scholarly journals An Integrative Synthetic Biology Approach to Interrogating Cellular Ubiquitin and Ufm Signaling

2020 ◽  
Vol 21 (12) ◽  
pp. 4231 ◽  
Author(s):  
Chuanyin Li ◽  
Tianting Han ◽  
Rong Guo ◽  
Peng Chen ◽  
Chao Peng ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Post Translational Modifications ◽  
E3 Ligases ◽  
Cloning Technique ◽  
Global Identification ◽  
Multiple Hit ◽  
Reading Frames ◽  
Synthetic Biology Approach

Global identification of substrates for PTMs (post-translational modifications) represents a critical but yet dauntingly challenging task in understanding biology and disease pathology. Here we presented a synthetic biology approach, namely ‘YESS’, which coupled Y2H (yeast two hybrid) interactome screening with PTMs reactions reconstituted in bacteria for substrates identification and validation, followed by the functional validation in mammalian cells. Specifically, the sequence-independent Gateway® cloning technique was adopted to afford simultaneous transfer of multiple hit ORFs (open reading frames) between the YESS sub-systems. In proof-of-evidence applications of YESS, novel substrates were identified for UBE3A and UFL1, the E3 ligases for ubiquitination and ufmylation, respectively. Therefore, the YESS approach could serve as a potentially powerful tool to study cellular signaling mediated by different PTMs.

scholarly journals Pan-cancer analysis of transcripts encoding novel open-reading frames (nORFs) and their potential biological functions

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chaitanya Erady ◽  
Adam Boxall ◽  
Shraddha Puntambekar ◽  
N. Suhas Jagannathan ◽  
Ruchi Chauhan ◽  
...  
Keyword(s):  
Transcript Level ◽  
Open Reading Frames ◽  
The Cancer Genome Atlas ◽  
Small Subset ◽  
Cancer Tissue ◽  
Post Translational Modifications ◽  
Cancer Genome Atlas ◽  
Systematic Identification ◽  
Reading Frames

AbstractUncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.

scholarly journals FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells

2020 ◽  
Vol 22 ◽  
pp. 885-899
Author(s):  
Fang He ◽  
Na Ni ◽  
Zongyue Zeng ◽  
Di Wu ◽  
Yixiao Feng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Mammalian Cells ◽  
Expression In Mammalian Cells ◽  
Synthetic Biology Approach

scholarly journals Proteins encoded by Novel ORFs have increased disorder but can be biochemically regulated and harbour pathogenic mutations

2019 ◽  
Author(s):  
N. Suhas Jagannathan ◽  
Narendra Meena ◽  
Kethaki Prathivadi Bhayankaram ◽  
Sudhakaran Prabakaran
Keyword(s):  
Biological Function ◽  
Structural Disorder ◽  
Open Reading Frames ◽  
Deleterious Mutations ◽  
Computational Tools ◽  
Post Translational Modifications ◽  
Novel Proteins ◽  
Regulatory Changes ◽  
Reading Frames ◽  
Novel Protein

Abstract Recent advances in proteogenomics indicate that protein or protein-like products can be encoded by previously uncharacterized Open Reading Frames (ORFs) that we define as Novel Open Reading Frames (nORFs)1​ ​,2​ ​. Although it is yet unclear if these protein or protein-like products could possess any significant biological function hopes have been raised to target them for anticancer and antimicrobial therapy 3​ ,4​. In this study, we used computational tools to systematically investigate these novel protein sequences for their propensities toward structural disorder, post-translational modifications (PTM) and mutational densities. We found that these novel proteins have significantly higher disorder and similar PTM frequencies compared to known proteins. Although these regions were found to harbour deleterious mutations, we did not observe any correlation between the pathogenicity of mutations and their location (ordered/disordered) within these novel proteins. This study suggests that these nORFs encode an important class of proteins, that could undergo sequence, structural or regulatory changes during complex diseases, and hence warrant further study.

Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach

2018 ◽  
Vol 7 (5) ◽  
pp. 1447-1455 ◽  
Author(s):  
Marco Santorelli ◽  
Daniela Perna ◽  
Akihiro Isomura ◽  
Immacolata Garzilli ◽  
Francesco Annunziata ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Mammalian Cells ◽  
Synthetic Biology Approach

scholarly journals Identification of Candidate Gammaherpesvirus 68 Genes Required for Virus Replication by Signature-Tagged Transposon Mutagenesis

2004 ◽  
Vol 78 (19) ◽  
pp. 10282-10290 ◽  
Author(s):  
Nathaniel J. Moorman ◽  
Chie Yu Lin ◽  
Samuel H. Speck
Keyword(s):  
Life Cycle ◽  
Virus Replication ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Transposon Insertion ◽  
Gammaherpesvirus 68 ◽  
Reading Frames

ABSTRACT Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (γHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the γHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type γHV68. From these analyses we identified 16 γHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other γHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.

scholarly journals Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation

1997 ◽  
Vol 328 (3) ◽  
pp. 807-813 ◽  
Author(s):  
Graham PACKHAM ◽  
Matthew BRIMMELL ◽  
L. John CLEVELAND
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Hormone Receptors ◽  
Steroid Hormone ◽  
Open Reading Frames ◽  
Steroid Hormone Receptors ◽  
Alternative Translation Initiation ◽  
Alternative Translation ◽  
Expression In Mammalian Cells ◽  
Reading Frames

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 protein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apoptosis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the structure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammalian cells. We demonstrate that, in addition to the previously identified 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a second 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have distinct subcellular localizations, suggesting that they are functionally distinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5ʹ sequences demonstrated that human and murine Bag-1 cDNAs contain longer open reading frames than originally suspected. We determined which open reading frames gave rise to the Bag-1 isoforms in human cells. Suprisingly, translation of neither protein initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag-1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently localized Bag-1 isoforms generated by alternative translation initiation, and Bag-1 proteins may play a dual role in regulating apoptosis and steroid hormone-dependent transcription.

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