in-situ-tem-of-biological-assemblies-in-liquid

<em>In situ</em> TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

erratum-in-situ-tem-of-biological-assemblies-in-liquid

Erratum: <em>In situ</em> TEM of Biological Assemblies in Liquid

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Virginia Tech Carilion Research Institute

Eukaryotic genomes are divided into independent transcriptional domains by DNA elements known as insulators. The gypsy insulator, a 350-bp element isolated from the Drosophila gypsy retrovirus, contains twelve degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Su(Hw) associates with over 500 non-gypsy genomic sites, the functions of which are largely unknown. Using a bioinformatics approach, we identified 37 putative Su(Hw) insulators (pSIs) that represent regions containing clustered matches to the gypsy insulator Su(Hw) consensus binding sequence. The majority of these pSIs contain fewer than four Su(Hw) binding sites, with only seven showing in vivo Su(Hw) association, as demonstrated by chromatin immunoprecipitation. To understand the properties of the pSIs, these elements were tested for enhancer-blocking capabilities using a transgene assay system. In a complementary set of experiments, effects of the pSIs on transcriptional regulation of genes at the natural genomic location were determined. Our data suggest that pSIs have complex genomic functions and, in some cases, establish insulators. These studies provide the first direct evidence that the Su(Hw) protein contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators.

Identification of genomic sites that bind the Drosophila suppressor of Hairy-wing insulator protein.

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.

Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.

Insulators define interactions between transcriptional control elements in eukaryotic genomes. The gypsy insulator found in the gypsy retrovirus binds the zinc-finger Suppressor of Hairy-wing [Su(Hw)] protein that associates with hundreds of non-gypsy regions throughout the Drosophila genome. Models of insulator function predict that the gypsy insulator forms chromatin loop domains through interactions with endogenous Su(Hw) insulators (SIs) to limit the action of transcriptional control elements. Here we study SI 62D and show that interactions occur between two SI 62D elements, but not between SI 62D and the gypsy insulator, limiting the scope of genomic gypsy insulator interactions. Enhancer blocking by SI 62D requires fewer Su(Hw)-binding sites than needed for gypsy insulator function, with these target regions having distinct zinc-finger requirements for in vivo Su(Hw) association. These observations led to an investigation of the role of the Su(Hw) zinc-finger domain in insulator function. Using a combination of in vitro and in vivo studies, we find that this domain makes sequence-dependent and -independent contributions to in vivo chromosome association, but is not essential for enhancer or silencer blocking. These studies extend our understanding of the properties of Su(Hw) and the endogenous genomic regions to which this protein localizes.

Investigation of the properties of non-gypsy suppressor of hairy-wing-binding sites.

Animals regulate gene expression at multiple levels, contributing to the complexity of the proteome. Among these regulatory events are post-transcriptional gene silencing, mediated by small non-coding RNAs (e.g. microRNAs), and adenosine-to-inosine (A-to-I) editing, generated by adenosine deaminases that act on double-stranded RNA (ADAR). Recent data suggest that these regulatory processes are connected at a fundamental level. A-to-I editing can affect Drosha processing or directly alter the microRNA (miRNA) sequences responsible for mRNA targeting. Here, we analyzed the previously reported adenosine deaminations occurring in human cDNAs, and asked if there was a relationship between A-to-I editing events in the mRNA 3' untranslated regions (UTRs) and mRNA:miRNA binding. We find significant correlations between A-to-I editing and changes in miRNA complementarities. In all, over 3000 of the 12 723 distinct adenosine deaminations assessed were found to form 7-mer complementarities (known as seed matches) to a subset of human miRNAs. In 200 of the ESTs, we also noted editing within a specific 13 nucleotide motif. Strikingly, deamination of this motif simultaneously creates seed matches to three (otherwise unrelated) miRNAs. Our results suggest the creation of miRNA regulatory sites as a novel function for ADAR activity. Consequently, many miRNA target sites may only be identifiable through examining expressed sequences.

