Functional Analysis of MicroRNAs as Regulators of Membrane Trafficking

Abstract

MicroRNAs (miRNAs) are a large family of small noncoding RNAs that extensively regulate gene expression in animals, plants and protozoa. The first miRNA was identified in the early 1990s, but it took almost a decade until miRNAs were recognized as key post-transcriptional regulators of gene expression. Despite the rapidly growing list of miRNA-regulated physiological and pathological processes, intracellular membrane trafficking has attracted little interest from scientific miRNA community. Membrane trafficking defines a complex network of pathways, including biosynthetic trafficking and endocytosis that are indispensable for normal cellular functions. Previous studies have analyzed a few miRNAs involved in insulin secretion, however, no systematic investigation of miRNAs as important regulators of membrane trafficking has been performed. The overall aim of this study was to identify miRNAs and their biologically relevant target genes involved in the regulation of membrane trafficking. As tools to modulate miRNA functions, we used synthetic miRNA mimics (pre-miRs) and inhibitors (anti-miRs) to enhance (gain-of-function) and to suppress (loss-of-function) the activity of cellular miRNAs, respectively. As proof of principle, we demonstrated that increased activity of miR-17 family miRNAs accelerates the biosynthetic cargo protein (ts-O45-G) transport and reduces the cellular internalization of DiI-LDL ligand. Taking the advantage of available technological platforms, we designed a gain-of-function large-scale screening to identify miRNAs that affect biosynthetic ts-O45-G transport rate. We showed that 44 out of 470 tested miRNAs induced significant changes in cargo trafficking. Using image analysis platform, we further identified eight miRNAs (miR-30b, -382, -432, -517a, -517b, -517c, -637 and -765) that also showed significant effects on Golgi complex integrity. Importantly, the majority of identified miRNAs are not endogenously expressed in HeLa cells, indicating the need for validation studies in other experimental systems. To identify functionally relevant target genes, we selected miR-17 and miR-517a and performed genome-wide transcriptome analysis 12h, 24h and 48h after transfection with the respective pre-miRs. We identified TBC1D2 and LDLR genes as novel functional miR-17 targets and confirmed that they exert the miR-17-mediated regulation of endocytosis. Further studies are needed to identify target genes responsible for the miR-17-governed acceleration of ts-O45-G to the plasma membrane. In case of miR-517a, we found a set of target genes with functions in 12 membrane trafficking system, however, their functional interplay with miR-517a remains to be confirmed. Bioinformatics analysis of transcriptome profiling data confirmed that the presence of miRNA seed binding site in the 3´UTRs of human mRNAs is an important determinant for functional miRNA:mRNA interaction. Additionally, we demonstrated that the sets of transcripts downregulated at early time points after transfection with pre-miRs have substantially higher fractions of transcripts with miRNA binding sites in their 3´UTRs compared to the transcripts downregulated at late time points. We believe that these findings could contribute to the development of more accurate miRNA target prediction tool, also allowing identification of nonconserved miRNA targets. In conclusion, we have established an experimental platform that consists of (i) a functional screening module to identify miRNAs that affect membrane trafficking, (ii) a microarray module to identify miRNA target genes, (iii) a statistics and bioinformatics module for data analysis and integration and (iv) a target validation module to validate functional links between targets and miRNAs. Using this platform, we identified numerous miRNAs with novel functions in membrane trafficking system. Moreover, we identified and confirmed TBC1D2 and LDLR genes as novel functional targets of miR-17.