Zissimos Mourelatos

Microarray-based, high-throughput gene expression profiling of microRNAs

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.

An mRNA m7G Cap Binding-like Motif within Human Ago2 Represses Translation

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.

A yeast functional screen predicts new candidate ALS disease genes

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 aggregation-prone 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.

SMN interacts with a novel family of hnRNP and spliceosomal proteins

Spinal muscular atrophy (SMA) is a common neurodegenerative disease caused by deletion or loss‐of‐function mutations of the survival of motor neurons (SMN) protein. SMN is in a complex with several proteins, including Gemin2, Gemin3 and Gemin4, and it plays important roles in small nuclear ribonucleoprotein (snRNP) biogenesis and in pre‐mRNA splicing. Here, we characterize three new hnRNP proteins, collectively referred to as hnRNP Qs, which are derived from alternative splicing of a single gene. The hnRNP Q proteins interact with SMN, and the most common SMN mutant found in SMA patients is defective in its interactions with them. We further demonstrate that hnRNP Qs are required for efficient pre‐mRNA splicing in vitro. The hnRNP Q proteins may provide a molecular link between the SMN complex and splicing.

Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability

Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila melanogaster encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). Here we report that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). We found that Piwi proteins are expressed in Xenopus oocytes and we identified numerous Xenopus piRNAs. We report that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, csul/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. Our studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.

Mouse Piwi-interacting RNAs are 2'-O-methylated at their 3' termini.

Piwi-interacting RNAs (piRNAs) are a novel class of small RNAs that are expressed specifically and abundantly in male germ cells. Here we report that the 3′ termini of piRNAs are 2′-O-methylated; this modification may have important implications for the biogenesis and function of piRNAs.

MicroRNAs : Biogenesis and Molecular Functions

Small regulatory RNAs are essential and ubiquitous riboregulators that are the key mediators of RNA interference (RNAi). They include microRNAs (miRNAs) and short‐interfering RNAs (siRNAs), classes of ∼22 nucleotide RNAs. miRNAs and siRNAs bind to Argonaute proteins and form effector complexes that regulate gene expression; in animals, this regulation occurs primarily at the post‐transcriptional level. In this review, we will discuss our current understanding of how miRNA and siRNAs are generated and how they function to silence gene expression, focusing on animal and, in particular, mammalian miRNAs.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

Gemin5, a Novel WD Repeat Protein Component of the SMN Complex That Binds Sm Proteins

The survival of motor neurons (SMN) protein is the product of the disease gene of spinal muscular atrophy and is found both in the cytoplasm and the nucleus, where it is concentrated in gems. SMN is part of a multi-protein complex that includes Gemin2, Gemin3, and Gemin4. The SMN complex plays an important role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and in the assembly of transcriptosomes. Here, we report the identification of an additional component of the SMN complex, a novel WD repeat protein termed Gemin5. Gemin5 binds SMN directly and is a component of the SMN complex. Furthermore, Gemin5 interacts with several of the snRNP core proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it participates in the activities of the SMN complex in snRNP assembly. Immunolocalization studies demonstrate that Gemin5 is found in the cytoplasm and in the nucleus, where it colocalizes with SMN in gems. The presence of 13 WD repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif near its carboxyl terminus indicate that it may form a large heteromeric complex and engage in multiple interactions.

Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell

Transformation of normal cells into malignant tumors requires the acquisition of six hallmark traits, e.g., self-sufficiency in growth signals, insensitivity to antigrowth signals and self-renewal, evasion of apoptosis, limitless replication potential, angiogenesis, invasion, and metastasis, which are common to all cancers (Hanahan and Weinberg 2000). These new cellular traits evolve from defects in major regulatory microcircuits that are fundamental for normal homeostasis. The discovery of microRNAs (miRNAs) as a new class of small non-protein-coding RNAs that control gene expression post-transcriptionally by binding to various mRNA targets suggests that these tiny RNA molecules likely act as molecular switches in the extensive regulatory web that involves thousands of transcripts. Most importantly, accumulating evidence suggests that numerous microRNAs are aberrantly expressed in human cancers. In this review, we discuss the emergent roles of microRNAs as switches that function to turn on/off known cellular microcircuits. We outline recent compelling evidence that deregulated microRNA-mediated control of cellular microcircuits cooperates with other well-established regulatory mechanisms to confer the hallmark traits of the cancer cell. Furthermore, these exciting insights into aberrant microRNA control in cancer-associated circuits may be exploited for cancer therapies that will target deregulated miRNA switches.