Expanded RNA-binding activities of mammalian Argonaute 2

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells

Reconstitution of human RNA interference in budding yeast

Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism

The terminal loop region controls microRNA processing by Drosha and Dicer

microRNAs are widely expressed, ∼22-nt-long regulatory RNAs. They are first transcribed as much longer primary transcripts, which then undergo a series of processing steps to yield the single-stranded, mature microRNAs, although the mechanisms are incompletely understood. Here, we show that the terminal loop region of human primary microRNA transcripts is an important determinant of microRNA biogenesis. Mutations that restrain the terminal loop region inhibit Drosha processing of primary microRNA transcripts as well as Dicer processing of precursor microRNA transcripts in vitro. The inhibition may result from lower enzyme turnover on the mutant transcripts. Consequently, the mutations reduce miRNA maturation in transfected human cells. We conclude that a flexible terminal loop region is critical for microRNA processing

Structural basis of microRNA length variety

The biogenesis of human microRNAs (miRNAs) includes two RNA cleavage steps in which the activities of the RNases Drosha and Dicer are involved. miRNAs of diverse lengths are generated from different genes, and miRNAs that are heterogeneous in length are produced from a single miRNA gene. We determined the solution structures of many miRNA precursors and analysed the structural basis of miRNA length diversity using a new measure: the weighted average length of diced RNA (WALDI). We found that asymmetrical structural motifs present in precursor hairpins are primarily responsible for the length diversity of miRNAs generated by Dicer. High-resolution northern blots of miRNAs and their precursors revealed that both Dicer and Drosha cleavages of imperfect specificity contributed to the miRNA length heterogeneity. The relevance of these findings to the dynamics of the dicing complex, mRNA regulation by miRNA, RNA interference and miRNA technologies are discussed

Phosphorylation of human Argonaute proteins affects small RNA binding

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5′-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5′ phosphate of the small RNA

Human box C/D snoRNAs with miRNA like functions: expanding the range of regulatory RNAs

Small nucleolar RNAs (snoRNAs) and microRNAs are two classes of non-protein-coding RNAs with distinct functions in RNA modification or post-transcriptional gene silencing. In this study, we introduce novel insights to RNA-induced gene activity adjustments in human cells by identifying numerous snoRNA-derived molecules with miRNA-like function, including H/ACA box snoRNAs and C/D box snoRNAs. In particular, we demonstrate that several C/D box snoRNAs give rise to gene regulatory RNAs, named sno-miRNAs here. Our data are complementing the increasing number of studies in the field of small RNAs with regulatory functions. In massively deep sequencing of small RNA fractions we identified high copy numbers of sub-sequences from >30 snoRNAs with lengths of ≥18 nt. RNA secondary structure prediction indicated for a majority of candidates a location in predicted stem regions. Experimental analysis revealed efficient gene silencing for 11 box C/D sno-miRNAs, indicating cytoplasmic processing and recruitment to the RNA silencing machinery. Assays in four different human cell lines indicated variations in both the snoRNA levels and their processing to active sno-miRNAs. In addition we show that box D elements are predominantly flanking at least one of the sno-miRNA strands, while the box C element locates within the sequence of the sno-miRNA guide strand

Expanding the MicroRNA Targeting Code: Functional Sites with Centered Pairing

Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5′ end. Although pairing to the 3′ region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present “centered sites,” a class of miRNA target sites that lack both perfect seed pairing and 3′-compensatory pairing and instead have 11–12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg[superscript 2+]), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved.National Institutes of Health (U.S.)Damon Runyon Cancer Research Foundation. Fellowship Awar

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures

The Role of Dicer Protein Partners in the Processing of MicroRNA Precursors

One of the cellular functions of the ribonuclease Dicer is to process microRNA precursors (pre-miRNAs) into mature microRNAs (miRNAs). Human Dicer performs this function in cooperation with its protein partners, AGO2, PACT and TRBP. The exact role of these accessory proteins in Dicer activity is still poorly understood. In this study, we used the northern blotting technique to investigate pre-miRNA cleavage efficiency and specificity after depletion of AGO2, PACT and TRBP by RNAi. The results showed that the inhibition of either Dicer protein partner substantially affected not only miRNA levels but also pre-miRNA levels, and it had a rather minor effect on the specificity of Dicer cleavage. The analysis of the Dicer cleavage products generated in vitro revealed the presence of a cleavage intermediate when pre-miRNA was processed by recombinant Dicer alone. This intermediate was not observed during pre-miRNA cleavage by endogenous Dicer. We demonstrate that AGO2, PACT and TRBP were required for the efficient functioning of Dicer in cells, and we suggest that one of the roles of these proteins is to assure better synchronization of cleavages triggered by two RNase III domains of Dicer

Clarifying mammalian RISC assembly in vitro

<p>Abstract</p> <p>Background</p> <p>Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active <it>in vitro </it>and constitute non-canonical RISCs.</p> <p>Results</p> <p>The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates <it>in vitro </it>RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate <it>in vitro </it>pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes.</p> <p>Conclusions</p> <p>Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.</p