Abstract
SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouse AS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)-containing the invSINEB2 element-able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.
Highlights
The quantitative improvement of protein production in mammalian systems is a compelling need for the industrial manufacturing of commercially available enzymes, antibodies and supplements, and for gene therapy-based treatments of medical conditions
SINEUP molecular mechanism relies on its modular structure, composed of two fundamental domains: a Binding Domain (BD)—a region at the 50 of the long non-coding RNAs (lncRNAs) overlapping head to head to the 50 of the target mRNA—and an Effector Domain (ED)—constituted by an invSINEB2 repetitive element
The BD is crucial for target gene pairing and it confers molecular specificity, while the ED is the functional part of SINEUP required for loading the target transcript on polysomes and driving the translational increase [4]
Summary
The quantitative improvement of protein production in mammalian systems is a compelling need for the industrial manufacturing of commercially available enzymes, antibodies and supplements, and for gene therapy-based treatments of medical conditions. In 2012, Carrieri et al discovered a new class of lncRNAs, belonging to the category of natural antisense transcripts (NATs), that have the property to increase the protein translation of the target mRNA [4]. These transcripts were named SINEUP based on their ability to upregulate target protein translation by means of an invSINEB2 repeat, leaving unaltered the transcriptional levels of the target mRNA. TranSINE Therapeutics Limited (Cambridge, UK) has been founded to translate the SINEUP technology into the clinics as therapeutics for haploinsufficiency All together, these findings qualified SINEUP as a flexible tool able to upregulate the protein production of virtually any mRNA target of interest, affecting solely the translational levels. We describe in detail the fundamental steps in order to design, clone, and deliver SINEUP molecular tools in the cellular system of interest
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