Adenosine deamination in human transcripts generates novel microRNA binding sites.

We present a novel microfluidic platform to examine biological assemblies at high-resolution. We have engineered a functionalized chamber that serves as a "nanoscale biosphere" to capture and maintain rotavirus double-layered particles (DLPs) in a liquid environment. The chamber can be inserted into the column of a transmission electron microscope while being completely isolated from the vacuum system. This configuration allowed us to determine the structure of biological complexes at nanometer-resolution within a self-contained vessel. Images of DLPs were used to calculate the first 3D view of macromolecules in solution. We refer to this new fluidic visualization technology as in situ molecular microscopy.

Visualizing viral assemblies in a nanoscale biosphere.

The triple-layered rotavirus virion encases an 11-segmented, dsRNA genome and 11-12 copies of the viral polymerase (VP1). VP1 transcribes and replicates the genome while tethered beneath the VP2 core shell. Genome replication (i.e. minus-strand RNA synthesis) by VP1 occurs in association with core assembly. During this process, VP2 directly engages VP1, thereby (i) packaging the polymerase into a nascent core and (ii) triggering the enzyme to initiate minus-strand RNA synthesis on bound plus-strand RNA templates. Recent work has shed light on VP2 regions important for VP1 enzymic activity. In the current study, we sought to investigate VP2 subdomains involved in the encapsidation of VP1 into recombinant virus-like particles (VLPs), which are formed of VP2 and the middle layer virion protein (VP6). We showed that strain SA11 VLPs efficiently encapsidated SA11 VP1, but not the genetically divergent Bristol VP1. VLPs made with an SA11 VP2 mutant lacking residues 1-10 of the amino-terminal domain (NTD) were still able to encapsidate VP1; however, removal of the entire NTD (residues 1-102) completely abolished polymerase packaging. We also showed that a chimeric VP2 protein containing the NTD and dimer-forming subdomain of strain Bristol VP2 can efficiently encapsidate SA11 VP1. These results suggest that the VP2 NTD and dimer-forming subdomain play important, albeit non-specific, roles in both VP1 packaging and activation. When combined with previous work, the results of this study support the notion that the same VP2 regions that engage VP1 during activation are also involved in packaging the enzyme into the core.

Rotavirus core shell subdomains involved in polymerase encapsidation into virus-like particles.

Delivery of small interfering RNA (siRNA) targeted to specific cell types is a significant challenge for the development of RNA interference-based therapeutics. Recently, PTD-DRBD, a double-stranded RNA binding domain (DRBD) fused to the TAT protein transduction domain (PTD), was shown to be effective at delivering siRNA in a non-cell type-specific manner. Here, we evaluated the potential of DRBD as a general protein platform for targeted small interfering RNA (siRNA) delivery. We found that a single DRBD was insufficient to stably complex siRNA when fused to targeting peptides other than PTD, which facilitated nonspecific nucleic acid binding. In contrast to PTD-DRBD, fusion proteins containing two DRBDs (2× DRBD) yielded specific and stable siRNA binding. These proteins could mediate the cellular uptake of siRNA in vitro, though compared with PTD-DRBD gene silencing was attenuated by endosomal entrapment. Our findings suggest that unlike a single DRBD, 2× DRBD inhibits siRNA escape into the cytoplasm and/or induces an internalization pathway distinct from that of PTD-DRBD. Collectively, these data indicate that while 2× DRBD retains siRNA-binding activity when fused to different cell surface-interacting peptides, the utility of 2× DRBD for cell-specific RNA interference is limited without further protein engineering to enhance the bioavailability of the delivered siRNAs.Molecular Therapy - Nucleic Acids (2012) 1, e53; doi:10.1038/mtna.2012.43; published online 13 November 2012.

Brian L. Gilmore

Virginia Tech Carilion Research Institute

In situ TEM of Biological Assemblies in Liquid

Erratum: In situ TEM of Biological Assemblies in Liquid