Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis.

Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.

Centrioles want to move out and make cilia.

Cilia formation in mammalian cells requires basal bodies that are either derived from centrioles that transition from their cytoplasmic role in centrosome organization or that form en masse in multiciliated cells. Several recent studies have begun to uncover the links between centriole duplication and their transformation to basal bodies.

New Tetrahymena basal body protein components identify basal body domain structure.

Basal bodies organize the nine doublet microtubules found in cilia. Cilia are required for a variety of cellular functions, including motility and sensing stimuli. Understanding this biochemically complex organelle requires an inventory of the molecular components and the contribution each makes to the overall structure. We define a basal body proteome and determine the specific localization of basal body components in the ciliated protozoan Tetrahymena thermophila. Using a biochemical, bioinformatic, and genetic approach, we identify 97 known and candidate basal body proteins. 24 novel T. thermophila basal body proteins were identified, 19 of which were localized to the ultrastructural level, as seen by immunoelectron microscopy. Importantly, we find proteins from several structural domains within the basal body, allowing us to reveal how each component contributes to the overall organization. Thus, we present a high resolution localization map of basal body structure highlighting important new components for future functional studies.

A role for huntington disease protein in dendritic RNA granules.

Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry, and establishing long term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation, and small RNA-mediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and co-traffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3'-untranslated region sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules.

Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing.

Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens.

Uterine Inflammatory Myofibroblastic Tumors Frequently Harbor ALK Fusions With IGFBP5 and THBS1.

Inflammatory myofibroblastic tumor (IMT) can occur in a number of anatomic sites, including the uterus. Like its soft tissue counterpart, uterine IMT frequently expresses ALK and harbors ALK genetic rearrangements. The aim of this study is to fully characterize the genetic fusions that occur in uterine IMT. We studied 11 uterine IMTs with typical histology and 8 uterine myxoid smooth muscle tumors (5 leiomyomas, 1 smooth muscle tumor of uncertain malignant potential, and 2 leiomyosarcomas) in which the differential of IMT was considered, using a RNA-sequencing-based fusion assay to detect genetic fusions involving ALK, ROS1, RET, NTRK1/3, and other genes. ALK was expressed in 10 of 11 IMTs and 1 tumor initially categorized as a myxoid leiomyoma (granular cytoplasmic staining with paranuclear accentuation). Fusion transcripts involving ALK were identified in 9 of 10 ALK immunopositive IMTs, with 3 harboring IGFBP5-ALK, 3 harboring THBS1-ALK, 2 harboring FN1-ALK, and 1 harboring TIMP3-ALK. Among the smooth muscle tumors, IGFBP5-ALK fusion transcript was identified in only 1 ALK immunopositive case. Further review revealed that although a diagnosis of IMT was considered for the ALK immunopositive myxoid leiomyoma, this diagnosis was not initially rendered only because fluorescence in situ hybridization analysis was interpreted as negative for ALK genetic rearrangement; this case is best reclassified as an IMT. Notably, all the ALK fusions identified in our study included the transmembrane domain-encoding exon 19 of ALK. Our findings confirm the high frequency of ALK fusions in uterine IMT, with an enrichment of novel 5' ALK fusion partners (IGFBP5, THBS1, and TIMP3) and exon 19-containing ALK fusion. Given that IGFBP5 and FN1 are both situated on the same chromosome as ALK, fluorescence in situ hybridization analysis for ALK rearrangement may not be reliable and a negative result should not exclude a diagnosis of uterine IMT if the histologic features and ALK immunostaining findings are supportive.

Huntington's Disease Protein Huntingtin Associates with its own mRNA.

BACKGROUND: The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. OBJECTIVE: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. METHODS: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. RESULTS: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3' UTR. CONCLUSIONS: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases.

Huntingtin mediates dendritic transport of ß-actin mRNA in rat neurons.

Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in regulating protein synthesis within restricted sub-cellular domains. We recently detected the Huntington's disease protein huntingtin (Htt) in dendritic RNA granules; however, the functional significance of this localization is not known. Here we report that Htt and the huntingtin-associated protein 1 (HAP1) are co-localized with the microtubule motor proteins, the KIF5A kinesin and dynein, during dendritic transport of ß-actin mRNA. Live cell imaging demonstrated that ß-actin mRNA is associated with Htt, HAP1, and dynein intermediate chain in cultured neurons. Reduction in the levels of Htt, HAP1, KIF5A, and dynein heavy chain by lentiviral-based shRNAs resulted in a reduction in the transport of ß-actin mRNA. These findings support a role for Htt in participating in the mRNA transport machinery that also contains HAP1, KIF5A, and dynein.

Localization of BDNF mRNA with the Huntington's disease protein in rat brain.

BACKGROUND: Studies have implicated reduced levels of brain-derived neurotrophic factor (BDNF) in the pathogenesis of Huntington's disease. Mutant huntingtin (Htt) protein was previously reported to decrease BDNF gene transcription and axonal transport of BDNF. We recently showed that wild-type Htt is associated with the Argonaute 2 microRNA-processing enzyme involved in gene silencing. In dendrites, Htt co-localizes with components of neuronal granules and mRNAs, indicating that it might play a role in post-transcriptional processing/transport of dendritic mRNAs. RESULTS: We conducted imaging experiments in cultured cortical neurons to demonstrate the co-localization of endogenous Htt and BDNF mRNA in fixed cells, and co-trafficking of BDNF 3'UTR mRNA with endogenous and fluorescently tagged Htt in live neurons. We used an enhanced technique that combines FISH and immunofluorescent staining to co-localize BDNF mRNA with Htt, Ago2, CPEB and dynein in thick vibratome sections of the rat cortex. CONCLUSIONS: In cultured neurons and sections of the rat cortex, we found BDNF mRNA associated with Htt and components of neuronal RNA granules, which are centers for regulating RNA transport and local translation. Htt may play a role in post-transcriptional transport/targeting of mRNA for BDNF, thus contributing to neurotrophic support and neuron survival.

The two SAS-6 homologs in Tetrahymena thermophila have distinct functions in basal body assembly.

Cilia and flagella are structurally and functionally conserved organelles present in basal as well as higher eukaryotes. The assembly of cilia requires a microtubule based scaffold called a basal body. The ninefold symmetry characteristic of basal bodies and the structurally similar centriole is organized around a hub and spoke structure termed the cartwheel. To date, SAS-6 is one of the two clearly conserved components of the cartwheel. In some organisms, overexpression of SAS-6 causes the formation of supernumerary centrioles. We questioned whether the centriole assembly initiation capacity of SAS-6 is separate from or directly related to its structural role at the cartwheel. To address this question we used Tetrahymena thermophila, which expresses two SAS-6 homologues, TtSAS6a and TtSAS6b. Cells lacking either TtSAS6a or TtSAS6b are defective in new basal body assembly. TtSas6a localizes to all basal bodies equally, whereas TtSas6b is enriched at unciliated and assembling basal bodies. Interestingly, overexpression of TtSAS6b but not TtSAS6a, led to the assembly of clusters of new basal bodies in abnormal locations. Our data suggest a model where TtSAS6a and TtSAS6b have diverged such that TtSAS6a acts as a structural component of basal bodies, whereas TtSAS6b influences the location of new basal body assembly.