Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

To further our understanding of the RNAi machinery within the human nucleus, we analyzed the chromatin and RNA binding of Argonaute 2 (AGO2) within human cancer cell lines. Our data indicated that AGO2 binds directly to nascent tRNA and 5S rRNA, and to the genomic loci from which these RNAs are transcribed, in a small RNA- and DICER-independent manner. AGO2 chromatin binding was not observed at non-TFIIIC-dependent RNA polymerase III (Pol III) genes or at extra-TFIIIC (ETC) sites, indicating that the interaction is specific for TFIIIC-dependent Pol III genes. A genome-wide analysis indicated that loss of AGO2 caused a global increase in mRNA expression level among genes that flank AGO2-bound tRNA genes. This effect was shown to be distinct from that of the disruption of DICER, DROSHA, or CTCF. We propose that AGO2 binding to tRNA genes has a novel and important regulatory role in human cells.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

Silencing synaptic communication between random interneurons during Drosophila larval locomotion

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons. These larvae were screened for aberrant behavior at an elevated temperature (31-32°C). Among larvae with abnormal locomotion or sensory-motor responses, some had very small numbers of GFP-labeled temperature-sensitive interneurons. Labeled ascending interneurons projecting from the abdominal ganglia to specific brain neuropile compartments emerged as candidates for mediation of larval locomotion. Random targeting of small sets of neurons for functional evaluation, together with anatomical mapping of their processes, provides a tool for identifying the regions of the central nervous system that are required for normal locomotion. We discuss the limitations and advantages of this approach to discovery of interneurons that regulate motor behavior.     

Post‐Project Appraisals of River Restoration in Advanced University Instruction

Post‐project appraisals (PPAs) are systematic assessments of built restoration projects, which provide feedback on performance of restoration approaches to improve future restoration efforts. Unfortunately, most restoration projects are not subject to systematic assessment because of lack of institutional arrangements to sustain long‐term evaluation and the orientation of most funding agencies towards project implementation rather than “studies.” As semester‐long courses on river restoration increasingly appear in university curricula at the graduate and advanced undergraduate level, independent student research projects for such courses can provide a mechanism for building a database of PPAs (and components thereof) and providing the students with a powerful learning experience. In two UC Berkeley courses, we require independent student projects involving original field research, peer review of first drafts, instructor (and often outside) review of second drafts, and presentation of results to a public symposium. Since 1995, the revised, final papers have been added to the University of California library, where they constitute one of the largest collections of restoration‐related studies currently available for any region: over 300 restoration‐related studies, of which 80 are PPAs or components thereof. Since 2003, the papers have been posted on‐line, with 40,000 full text downloads through 2010. Some term projects have directly influenced river restoration programs, inducing changes in salmon habitat enhancement project design, documenting failure of projects based on inappropriate restoration approaches, and contributing to systematic assessments of step‐pool and compound channel designs in urban areas. Student evaluations cite the term projects as valuable learning experiences.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

An N-linked glycoprotein with alpha(2,3)-linked sialic acid is a receptor for BK virus

BK virus (BKV) is a common human polyomavirus infecting >80% of the population worldwide. Infection with BKV is asymptomatic, but reactivation in renal transplant recipients can lead to polyomavirus-associated nephropathy. In this report, we show that enzymatic removal of alpha(2,3)-linked sialic acid from cells inhibited BKV infection. Reconstitution of asialo cells with alpha(2,3)-specific sialyltransferase restored susceptibility to infection. Inhibition of N-linked glycosylation with tunicamycin reduced infection, but inhibition of O-linked glycosylation did not. An O-linked-specific alpha(2,3)-sialyltransferase was unable to restore infection in asialo cells. Taken together, these data indicate that an N-linked glycoprotein containing alpha(2,3)-linked sialic acid is a critical component of the cellular receptor for BKV.     

Involvement of cytoskeletal components in BK virus infectious entry

Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.     

Modeling a sialic acid binding pocket in the external loops of JC virus VP1

JC virus (JCV) is a common human polyomavirus that infects over 70% of the population worldwide. JCV has a restricted cell tropism that is caused partly by the initial interaction between the virus and sialic acid-containing host cell receptors. To identify the molecular interactions between the virus and its cellular receptor, we used a combined approach of site-directed mutagenesis and homology-based molecular modeling. A model of the major viral capsid protein VP1 based on sequence alignment with other closely related polyomaviruses allowed us to target specific amino acids in the extracellular loops of VP1 for mutagenesis. An analysis of the growth rates of 17 point mutants led to the identification of VP1 amino acids that are critical in virus-host cell receptor interactions. Molecular dynamics simulations were then used to build and confirm a model of the interaction between VP1 and the sialic acid component of the JCV receptor.     

Identification of a gonadotropin-releasing hormone receptor orthologue in Caenorhabditis elegans

Background  

The Caenorhabditis elegans genome is known to code for at least 1149 G protein-coupled receptors (GPCRs), but the GPCR(s) critical to the regulation of reproduction in this nematode are not yet known. This study examined whether GPCRs orthologous to human gonadotropin-releasing hormone receptor (GnRHR) exist in C. elegans.