that are highly responsive to changes in the expression of almost any of the miRNAs. In contrast, about 20% of the tested genes remain insensitive to a broad range of miRNA manipulations. The set of insensitive genes is strongly enriched with genes that belong to the translation machinery. These trends are shared by different cell types. We conclude that the stochastic nature of miRNAs reveals unexpected robustness of gene expression in living cells. By applying a systematic probabilistic approach some key design principles of cell states are revealed, emphasizing in particular, the immunity of the translational machinery vis-a-vis miRNA manipulations across cell types. We propose COMICS as a valuable platform for assessing the outcome of miRNA regulation of cells in health and disease.

Small RNA sequences derived from pre-microRNAs in the supraspliceosome.

MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the expression and translation of genes in healthy and diseased tissues. Herein, we characterize short RNAs from human HeLa cells found in the supraspliceosome, a nuclear dynamic machine in which pre-mRNA processing occurs. We sequenced small RNAs (<200 nt) extracted from the supraspliceosome, and identified sequences that are derived from 200 miRNAs genes. About three quarters of them are mature miRNAs, whereas the rest account for various defined regions of the pre-miRNA, and its hairpin-loop precursor. Out of these aligned sequences, 53 were undetected in cellular extract, and the abundance of additional 48 strongly differed from that in cellular extract. Notably, we describe seven abundant miRNA-derived sequences that overlap non-coding exons of their host gene. The rich collection of sequences identical to pre-miRNAs at the supraspliceosome suggests overlooked nuclear functions. Specifically, the abundant hsa-mir-99b may affect splicing of LINC01129 primary transcript through base-pairing with its exon-intron junction. Using suppression and overexpression experiments, we show that hsa-mir-7704 negatively regulates the level of the lncRNA HAGLR. We claim that in cases of extended base-pairing complementarity, such supraspliceosomal pre-miRNA sequences might have a role in transcription attenuation, maturation and processing.