scholarly journals Combined Use of MS2 and PP7 Coat Fusions Shows that TIA-1 Dominates hnRNP A1 for K-SAM Exon Splicing Control

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
Marie-Claude Gesnel ◽  
Fabienne Del Gatto-Konczak ◽  
Richard Breathnach
Keyword(s):  
Coat Protein ◽  
Binding Site ◽  
The Other ◽  
Coat Proteins ◽  
Hnrnp A1 ◽  
Bacteriophage Ms2 ◽  
Exon Splicing ◽  
Mrna Molecule ◽  
Combined Use

Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.

scholarly journals Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

2020 ◽  
Vol 295 (25) ◽  
pp. 8401-8412 ◽  
Author(s):  
David B. Melville ◽  
Sean Studer ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Binding Site ◽  
Protein Complex ◽  
Small Gtpase ◽  
The Other ◽  
Complex Ii ◽  
Gtp Binding ◽  
Lipoprotein Secretion ◽  
Coat Protein Complex

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

scholarly journals Novel Interactions of ESCRT-III with LIP5 and VPS4 and their Implications for ESCRT-III Disassembly

2008 ◽  
Vol 19 (6) ◽  
pp. 2661-2672 ◽  
Author(s):  
Soomin Shim ◽  
Samuel A. Merrill ◽  
Phyllis I. Hanson
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Essential Role ◽  
Multivesicular Body ◽  
The Other ◽  
Aaa Atpase ◽  
Direct Binding ◽  
Novel Interactions

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2–1, and CHMP3/hVps24 but not CHMP4A/hSnf7–1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with “MIT interacting motifs” (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

scholarly journals Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

scholarly journals Overcoming the design, build, test bottleneck for synthesis of nonrepetitive protein-RNA cassettes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Noa Katz ◽  
Eitamar Tripto ◽  
Naor Granik ◽  
Sarah Goldberg ◽  
Orna Atar ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Coat Proteins ◽  
Structural Determinants ◽  
Design Build ◽  
Machine Learning Approach ◽  
Successful Design ◽  
Experimental Findings ◽  
Rna Interaction

AbstractWe apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qβ (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.

Association of the IVR Gene with Virus Localization and Resistance

1995 ◽  
Author(s):  
Gad Loebenstein ◽  
William Dawson ◽  
Abed Gera
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Plant Viruses ◽  
Full Length ◽  
N Gene ◽  
Complete Suppression ◽  
Coat Proteins ◽  
Nicotiana Glutinosa ◽  
Nicotiana Debneyi ◽  
Full Length Gene

We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.

E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

Association of Cea Test and Liver Scan in Detection of Hepatic Metastases of Gastrointestinal Carcinoma

1981 ◽  
Vol 67 (6) ◽  
pp. 553-558 ◽  
Author(s):  
Gian Luigi Buraggi ◽  
Emilio Bombardieri ◽  
Maria Rita Castellani ◽  
Antonio Rodari ◽  
Flavio Crippa
Keyword(s):  
Gastrointestinal Tract ◽  
Hepatic Metastases ◽  
The Other ◽  
Gastrointestinal Tumors ◽  
Diagnostic Specificity ◽  
Gastrointestinal Carcinoma ◽  
Diagnostic Use ◽  
Combined Use ◽  
Combined Test ◽  
Liver Scan

The authors evaluate the combined use of liver scan and the CEA test in the diagnosis of hepatic metastases of carcinoma of the gastrointestinal tract. Association of the two tests is justified by the fact that the liver scan is very specific but not very sensitive, whereas the CEA test is more sensitive and not very specific. The sensitivity of the CEA test, on the other hand, can be increased by increasing the threshold of normality. However, the associated diagnostic use of the liver scan and the CEA test gives a loss of specificity with respect to the use of the liver scan alone. The present study, carried out on a series of 376 patients affected by gastrointestinal tumors of which 79 were of the stomach (9 with hepatic metastases), 133 of the colon and higher sigmoid (25 with hepatic metastases), and 164 of the lower sigmoid and rectum (29 with hepatic metastases), proposed to establish by use of a statistical method the optimal threshold of the CEA test that would give the best diagnostic specificity of the combined CEA test and liver scan without any relevant loss of sensitivity. A threshold of 26 ng/ml of the CEA test gave a specificity of 92 %, a sensitivity of 80 %, and an accuracy of 90 %. The authors think that in the detection of liver metastases of gastrointestinal tumors, the combined test can be more helpful the less the probability, for a given patient, for other metastatic localizations.

scholarly journals Assembly of Two Independent Populations of Flock House Virus Particles with Distinct RNA Packaging Characteristics in the Same Cell

2006 ◽  
Vol 81 (2) ◽  
pp. 613-619 ◽  
Author(s):  
P. Arno Venter ◽  
Anette Schneemann
Keyword(s):  
Coat Protein ◽  
Rna Virus ◽  
Protein Translation ◽  
Exchange Chromatography ◽  
Coat Proteins ◽  
Flock House Virus ◽  
Rna Packaging ◽  
Virus Particles ◽  
Bipartite Genome ◽  
Positive Strand Rna

ABSTRACT Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.

scholarly journals Sec16 influences transitional ER sites by regulating rather than organizing COPII

2013 ◽  
Vol 24 (21) ◽  
pp. 3406-3419 ◽  
Author(s):  
Nike Bharucha ◽  
Yang Liu ◽  
Effrosyni Papanikou ◽  
Conor McMahon ◽  
Masatoshi Esaki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Pichia Pastoris ◽  
Protein Complex ◽  
The Other ◽  
Yeast Pichia Pastoris ◽  
Functional Regions ◽  
Conserved Domain ◽  
Copii Vesicles ◽  
Negative Regulatory Role

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

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