Proteomic Analysis Reveals CACN-1 Is a Component of the Spliceosome in Caenorhabditis elegans

Cell migration is essential for embryonic development and tissue formation in all animals. cacn-1 is a conserved gene of unknown molecular function identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode worm Caenorhabditis elegans. In this study we take a proteomics approach to understand CACN-1 function. To isolate CACN-1−interacting proteins, we used an in vivo tandem-affinity purification strategy. Tandem-affinity purification−tagged CACN-1 complexes were isolated from C. elegans lysate, analyzed by mass spectrometry, and characterized bioinformatically. Results suggest significant interaction of CACN-1 with the C. elegans spliceosome. All of the identified interactors were screened for distal tip cell migration phenotypes using RNAi. Depletion of many of these factors led to distal tip cell migration defects, particularly a failure to stop migrating, a phenotype commonly seen in cacn-1 deficient animals. The results of this screen identify eight novel regulators of cell migration and suggest CACN-1 may participate in a protein network dedicated to high-fidelity gonad development. The composition of proteins comprising the CACN-1 network suggests that this critical developmental module may exert its influence through alternative splicing or other post-transcriptional gene regulation

Genome-scale Proteome Quantification by DEEP SEQ Mass Spectrometry

Advances in chemistry and massively parallel detection underlie DNA sequencing platforms that are poised for application in personalized medicine. In stark contrast, systematic generation of protein-level data lags well-behind genomics in virtually every aspect: depth of coverage, throughput, ease of sample preparation, and experimental time. Here, to bridge this gap, we develop an approach based on simple detergent lysis and single-enzyme digest, extreme, orthogonal separation of peptides, and true nanoflow LC-MS/MS that provides high peak capacity and ionization efficiency. This automated, deep efficient peptide sequencing and quantification (DEEP SEQ) mass spectrometry platform provides genome-scale proteome coverage equivalent to RNA-seq ribosomal profiling and accurate quantification for multiplexed isotope labels. In a model of the embryonic to epiblast transition in murine stem cells, we unambiguously quantify 11,352 gene products that span 70% of Swiss-Prot and capture protein regulation across the full detectable range of high-throughput gene expression and protein translation

An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication

Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis, B cell lymphomas, and nasopharyngeal carcinoma. Many of the genes required for EBV virion morphogenesis are found in all herpesviruses, but some are specific to gammaherpesviruses. One of these gamma-specific genes, BLRF2, encodes a tegument protein that has been shown to be essential for replication in other gammaherpesviruses. In this study, we identify BLRF2 interacting proteins using binary and co-complex protein assays. Serine/Arginine-rich Protein Kinase 2 (SRPK2) was identified by both assays and was further shown to phosphorylate an RS motif in the BLRF2 C-terminus. Mutation of this RS motif (S148A+S150A) abrogated the ability of BLRF2 to support replication of a murine gammaherpesvirus 68 genome lacking the BLRF2 homolog (ORF52). We conclude that the BLRF2 RS motif is phosphorylated by SRPK2 and is important for viral replication

Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT

Short‐range multispectral imaging is an inexpensive, fast, and accurate approach to estimate biodiversity in a temperate calcareous grassland

Image sensing technologies are rapidly increasing the cost‐effectiveness of biodiversity monitoring efforts. Species differences in the reflectance of electromagnetic radiation can be used as a surrogate estimate plant biodiversity using multispectral image data. However, these efforts are often hampered by logistical difficulties in broad‐scale implementation. Here, we investigate the utility of multispectral imaging technology from commercially available unmanned aerial vehicles (UAVs, or drones) in estimating biodiversity metrics at a fine spatial resolution (0.1–0.5 cm pixel resolution) in a temperate calcareous grassland in Oxfordshire, UK. We calculate a suite of moments (coefficient of variation, standard deviation, skewness, and kurtosis) for the distribution of radiance from multispectral images at five wavelength bands (Blue 450 ± 16 nm; Green 560 ± 16 nm; Red 650 ± 16 nm; Red Edge 730 ± 16 nm; Near Infrared 840 ± 16 nm) and test their effectiveness at estimating ground‐truthed biodiversity metrics from in situ botanical surveys for 37–1 × 1 m quadrats. We find positive associations between the average coefficient of variation in spectral radiance and both the Shannon–Weiner and Simpson's biodiversity indices. Furthermore, the average coefficient of variation in spectral radiance is consistent and highly repeatable across sampling days and recording heights. Positive associations with biodiversity indices hold irrespective of the image recording height (2–8 m), but we report reductions in estimates of spectral diversity with increases to UAV recording height. UAV imaging reduced sampling time by a factor of 16 relative to in situ botanical surveys. We demonstrate the utility of multispectral radiance moments as an indicator of biodiversity in this temperate calcareous grassland at a fine spatial resolution using a widely available UAV monitoring system with a coarse spectral resolution. The use of UAV technology with multispectral sensors has far‐reaching potential to provide cost‐effective and high‐resolution monitoring of biodiversity

