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Platelet-Derived Growth Factor
Published in Jason Kelley, Cytokines of the Lung, 2022
James P. Fabisiak, Jason Kelley
Nucleotide sequencing of the lengthy 5′ flanking region of the c-sis gene revealed a traditional TATAAA promoter element 24-base pairs (bp) upstream from the mRNA start site, a putative Sp1 binding site, but no CAAT box (van den Ouweland et al., 1987; Rao et al., 1988). It does encode sequences that act as translational inhibitors (Rao et al., 1988). A single atypical polyadenylation site within the 3′ UTR is in concordance with the presence of a single species of mRNA transcript of 3.8–4.0 kilobases (kb) seen in positive cells. The 3′ UTR contains (A + U)-rich consensus sequences similar to those described by Shaw and Kamen (1986), which appear to function in determining the stability of several rapidly degraded cytokine transcripts, through the action of a specific exonuclease. More recently, the recognition of other positive and negative regulatory elements within the 5′ UTR have suggested additional mechanisms for regulation of PDGF-B gene expression (Rao et al., 1986; Pech et al., 1989; Franklin et al., 1991).
Order Mononegavirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Figure 31.2 demonstrates the genomic structure of the most popular families of the order. The size of the genomes varies in wide range from 8–19 kb. The genomes demonstrate the characteristic gene order 3’-UTR—core protein genes—envelope protein genes—RNA-dependent RNA polymerase gene—5’-UTR (3’-N-P-M-G-L-5’), with some exceptions. The infectious helical ribonucleocapsids are enveloped into virions.
Gene Expression
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Some results with nonviral lipid delivery systems show that only a small fraction (~1%) of DNA introduced into the cell cytoplasm actually reaches the nucleus. There, DNA must be transcribed into mRNA which, upon further changes, migrates into cytoplasm to ribosomes where protein synthesis commences (Chen et al., 1993). It may be advantageous if gene expression would begin in the cytoplasm. Indeed, some vectors, such as bacteriophage T7 which encodes for an enzyme T7 RNA polymerase which can transcribe DNA into RNA in the cytosol of mammalian cells with high transcriptional activity, can express cDNA in the cytoplasm. For such a system to work, a T7 RNA polymerase protein must be present in the cytoplasm along with the cDNA of interest. It can be introduced together with DNA, encoded in the DNA, or both. Expression of both genes is induced by T7 RNA polymerase promoters and rapid cytoplasmic gene expression independent of nuclear transcription factors was observed. Transcripts are not capped and an IRES sequence is inserted into the 5’UTR for efficient translation of transcripts. Although high levels of expression can be achieved, it is only transient because polymerase is degraded. By using a T7 autogene, however, a continuous synthesis of T7 RNA polymerase can be achieved, and expression for a week was reported (Gao and Huang, 1993). Such an approach may be useful when a fast, transient, and high level of transgene expression is preferred.
Emergence of mRNA vaccines in the management of cancer
Published in Expert Review of Vaccines, 2023
Mohamad Irfan Mohamad Razif, Nabilah Nizar, Nur Hannah Zainal Abidin, Syasya Nasuha Muhammad Ali, Wan Nurul Najihah Wan Zarimi, Junaidi Khotib, Deny Susanti, Muhammad Taufiq Mohd Jailani, Muhammad Taher
After identifying the antigen of choice of the target protein, several steps of gene sequencing, synthesizing, and cloning into the DNA template plasmid are performed [15]. An mRNA-based cancer vaccine production process is initiated by designing a DNA template or pDNA that consists of the ORF, flanking 5’- and 3’-UTRs as well as a primer binding site that contains available RNA polymerase recognition sites to initiate in vitro transcription [14]. Moreover, they also stated that the efficacy of translation of the target protein can be improved by using the codon concurrency that affects the amino acids on the mRNA. The sequence could also be designed in silico producing a variety of antigen sequences that have efficient leader sequences, optimal codon usage, increased neutralization, and reduced cross-reactivity [15]. An interesting method that could be used to increase the efficacy of protein expression or mRNA translation is by substitution of rare codons with regularly used identical codons [16]. The UTRs play an important role in regulating the protein expression, rates of degradation and translation of mRNA by interacting with different RNA-binding proteins [14]. It is also stated that the 5’-UTR initiates the translation and formation of preinitiation complexes and stabilizes the mRNA. However, the efficacy of translation can be improved by shortening the length of 3’-UTR. An optimal template sequence with high stability and translating efficacy is ideal in the formulation of mRNA vaccines.
Rewiring of miRNA-mRNA bipartite co-expression network as a novel way to understand the prostate cancer related players
Published in Systems Biology in Reproductive Medicine, 2023
Mohammad Mehdi Naghizadeh, Behnaz Bakhshandeh, Farshid Noorbakhsh, Marjan Yaghmaie, Ali Masoudi-Nejad
There were several factors that disturb the function of the genes, leading the normal cells to tumors and making cancer a complex disease. MicroRNAs, as endogenous and small non-coding RNAs, play critical roles in gene regulation of cell development, cell proliferation, cell survival, and apoptosis functions, either oncogenes or tumor suppressors under certain conditions (He and Hannon 2004; Bentwich et al. 2005; Meltzer 2005). The stimulatory, inhibitory, or promoting role of miRNAs is lesser known but, the miRNA binding mechanism could determine this role. Inhibitory action of miRNA is likely by way of direct DNA or mRNA 3'UTR binding (Jopling et al. 2005) and through a combination of the mechanisms including direct mRNA degradation, deadenylation, initiation repression, ribosomal stalling, ribosomal drop-off, co- translational degradation, and translation repression following DNA binding. However, it's direct binding to DNA directly or to mRNA 5’UTR may promote transcription (Zhang et al. 2013).
A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells
Published in mAbs, 2018
Emanuele Sasso, Debora Latino, Guendalina Froechlich, Mariangela Succoio, Margherita Passariello, Claudia De Lorenzo, Alfredo Nicosia, Nicola Zambrano
A new class of natural and synthetic long non-coding RNAs has been recently described, that increases the translation of specific mRNA targets.14 SINEUP RNAs work by a 5′ head-to-head divergent perfect complementarity with the target mRNA. Namely, the region encompassing the 5′UTR and the first AUG codon of the target mRNA is bound by the 5′ end of the long non-coding RNA. The activity of the SINEUP transcripts depends on two main domains: a target-specific binding domain and a universal effector domain containing a modular structure with inverted SINE elements, belonging to the B2 subclass (SINEB2 element) and partial Alu element.15 Moreover, it has been reported that SINEUPs work in several mammalian cells, including mouse, hamster, monkey and human cell lines. To date, a range of 1.5 to 3 times increases in recombinant protein production by SINEUP have been reported for both cytoplasmic and secreted proteins, according to cell- and target-specificity.16 A first attempt to increase mAb production through SINEUP reported by Yao et al. shows a potential usefulness of the system even if with a seemingly limited efficacy, with 50% increases in production of antibodies.17 In this paper, we implemented SINEUP technology to boost semi-stable production of mAbs in HEK293E cells, demonstrating its validity, in terms of improved antibody production, without losses in post-translation modifications and binding ability of the SINEUP-produced antibodies.