Gemin3 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 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. bros 1999;Seggerson et al. 2002;Zeng et al. 2002;Doench et al. 2003). The manner by which a miRNA or siRNA base pairs with its mRNA target correlates with its function: if the complementarity between a miRNA and its target is extensive, the RNA target is cleaved (Hutvagner and Zamore 2002;Llave et al. 2002;Rhoades et al. 2002;Tang et al. 2003;Xie et al. 2003); if the complementarity is partial, the stability of the target mRNA in not affected but its translation is repressed (Olsen and Ambros 1999;Seggerson et al. 2002;Zeng et al. 2002;Doench et al. 2003). However, how general this correlation is and the factors and mechanisms that determine the function of any given miRNA are unknown.

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregationprone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery. I n the future, personalized genome sequencing will become routine, empowering us to define the genetic basis of many human diseases. Currently, however, complete genome sequencing for individuals to discover rare pathogenic mutations is still too costly and time consuming. Thus, more creative approaches are needed to accelerate the discovery of disease genes. Moreover, even once genes are revealed, the need for innovative approaches to elucidate causality remains critical.ALS, also known as Lou Gehrig's disease, is a devastating adultonset neurodegenerative disease that attacks upper and lower motor neurons (1). A progressive and ultimately fatal muscle paralysis ensues, usually causing death within 2-5 y of disease onset. ALS is mostly sporadic, but ∼10% of cases are familial. Pathogenic mutations in several genes have been linked to familial and sporadic ALS, including SOD1, TARDBP, FUS/TLS, VAPB, OPTN, VCP, and others (2). Two of these genes, TARDBP (TDP-43) and FUS/TLS (FUS) are notable because they encode related RNA-binding proteins that harbor a prion-like domain (3-6). Moreover, both of these proteins have been identified as components of pathological inclusions in neurons of patients with ALS (7-9). Indeed, an emerging concept suggested by the association of FUS and TDP-43 to ALS is that defects in RNA metabolism might contribute to disease pathogenesis. These observations suggested an intriguing possibility: Could TDP-43 and FUS be just the tip of an iceberg? In other words, could other human RNA-binding proteins with properties similar to th...

MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.

microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m(7)G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. Here we identify a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m(7)G cap-binding domain of eIF4E, an essential translation initiation factor. We identify conserved aromatic residues within the MC motif of human Ago2 that are required for binding to the m(7)G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. We propose that Ago2 represses the initiation of mRNA translation by binding to the m(7)G cap of mRNA targets, thus likely precluding the recruitment of eIF4E.