Exogenous expression of a dominant negative RORa1 vector in muscle cells impairs differentiation: RORa1 directly interacts with p300 and MyoD

ROR/RZR is an orphan nuclear receptor that has no known ligand in the 'classical sense'. In the present study we demonstrate that RORalpha is constitutively expressed during the differentiation of proliferating myoblasts to post-mitotic multinucleated myotubes, that have acquired a contractile phenotype. Exogenous expression of dominant negative RORalpha1DeltaE mRNA in myogenic cells significantly reduces the endogenous expression of RORalpha1 mRNA, represses the accumu-lation and delays the activation of mRNAs encoding MyoD and myogenin [the muscle-specific basic helix-loop-helix (bHLH) proteins] and p21(Waf- 1/Cip-1) (a cdk inhibitor). Immunohistochemistry demonstrates that morpho-logical differentiation is delayed in cells expressing the RORDeltaE transcript. Furthermore, the size and development of mutlinucleated myotubes is impaired. The E region of RORalpha1 interacts with p300, a cofactor that functions as a coactivator in nuclear receptor and MyoD-mediated transactivation. Consistent with the functional role of RORalpha1 in myogenesis, we observed that RORalpha1 directly interacts with the bHLH protein MyoD. This interaction was mediated by the N-terminal activation domain of the bHLH protein, MyoD, and the RORalpha1 DNA binding domain/C region. Furthermore, we demonstrated that p300, RORalpha1 and MyoD interact in a non- competitive manner. In conclusion, this study provides evidence for a biological role and positive influence of RORalpha1 in the cascade of events involved in the activation of myogenic-specific markers and cell cycle regulators and suggests that crosstalk between theretinoid- relatedorphan (ROR) nuclear receptors and the myogenic bHLH proteins has functional consequences for differentiation

Implication of the F-Box Protein FBXL21 in Circadian Pacemaker Function in Mammals

In mammals, the circadian clock relies on interlocked feedback loops involving clock genes and their protein products. Post-translational modifications control intracellular trafficking, functionality and degradation of clock proteins and are keys to the functioning of the clock as recently exemplified for the F-Box protein Fbxl3. The SCFFbxl3 complex directs degradation of CRY1/2 proteins and Fbxl3 murine mutants have a slower clock. To assess whether the role of Fbxl3 is phylogenetically conserved, we investigated its function in the sheep, a diurnal ungulate. Our data show that Fbxl3 function is conserved and further reveal that its closest homologue, the F-Box protein Fbxl21, also binds to CRY1 which impairs its repressive action towards the transcriptional activators CLOCK/BMAL1. However, while Fbxl3 appears to be ubiquitously expressed, Fbxl21 expression is tissue-specific. Furthermore, and in sharp contrast with Fbxl3, Fbxl21 is highly expressed within the suprachiasmatic nuclei, site of the master clock, where it displays marked circadian oscillations apparently driven by members of the PAR-bZIP family. Finally, for both Fbxl3 and Fbxl21 we identified and functionally characterized novel splice-variants, which might reduce CRY1 proteasomal degradation dependent on cell context. Altogether, these data establish Fbxl21 as a novel circadian clock-controlled gene that plays a specific role within the mammalian circadian pacemaker

IER5, a dna damage response gene, is required for notch-mediated induction of squamous cell differentiation

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including IER5, which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. IER5 is epistatic to PPP2R2A, a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation