Pooled Sequencing Analysis of Geese (Anser cygnoides) Reveals Genomic Variations Associated With Feather Color

During the domestication of the goose a change in its feather color took place, however, the molecular mechanisms responsible for this change are not completely understood. Here, we performed whole-genome resequencing on three pooled samples of geese (feral and domestic geese), with two distinct feather colors, to identify genes that might regulate feather color. We identified around 8 million SNPs within each of the three pools and validated allele frequencies for a subset of these SNPs using PCR and Sanger sequencing. Several genomic regions with signatures of differential selection were found when we compared the gray and white feather color populations using the FST and Hp approaches. When we combined previous functional studies with our genomic analyses we identified 26 genes (KITLG, MITF, TYRO3, KIT, AP3B1, SMARCA2, ROR2, CSNK1G3, CCDC112, VAMP7, SLC16A2, LOC106047519, RLIM, KIAA2022, ST8SIA4, LOC106044163, TRPM6, TICAM2, LOC106038556, LOC106038575, LOC106038574, LOC106038594, LOC106038573, LOC106038604, LOC106047489, and LOC106047492) that potentially regulate feather color in geese. These results substantially expand the catalog of potential feather color regulators in geese and provide a basis for further studies on domestication and avian feather coloration

Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold

AbstractThe molecular coupling of CAS and Crk in response to integrin activation is an evolutionary conserved signaling module that controls cell proliferation, survival and migration. However, when deregulated, CAS/Crk signaling also contributes to cancer progression and developmental defects in humans. Here we highlight recent advances in our understanding of how CAS/Crk complexes assemble in cells to modulate the actin cytoskeleton, and the molecular mechanisms that regulate this process. We discuss in detail the spatiotemporal dynamics of CAS/Crk assembly and how this scaffold recruits specific effector proteins that couple integrin signaling networks to the migration machinery of cells. We also highlight the importance of CAS/Crk signaling in the dual regulation of cell migration and survival mechanisms that operate in invasive cells during development and pathological conditions associated with cancer metastasis

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes

Modulations in the host cell proteome by the hantavirus nucleocapsid protein.

Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell

Filter binding and Biolayer interferometry to study the interaction of NP with wild type and mutant sequences of the VCP mRNA 5’ UTR.

Filter binding and Biolayer interferometry to study the interaction of NP with wild type and mutant sequences of the VCP mRNA 5’ UTR.</p

Binding of NP with wild type and mutant sequences of VCP mRNA 5’ UTR using filter binding analysis and biolayer interferometry.

Representative binding profiles for the interaction of NP with the wild type sequence of VCP mRNA 5’ UTR (panels A & B), randomized sequence of VCP mRNA 5’ UTR (panels C&D) and point mutant of VCP mRNA 5’ UTR (E&F). Binding profiles generated by filter binding analysis and biolayer interferometry are shown on the left side and right side, respectively. The binding profiles were generated as discussed in materials and methods. The red and blue lines in binding profiles generated by biolayer interferometry (B, D & F) represent the binding analysis carried out at two different concentrations of NP.</p

Filter binding analysis to study the interaction of NP with the mutant sequences of the VCP mRNA 5’ UTR at different salt concentrations.

Filter binding analysis to study the interaction of NP with the mutant sequences of the VCP mRNA 5’ UTR at different salt concentrations.</p

Alterations in the host proteome by the co-expression of hantavirus NP.

(A). Model showing the NP-mediated translation initiation mechanism. The nucleocapsid protein is shown by letter “N”. (B). 2D-DIGE analysis of HUVEC lysates expressing either GFP or Sin Nombre virus nucleocapsid protein (SNVN). The cell lysates from GFP and NP expressing cells were labelled with cy3 (green) and cy5(red), respectively. The overlay of cy3 (green) and cy5 (red) images is shown on the right (GFP/SNVN). (C). Zoomed view of the overlay image showing 54 well resolved encircled protein spots. The protein ladder and PH scale are shown. (D). The cy5 (red) and cy3 (green) signals of each protein spot in panel C was calculated and the ratio of the signals was reported in panel D. The spots were excised from the gel and protein identification was carried out by mass spectrometry (see Materials and methods for details). (E). The string analysis of the host factors whose intrinsic protein levels were higher in NP expressing cells. Nodes represent the proteins, and edges represent the protein-protein associations. The associations are meant to be meaningful, indicating that proteins could jointly contribute to a shared function without necessarily meaning that they are physically binding to each other. The known interaction from curated databases (—) and experimentally verified data sets (—) are shown. The predicted interaction from gene neighborhood (—), gene fusions (—), and gene co-occurrences (—) are shown. Other interactions based on textmining (—), co-expression (—) and protein homology (—) are shown.</p

Binding profiles for the interaction of NP with the mutant sequences of VCP mRNA 5’ UTR using filter binding approach.

(A) Pictorial representation of the VCP mRNA 5’ UTR and its mutants. Shown are the representative binding profiles for the interaction of NP with the mutant 1 (B), mutant 2 (C), mutant 3 (D) and mutant 4 (E) in the RNA binding buffer at 80 mM NaCl. The binding profiles were generated using filter binding analysis, as mentioned in materials and methods.</p