miRNPs: a novel class of ribonucleoproteins containing numerous microRNAsGemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large approximately 15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem-loop structures.
A combined computational-experimental approach predicts human microRNA targetsA new paradigm of gene expression regulation has emerged recently with the discovery of microRNAs (miRNAs). Most, if not all, miRNAs are thought to control gene expression, mostly by base pairing with miRNA-recognition elements (MREs) found in their messenger RNA (mRNA) targets. Although a large number of human miRNAs have been reported, many of their mRNA targets remain unknown. Here we used a combined bioinformatics and experimental approach to identify important rules governing miRNA-MRE recognition that allow prediction of human miRNA targets. We describe a computational program, "DIANA-microT", that identifies mRNA targets for animal miRNAs and predicts mRNA targets, bearing single MREs, for human and mouse miRNAs.
Microarray-based, high-throughput gene expression profiling of microRNAsAn mRNA m7G Cap Binding-like Motif within Human Ago2 Represses TranslationA human, ATP-independent, RISC assembly machine fueled by pre-miRNARNA interference (RNAi) is mediated by RNA-induced silencing complexes (RISCs), which are guided by microRNAs (miRNAs) or short interfering RNAs (siRNAs) to cognate RNA targets. In humans, the catalytic engine of RISC is a ribonucleoprotein formed by the Argonaute2 (Ago2) protein and either miRNA (miRNP) or siRNA (siRNP). The Dicer nuclease produces mature miRNAs and siRNAs from pre-miRNAs and double-stranded RNA (dsRNA), respectively, and associates with Ago2. Here, we studied the assembly of human RISC by presenting pre-miRNA to immunopurified complexes that contain Ago2, Dicer, and TRBP. Mature miRNAs were produced in an ATP-independent manner and guided specific cleavage of cognate RNA targets in a pattern that is typical of RISC. This de novo formed RISC activity dissociated from Dicer. The asymmetry of the RISC loading process was fully recapitulated in this system, which, however, could not efficiently assemble RISC from siRNA duplexes. Our findings demonstrate that, in humans, a miRNA loading complex (miRLC) is formed by Ago2 and Dicer prior to their encounter with pre-miRNA. We suggest that the miRLC couples the processing of the pre-miRNA substrate to the unwinding of the product and that after loading of the mature miRNA to Ago2, the miRLC disassembles and the miRNP is released.