Post-transcriptional regulation of miRNA biogenesis

MicroRNAs (miRNAs) are short non-coding RNA molecules of ~22 nt in length that function as regulators of gene expression. They bind to the 3’ UTR of target mRNAs in a non-perfect complimentary manner, leading to either their degradation or translational repression. Over half of all protein coding genes contain miRNA target sites, meaning that miRNAs are involved in the regulation of virtually every cellular pathway. Because of this, miRNAs themselves are under strict regulation, often expressed in temporal or tissue-specific manners. MiRNAs are transcribed as long primary transcripts (pri-miRs) with stem-loop structures that are processed in the nucleus into ~70nt hairpins (pre-miRs), exported into the cytoplasm and cleaved into miRNA duplexes. MiRNA duplexes are incorporated into Ago proteins which retain single stranded mature miRNA in a functional RISC complex. Regulation of miRNA biogenesis occurs both at a transcriptional and post-transcriptional level. Various proteins that function in the post-transcriptional regulation of miRNA biogenesis have been described. However, in recent years, proteome-wide studies have expanded our knowledge of RNAbinding proteins (RBPs). Many non-canonical RBPs with unknown roles in RNA metabolism have been identified. This expanded repertoire of RBP represents a new pool of potential miRNA biogenesis regulators. One group of interest amongst newly identified non-canonical RBPs are metabolic enzymes which are beginning to show a role in RNA metabolism, providing a link between gene regulation and cellular metabolism that is not yet fully understood. This study has focused on better understanding the post-transcriptional regulation of miRNA biogenesis in several systems. Firstly, protein factors involved in the regulation of miRNA cluster 14q32 were sought. This cluster represents the largest mammalian miRNA cluster and is important in mediating the response to ischemia. In this study, two proteins, cold-induced RNA binding protein (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB), were identified as post-transcriptional regulators of selected 14q32 miRNAs and further validated in cellular systems. Next, the mechanism underlying the control of miRNA biogenesis by known RBP regulator, cell lineage abnormal 28a (Lin28a), was explored in further detail. Lin28a represents the best-studied example of post-transcriptional miRNA regulation, influencing both nuclear and cytoplasmic let-7 processing as well as mediating pre-let- 7 uridylation and subsequent degradation. Lin28a can also regulate neuro-specific miR-9 which differs in sequence and structure from let-7. Here we showed that both miRNA precursors interact with different domains of Lin28a and the mechanisms regulation are different. This shows how a single RBP can have extensive effects in the regulation of miRNA biogenesis in different cellular contexts. Finally, in this study the inhibition by oleic acid (OA) of HuR and MSI2 binding to pri-miR-7 was explored. MSI2 in a complex with HuR inhibits biogenesis of neuroenriched miR-7. OA is the most abundant fatty acid and had previously been shown to inhibit the RNA-binding of MSI2 homolog, MSI1. Here this knowledge was extended to show that OA also inhibits MSI2 and HuR binding to pri-miR-7, increasing its processing. This gives an example of a mechanism regulating the interaction of miRNA precursors with regulatory proteins, modulating the effect of these proteins on miRNA biogenesis. This also provided additional links between metabolism and regulation of RNA processing. In general, these investigations add to the current knowledge on post-transcriptional regulation of miRNAs and highlight the complexity of this system. Different mechanisms governing regulation of miRNA processing coexists in cells. Therefore, better understanding the individual factors at play will allow to generate a better picture of the regulatory networks mediating miRNA biogenesis.