Evidence for Positive Selection on a Number of MicroRNA Regulatory Interactions during Recent Human Evolution

MicroRNA (miRNA)–mediated gene regulation is of critical functional importance in animals and is thought to be largely constrained during evolution. However, little is known regarding evolutionary changes of the miRNA network and their role in human evolution. Here we show that a number of miRNA binding sites display high levels of population differentiation in humans and thus are likely targets of local adaptation. In a subset we demonstrate that allelic differences modulate miRNA regulation in mammalian cells, including an interaction between miR-155 and TYRP1, an important melanosomal enzyme associated with human pigmentary differences. We identify alternate alleles of TYRP1 that induce or disrupt miR-155 regulation and demonstrate that these alleles are selected with different modes among human populations, causing a strong negative correlation between the frequency of miR-155 regulation of TYRP1 in human populations and their latitude of residence. We propose that local adaptation of microRNA regulation acts as a rheostat to optimize TYRP1 expression in response to differential UV radiation. Our findings illustrate the evolutionary plasticity of the microRNA regulatory network in recent human evolution

rs683 modulates endogenous <i>TYRP1</i> targeting by miR-155 in SK-MEL-19 cells.

<p>(A) Genotyping the rs683 locus in SK-MEL-19 cells. The region around rs683 was amplified from SK-MEL-19 genomic DNA and sequenced. Sequence traces (shown) revealed rs683 heterozygosity at the <i>TYRP1</i> locus, as indicated. (B) Ectopic miR-155 expression reduces <i>TYRP1</i> protein levels. <i>TYRP1</i> levels in the skin-derived melanoma cell line, SK-Mel-19 were analyzed by performing immunoblotting on cell lysates from miR-CTL transfected or cells transfected with increasing amounts of miR-155 as indicated. Densitometric quantitation of <i>TYRP1</i> levels is indicated as protein levels relative to the mock transfectants. (C) miR-155 reduces <i>TYRP1</i> mRNA levels in SK-Mel19. SK-MEL19 cells were mock transfected or transfected with the indicated miR-CTL or miR-155. mRNA was extracted and <i>TYRP1</i> levels assessed by qPCR. Results are plotted relative to miR-CTL-treated cells. Bars are the mean ± standard deviation of triplicate experiments. The differences in expression between miR-155 transfected cells and either mock or miR-CTL are statistically significant (P<0.01, Students <i>t</i> test). (D) Targeting of the derived allele by miR-155. SK-Mel-19 cells were transfected with increasing amounts of miR-155 as indicated. mRNA was then extracted and expression of the ancestral (blue) versus the derived allele (red) assessed by allele-specific TaqMan SNP qPCR. Results are plotted as the expression level of each <i>TYRP1</i> allele relative to controls (log2 transformed). Note that the transcripts carrying the derived allele were suppressed by miR-155 greater that 2-fold, whereas transcripts carrying the ancestral allele were only modestly affected.</p

PTEN regulates cilia through Dishevelled

Cilia are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia enable fluid movement over epithelial surfaces, while primary (sensory) cilia play roles in cellular signalling. The molecular events underlying cilia dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin homologue (PTEN) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN plays an important role in multicilia formation and cilia disassembly by controlling the phosphorylation of Dishevelled (DVL), another ciliogenesis regulator. DVL is a central component of WNTsignalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN. Our studies identify a novel protein substrate for PTEN that couples PTEN to regulation of cilia dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN-related pathologies

Next-generation RNA sequencing of archival formalin-fixed paraffin-embedded urothelial bladder cancer

Molecular profiling of individual cancers is key to personalised medicine. While sequencing technologies have required stringent sample collection and handling, recent technical advances offer sequencing from tissues collected in routine practice and tissues already stored in archives. In this paper, we establish methods for whole-transcriptome RNA sequencing (RNA-seq) from formalin-fixed paraffin-embedded tissues. We obtain average RNA-seq reads of >100 million per sample using the Illumina HiSeq2000 platform. We find high concordance with results from matching fresh frozen samples (>0.8 Spearman correlation). For validation, we compared low- and high-grade bladder cancer transcriptomes in 49 tumour samples after transurethral resection of bladder tumour. We found 947 differentially expressed protein-coding genes. While high-grade lesions exhibited distinct intertumour transcriptome heterogeneity, the transcriptome of low-grade tumours was homogeneous. PATIENT SUMMARY: In this report, we show that it is now possible to use universally available bladder cancer samples that have been fixed in formalin to perform high-quality transcriptome analysis. This ability will facilitate the development of transcriptome-wide tests based on gene expression correlated with clinical outcome