THE PLASTICITY OF MIRNA POOL: A NOVEL APPROACH TO REVEAL MECHANISMS BEHIND MIRNA TURNOVER

MicroRNAs (miRNAs) are a small (18-25nt long), evolutionary conserved, class of non-coding RNAs that appears as a major regulatory component of gene expression, implicated in virtually all known physiological and pathological processes. They act at post-transcriptional level by silencing the expression of a multitude of target mRNAs through various mechanisms, including target degradation and protein synthesis inhibition. As a result, the regulation of the miRNA pool is one of the critical events in the definition of cell identity and behavior both in physiology and disease. Although large efforts have been put in understanding how miRNA transcription and biogenesis are regulated, to date very little is known on what happens to miRNAs once they exerted their repressive function, if they are recycled on other target molecules or degraded, and how fast they are turned over. Typically, miRNAs are thought to be stable molecules with long half-lives. Nevertheless, in last years it emerged that some miRNAs could be also turned over rapidly upon different cellular conditions. To clarify how miRNAs decay in mammalian cells, we developed a new tailored approach based on in vivo RNA labeling (4sU pulse-chase) and high-throughput sequencing, which allows to investigate the modes and the mechanisms of miRNAs decay without perturbing global miRNA levels or miRNA processing. By this approach, we precisely measured miRNA decay rates in exponentially growing 3T9 mouse fibroblasts. Overall, miRNA turnover appeared heterogeneous; hence, miRNAs are not just stable molecules as previously thought. We could distinguish two pools of miRNA by decay: a group of miRNAs that are, indeed, very stable molecules (T1/2 >24h, ‘slow’); and another group composed of miRNAs quickly turned over in the cell (T1/2 <14h, ‘fast’). We further exploited RNA labeling by 4sU to quantitatively measure the biosynthetic rate (transcription) of miRNAs alongside. By integrating decay rates with biosynthesis, we developed a mathematical model to infer how different decays impact on miRNA regulation during cell transitions: fast miRNAs quickly reach a plateau of accumulation and are downregulated in few hours as compared to slow miRNAs. These findings were recapitulated in a specific biological process, namely the regulation of miRNAs by serum stimulation of quiescent fibroblast, which is characterized by a consistent 15 change in gene and miRNA expression in absence of cell division (hence, miRNA cannot be diluted). Indeed, serum ‘up’ and ‘down’ regulated miRNAs were characterized by marked difference in turnover rate, compatible with the kinetic of their changes and often coupled with transcriptional regulation, pointing out how multiple mechanisms concomitantly control miRNAs and their activity in mammalian cells. Mechanistically, our analyses suggested that the ratio between high affinity targets and miRNA abundance [which we termed target per miRNA (TPM) value] is a key determinant in the definition of the type of miRNA decay, supporting the ‘target-induced miRNA decay’ (TIMD) as a common mechanism that promotes miRNA degradation. This contention is also supported by a clear parallelism between decay-associated miRNA isoforms (trimmed and tailed sequence variants) and miRNA degradation dynamics, implying that i) the distribution of miRNA isoforms is reflecting the type of decay of miRNAs, and ii) specific enzymatic activities (i.e. nucleases, transferases) are in place and mediate miRNA degradation. So far, no endogenous target has been directly linked to TIMD mechanism. Our analyses provided a list of potential targets involved in TIMD, which could be critical in clarifying the role played by such mechanism in physiology. Preliminary experiments were performed on one of such target, which is much expressed upon serum stimulation of quiescent fibroblasts and highly complementary to a miRNA suddenly downregulated. Exploiting CRISPR/cas9 based genome engineering, we specifically affected miRNA:target interaction, keeping transcript level and protein functionality unaltered, ensuing in a consequent effect on miRNA regulation (loss of downregulation upon serum stimulation), fully supporting TIMD as an endogenous mechanism in control of miRNA functions.