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1.  Correction: Dynein Associates with oskar mRNPs and Is Required For Their Efficient Net Plus-End Localization in Drosophila Oocytes 
PLoS ONE  2014;9(1):10.1371/annotation/1d5f32a4-54f6-4633-a8b4-f0fae0627507.
PMCID: PMC3880379
2.  Dynein Associates with oskar mRNPs and Is Required For Their Efficient Net Plus-End Localization in Drosophila Oocytes 
PLoS ONE  2013;8(11):e80605.
In order for eukaryotic cells to function properly, they must establish polarity. The Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc), the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. Here we show that Dynein heavy chain (Dhc), the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. Our results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1.
PMCID: PMC3823658  PMID: 24244700
3.  Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins 
The Journal of Cell Biology  2007;178(5):733-740.
Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.
PMCID: PMC2064538  PMID: 17709427
4.  Sigma Receptor Ligand, (+)-Pentazocine, Suppresses Inflammatory Responses of Retinal Microglia 
To evaluate the effects of the σ 1 receptor (σR1) agonist, (+)-pentazocine, on lipopolysaccharide (LPS)–induced inflammatory changes in retinal microglia cells.
Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (+)-pentazocine and with or without the σR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor α (TNF-α), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot.
The σR1 protein was expressed in retinal microglia. Incubation with LPS and/or (+)-pentazocine did not alter cell viability or σR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-α, IL-10, MCP-1, and NO. Pretreatment with (+)-pentazocine inhibited the LPS-induced morphologic changes. Release of TNF-α, IL-10, MCP-1, and NO was reduced with (+)-pentazocine. Intracellular ROS formation was suppressed with (+)-pentazocine. Phosphorylation of extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (+)-pentazocine. The σR1 antagonist BD1063 blocked the (+)-pentazocine–mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (+)-pentazocine–mediated suppression of LPS-induced TNF-α, IL-10, MCP-1, NO, and intracellular ROS release.
Treatment with (+)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (+)-pentazocine may exert neuroprotective effects through manipulation of microglia.
The sigma Receptor Ligand, (+)-Pentazocine, has shown neuroprotective properties in models of neurodegenerative disease. Our study found that (+)-pentazocine suppressed inflammatory responses of retinal microglia. (+)-Pentazocine holds therapeutic potential for ophthalmic diseases such as glaucoma.
PMCID: PMC4042630  PMID: 24812552
microglia; neuron–glia interactions; optic nerve; sigma receptor
5.  Spatial regulation of translation through RNA localization 
RNA localization is a mechanism to post-transcriptionally regulate gene expression. Eukaryotic organisms ranging from fungi to mammals localize mRNAs to spatially restrict synthesis of specific proteins to distinct regions of the cytoplasm. In this review, we provide a general summary of RNA localization pathways in Saccharomyces cerevisiae, Xenopus, Drosophila and mammalian neurons.
PMCID: PMC3412389  PMID: 22912650
6.  A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle 
The Journal of Cell Biology  2007;176(6):831-841.
Mutations in human survival motor neurons 1 (SMN1) cause spinal muscular atrophy (SMA) and are associated with defects in assembly of small nuclear ribonucleoproteins (snRNPs) in vitro. However, the etiological link between snRNPs and SMA is unclear. We have developed a Drosophila melanogaster system to model SMA in vivo. Larval-lethal Smn-null mutations show no detectable snRNP reduction, making it unlikely that these animals die from global snRNP deprivation. Hypomorphic mutations in Smn reduce dSMN protein levels in the adult thorax, causing flightlessness and acute muscular atrophy. Mutant flight muscle motoneurons display pronounced axon routing and arborization defects. Moreover, Smn mutant myofibers fail to form thin filaments and phenocopy null mutations in Act88F, which is the flight muscle–specific actin isoform. In wild-type muscles, dSMN colocalizes with sarcomeric actin and forms a complex with α-actinin, the thin filament crosslinker. The sarcomeric localization of Smn is conserved in mouse myofibrils. These observations suggest a muscle-specific function for SMN and underline the importance of this tissue in modulating SMA severity.
PMCID: PMC2064057  PMID: 17353360
7.  Cross-Talk between Snurportin1 SubdomainsD⃞ 
Molecular Biology of the Cell  2005;16(10):4660-4671.
The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. After formation of an Sm-core and a trimethylguanosine (TMG) cap, the RNPs are transported into the nucleus via the import adaptor snurportin1 (SPN) and the import receptor importin-β. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin-β, and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin-β binding domain (IBB) disrupted the interaction with importin-β, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are required for TMG binding. However, SPN can be imported into the nucleus without cargo. Interestingly, SPN targets to Cajal bodies when U2 but not U1 snRNPs are imported as cargo. SPN also relocalizes to Cajal bodies upon treatment with leptomycin B. Finally, we uncovered an interaction between the N- and C-terminal domains of SPN, suggesting an autoregulatory function similar to that of importin-α.
PMCID: PMC1237072  PMID: 16030253

Results 1-7 (7)