Comparison of IRES and 2A peptides in the simultaneous expression of multiple target genes

IRES & 2A peptide competition

Guide

Hello! Hello everyone! There is a problem. I don't know if you have considered it. In order to study the functional mechanism of a single gene or to achieve multi-gene co-expression, how to achieve multiple simultaneous expression of multiple target genes?

In fact, polycistronic expression schemes are currently widely used, that is, using internal ribosome entry sites (IRES) or self-cleaving 2A peptides (2A) to connect multiple A gene that achieves the purpose of expressing multiple polycistronics on a single vector.

So, what are the characteristics of IRES and 2A peptides? Let’s summarize it today, let me slowly come~

IRES

First, it was found that although translation of eukaryotic mRNA requires a 5' cap to mediate ribosome binding, there are some exceptions in eukaryotes and viruses, such as the fact that some genes have a shorter RNA sequence at the 5' end ( About 150-250 bp), such RNA sequences can be folded into a structure similar to the starting tRNA , thereby mediating ribosomal RNA binding and initiating protein translation. This untranslated RNA is called an internal ribosome entry site sequence ( Internal ribosome entry site, IRES).

The IRES element has the function of independently recruiting ribosomes regardless of the hat structure, and thus can initiate translation of downstream genes. Is there a bright light? One can use this element for co-expression of polycistronics.

Figure 1.A diagram of the Dicistroviridae RNA genome. Dicistroviruses are naturally dicistronic with two open reading frames that are translated by independent IRESs. The non-structural proteins are translated by the 5'IRES where the structural proteins are translated by the IGR IRES. A genome-linked viral protein (Vpg) and the poly(A) tail (An ) are indicated ^[1] .

The internal ribosome entry site (IRES) is a translational enhancer naturally present in a range of mRNAs that mediates the internal initiation of translation when present between genes of interest (Figure 2). Thus, IRES can drive translation of several genes encoded by the same mRNA by designing a polycistronic expression cassette similar to a bacterial operon.

The two genes in front of and behind the IRES element are relatively independent and can express the intact protein of interest. However, due to the large structure of the IRES, its application is often limited by the capacity of the vector, and the IRES is relatively weak in the ability to initiate translation of the gene behind it .

Figure 2. Cap-dependent and internal ribosome entry site-dependent initiation, two alternative mechanisms of translation. A: The so-called cap-dependent ribosome scanning mechanism predicts that ribosome 40S subunit binds to the mRNA 5' end. Ribosome binding requires the Initiation factor 4F (eIF-4F, composed of the three proteins eIF-4E, -4A and -4G). Then the mRNA is unwound under the control of the helicases eIF-4A and -4B, allowing the ribosome to scan the mRNA until Recognition of an initiation codon (classically AUG); B: When an Internal ribosome entry site (IRES) is present in the mRNA 5' untranslated region, IRES trans-acting factors (ITAFs) allow ribosome 40S internal recruitment, independently of the presence of Cap and eIF-4F. The IRES-dependent mechanism occurs in the case of picornavirus uncapped mRNAs as well as for cellular capped mRNAs ^[2] .

2A peptide

However, as mentioned above, IRES has a problem of co-expression of two genes with unbalanced expression levels. To solve this problem, the self-cleaving 2A peptide construction strategy has received more and more attention in recent years.

The 2A peptide was found in Foot-and-Mouth Disease Virus (FMDV), a "self-cleaving" small peptide identified therefrom with an average length of 18-22 amino acids. Initially, it was speculated that virally encoded proteins or host cell proteins might not be cleaved. However, this receptor mediates the synthesis of a trimer of the isovalerylproline peptide on the C-terminal 2A peptide, resulting in immediate cleavage between the 2A peptides, with the downstream peptide having the proline N-terminus (Figure 3). .

Figure 3. Schematic representation of cleavage occurring in a peptide translated in foot-and-mouth disease virus (FMDV). 1D, 2A and 2B indicate contiguous endogenous peptides translated in FMDV. The arrowhead indicates cleavage site ^[3] .

The high-level structure formed by the 2A element in the translation process causes steric hindrance to the ribosome peptidyl transferase center, resulting in the inability to form a normal peptide chain linkage, but at the same time the ribosome can continue to translate the downstream protein, thereby forming a similar proteolysis. The action of the enzyme cis-cuts the two proteins before and after.

The 2A peptide structure is short and the expression balance of the upstream and downstream genes is good . However, due to its unique "shear" mechanism, a final upstream protein with a 2A peptide tail and a downstream protein with a proline at the N-terminus are obtained, respectively. The additional 2A peptide structure may be for the target protein. The function has a certain impact.

Figure 4. (A) Two individual polypeptides can be generated from one transcript using 2A to link the individual genes. (B) 2A peptide sequence and its cleavage site (G/P) ^[4]

In summary, IRES "simultaneous transcription, independent translation", 2A peptide "simultaneous transcription, simultaneous translation" , both for simultaneous expression of multiple proteins. However, due to the advantages and disadvantages of both IRES and 2A peptides, it is often necessary to construct two or more different expression vectors for comparison in in vitro experiments in order to find the most suitable expression protocol.

BIO's many years of genetically engineered mouse design experience can be designed according to your specific genetically modified mouse program. If you have any questions, comments or suggestions, welcome to discuss!

references:

[1]Tricks an IRES uses to enslave ribosomes.

[2]Internal ribosome entry site-based vectors for combined gene therapy.

[3]High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice.

[4]Use of the Foot-and-Mouth Disease Virus 2A Peptide Co-Expression System to Study Intracellular Protein Trafficking in Arabidopsis

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