SUMMARY Type 2 diabetes mellitus (T2DM) is a complex disease characterized by the inability of the insulin-producing β-cells in the endocrine pancreas to overcome insulin resistance in peripheral tissues. To determine if microRNAs are involved in the pathogenesis of human T2DM, we sequenced the small RNAs of human islets from diabetic and non-diabetic organ donors. We identified a cluster of miRNAs in an imprinted locus on human chromosome 14q32 that is highly and specifically expressed in human β-cells and dramatically down-regulated in islets from T2DM organ donors. The down-regulation of this locus strongly correlates with hyper-methylation of its promoter. Using HITS-CLIP for the essential RISC-component Argonaute, we identified disease-relevant targets of the chromosome 14q32 microRNAs, such as IAPP and TP53INP1 that cause increased β-cell apoptosis upon over-expression in human islets. Our results support a role for microRNAs and their epigenetic control by DNA methylation in the pathogenesis of T2DM.

Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind piRNAs, a class of small germline RNAs whose biogenesis and functions are still largely elusive. We employed high throughput sequencing after crosslinking and immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrates that Miwi binds spermiogenic mRNAs directly, without utilizing piRNAs as guides, and independent biochemical analyses of testis mRNA-ribonucleoproteins (mRNPs) establishes that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.

Background and Aims While the importance of miRNA for the development and maintenance of several tissues is well established, its role in the intestine is unknown. Our aims were to determine the entire miRNA expression profile of the mammalian small intestine in a quantitative manner and to determine the contribution of miRNAs to intestinal development and homeostasis using genetic means. Methods We determined the complete miRNA transcriptome of the mouse intestinal epithelium using ultra-high throughput sequencing. We employed gene ablation of Dicer1 to generate mice deficient for all miRNAs in the mouse intestine. Results miRNA abundance varies over a large dynamic range in the mammalian small intestine, from one read per million to 250,000. Of the 453 miRNA families identified, mmu-miR-192 and mmu-let-7 are the most highly expressed. Morphologically, the epithelium of Dicer1loxP/loxP; Villin-Cre mutants mice is disorganized in both the small and large intestine, with a four-fold decrease in goblet cells in the colon and a dramatic increase in apoptosis in the crypts of both the large and small intestine. Furthermore, intestinal barrier function is dependent on the presence of miRNAs, and consequently Dicer1 deficient mice display intestinal inflammation with lymphocyte and neutrophil infiltration. Conclusion We have identified all intestinal miRNAs and shown using gene ablation of Dicer1 that miRNAs play a vital role in the differentiation and function of the intestinal epithelium.

Piwi-piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5 ′ -to-3 ′ directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing.