Circular mRNA Research Articles

Synthesizing Long and Repetitive Proteins Via a “Loopable Translator” System

The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we present a system that generates circular mRNA in vivo by circularly permuting the self-splicing thymidylate synthase (TD) intron from T4 bacteriophage, resulting in “loopable” translation. In this system, the order of exons and intron are rearranged such that the identical natural RNA splicing events result in a circular mRNA. The self-splicing group I intron was selected because it does not require an enzyme for its activity. We demonstrated that circularization successfully occurs in vivo, and under the optimal conditions (identified to date) the looping construct achieves 15% of the GFP production relative to the positive control. Successful mRNA circularization was confirmed by SDS-PAGE gel electrophoresis. The data suggest that protein yield may be limited by inefficient initiation, though ribosomes can perform many transits on a circular mRNA template per initiation event. This circularizing translation system may contribute to the production of protein-based polymers with diverse applications.

Circular RNA, the Key for Translation.

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m6A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5′ end-independent translation initiation. Today a new family of so-called “noncoding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5′ end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3′–5′ interaction have been reported, including m6A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

Continuous translation of circularized mRNA improves recombinant protein titer

Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A “self-cleavage” peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.

On the Ribosomal Density that Maximizes Protein Translation Rate.

During mRNA translation, several ribosomes attach to the same mRNA molecule simultaneously translating it into a protein. This pipelining increases the protein translation rate. A natural and important question is what ribosomal density maximizes the protein translation rate. Using mathematical models of ribosome flow along both a linear and a circular mRNA molecules we prove that typically the steady-state protein translation rate is maximized when the ribosomal density is one half of the maximal possible density. We discuss the implications of our results to endogenous genes under natural cellular conditions and also to synthetic biology.

Efficient backsplicing produces translatable circular mRNAs.

While the human transcriptome contains a large number of circular RNAs (circRNAs), the functions of most circRNAs remain unclear. Sequence annotation suggests that most circRNAs are generated from splicing in reversed orders across exons. However, the mechanisms of this backsplicing are largely unknown. Here we constructed a single exon minigene containing split GFP, and found that the pre-mRNA indeed produces circRNA through efficient backsplicing in human and Drosophila cells. The backsplicing is enhanced by complementary introns that form double-stranded RNA structure to bring splice sites in proximity, but such structure is not required. Moreover, backsplicing is regulated by general splicing factors and cis-elements, but with regulatory rules distinct from canonical splicing. The resulting circRNA can be translated to generate functional proteins. Unlike linear mRNA, poly-adenosine or poly-thymidine in 3′ UTR can inhibit circular mRNA translation. This study revealed that backsplicing can occur efficiently in diverse eukaryotes to generate circular mRNAs.

Insights from a Paradigm Shift: How the Poly(A)-Binding Protein Brings Translating mRNAs Full Circle

In recent years, our thinking of how the initiation of protein synthesis occurs has changed dramatically. Initiation was thought to involve only events occurring at or near the 5′-cap structure, which serves as the binding site for the cap-binding complex, a group of translation initiation factors (eIFs) that facilitate the binding of the 40 S ribosomal subunit to an mRNA. Because the poly(A)-binding protein (PABP) binds the poly(A) tail present at the 3′-terminus of an mRNA, it was long thought to play no role in translation initiation. In this review, I present evidence from my laboratory that has contributed to the paradigm shift in how we think of mRNAs during translation. The depiction of mRNAs as straight molecules in which the poly(A) tail is far from events occurring at the 5′-end has now been replaced by the concept of a circular mRNA where the interaction between PABP and the cap-binding complex bridges the termini of an mRNA and promotes translation initiation. The research from my laboratory supports the new paradigm that translation of most mRNAs requires a functional and physical interaction between the termini of an mRNA.

“Rolling out” High‐Molecular‐Weight Proteins, which Contain Repeating Polypeptide Motif, by Using Rolling Circle Amplification

Keep going round in circles: On a circular mRNA template and without an end in sight, E. coli ribosomes have no option but to keep on making a high-molecular-weight polypeptide with repeating units. In this Highlight, the recently published work by Abe and co-workers on this topic is discussed.

MRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein

During lytic infections, the herpes simplex virus (HSV) virion host shutoff (Vhs) endoribonuclease degrades many host and viral mRNAs. Within infected cells it cuts mRNAs at preferred sites, including some in regions of translation initiation. Vhs binds the translation initiation factors eIF4H, eIF4AI, and eIF4AII, suggesting that its mRNA degradative function is somehow linked to translation. To explore how Vhs is targeted to preferred sites, we examined the in vitro degradation of a target mRNA in rabbit reticulocyte lysates containing in vitro-translated Vhs. Vhs caused rapid degradation of mRNAs beginning with cleavages at sites in the first 250 nucleotides, including a number near the start codon and in the 5' untranslated region. Ligation of the ends to form a circular mRNA inhibited Vhs cleavage at the same sites at which it cuts capped linear molecules. This was not due to an inability to cut any circular RNA, since Vhs cuts circular mRNAs containing an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) at the same sites as linear molecules with the IRES. Cutting linear mRNAs at preferred sites was augmented by the presence of a 5' cap. Moreover, mutations that altered the 5' proximal AUG abolished Vhs cleavage at nearby sites, while mutations that changed sequences surrounding the AUG to improve their match to the Kozak consensus sequence enhanced Vhs cutting near the start codon. The results indicate that mutations in an mRNA that affect its translation affect the sites at which it is cut by Vhs and suggest that Vhs is directed to its preferred cut sites during translation initiation.

A Unique Articular Manifestation in a Child with Relapsing Polychondritis

<h3>Abstract</h3> Although proteolysis targeting chimera (PROTAC) technology that hijacking the ubiquitin-proteasome system (UPS) to artificially degrade protein is an emerging promising technology for many undruggable targets, it still faces several challenges, such as the difficulty to select high specificity molecule to protein of interest (POI), and only few of the E3-ligase are suitable for PROTAC mediated protein degradation. Protein-based PROTAC, termed BioPROTAC, owns the advantage of specificity but lacks membrane permeability. Here, we develop a new targeted protein degrading platform, which we termed RiboPROTAC, by encoding BioPROTAC protein degraders with circular mRNA (cmRNA) and delivering intracellularly. In this way, intracellular protein GFP and nuclear protein PCNA were successfully degraded. For PCNA targeting, not only the level of target protein is strongly decreased, but also the cell function related is strongly interfered, such as cell proliferation, apoptosis, as well as the cell cycle. What is more, for the first time, we observed that degrading PCNA with RiboPROTAC causes immunogenic cell death (ICD), although not strong, but was verified in vivo. Importantly, our in vivo results demonstrated that cmRNA encoded PCNA degrader exhibited significant tumor suppression effect, thus we provide the first proof of concept for the application of RiboPROTAC as potential mRNA therapeutic. We consider that RiboPROTAC is a new and superior PROTAC technology for targeting the undruggable targets.

Circular mRNA encoding for monomeric and polymeric green fluorescent protein.

Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences, and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, the author has developed a method for producing messenger RNAs on circular RNA templates. This circularization process is derived from a rearranged group I intron, from which circular RNA is produced through the splicing activity of autocatalytic group I RNA elements (Fig. 1; 1,2). Because the only cofactors required for splicing of the group I intron are magnesium and guanosine, the process can take place in a variety of organisms, making it amenable to a wide variety of protein expression systems (1–4).

Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo.

Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, we have produced circular messenger RNAs with infinite open reading frames. We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Analysis of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technology provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.

Circularization of mRNA by Eukaryotic Translation Initiation Factors

Communication between the 5′ cap structure and 3′ poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.

Splicing of a circular yeast pre-mRNA in vitro.

We have tested the fate of a circularized synthetic pre-mRNA transcript in a whole cell splicing extract of Saccharomyces cerevisiae. Our results demonstrate that this circular precursor RNA is able to induce spliceosome formation in vitro and that the products of the following splicing reaction are the lariat-shaped intron, and a mature circular mRNA. Thus, it would appear that free 5' and/or 3' ends are not obligatory for a splicing reaction to occur, although we find its efficiency to be strongly influenced by the presence or lack of free ends. To our knowledge, this is the first demonstration that a circular pre-mRNA molecule is recognized as a suitable substrate by an eukaryotic mRNA splicing apparatus.

Inability of circular mRNA to attach to eukaryotic ribosomes.

IN contrast to prokaryotic systems, initiation by eukaryotic ribosomes seems to be restricted to a single site near the 5′-terminus of the message. However, nucleotide sequence analyses have not revealed a common ‘recognition sequence’ within the 5′-proximal portion of eukaryotic messages. We have previously suggested that the position of an AUG codon within a eukaryotic message—rather than the surrounding nucleotide sequence—might be the primary feature defining an initiation site. We proposed1,2 that a 40S ribosomal subunit binds initially at or near the 5′-end of a message and migrates along the RNA chain until the first AUG codon is encountered, whereupon it stops, a 60S subunit joins, and polypeptide synthesis begins. Although this mechanism accounts for many of the peculiarities of eukaryotic translational initiation, it leaves unanswered the question of how ribosomes recognise the 5′-terminus of mRNA. The m7G cap which blocks the end of most eukaryotic messages3 is not, responsible for directing ribosomes to the 5′-terminus since ribosomes initiate at the 5′-proximal AUG even in uncapped or unmethylated RNAs4,5. The ability of ribosomes to select the 5′-end of a message irrespective of its chemical structure (that is, whether the terminus is m7GpppN…, GpppN… or pppN…) is difficult to explain. An intriguing possibility is that eukaryotic ribosomes attach to mRNA by a threading mechanism. A simple prediction of the threading mechanism is that circularisation of a message should abolish its ability to interact with ribosomes. The experiments described below fulfill this prediction. The possibility of threading by prokaryotic ribosomes was ruled out, several years ago, by the demonstration that Escherichia coli ribosomes were able to translate the circular DNA of phage fd6,7. Indeed, the simplest version of a threading mechanism (entry at the 5′-terminus of a message with translation beginning at the first AUG codon encountered by the ribosome) is contradicted by the ability of E. coli ribosomes to initiate at multiple internal sites in poly-cistronic messages, such as bacteriophage R17 RNA. Conversely, the threading mechanism is consistent with the mono-cistronic character of eukaryotic messages.