An IRES, or internal ribosome entry site, is a stretch of RNA that lets a cell’s protein-making machinery latch on to a messenger RNA molecule midstream, skipping the usual entry point at the very beginning. In the standard process, ribosomes recognize a chemical “cap” structure attached to one end of the message and scan forward until they find the right spot to start building a protein. An IRES bypasses that cap entirely, folding into a three-dimensional shape that grabs the ribosome directly and positions it at an internal location on the RNA. First discovered in viruses in the late 1980s, IRES elements have since turned up in some human genes and become essential tools in biotechnology and drug development.
Why Viruses Need a Back Door
Most of the messenger RNAs in your cells carry a small molecular tag on their front end called a 5′ cap. The cap acts like a boarding pass: it is recognized by a set of initiation factors that recruit the ribosome, the cellular machine that reads the RNA code and assembles proteins. Without the cap, the ribosome has no easy way to find the start of the message.
Many viruses face a problem here. Their RNA genomes either lack a cap or actively destroy the host’s cap-recognition system to shut down the cell’s own protein production. Picornaviruses, which include poliovirus and the common cold rhinoviruses, are a classic example. Their genomes carry long, highly structured untranslated regions at the front end that contain the IRES, a segment roughly 400 nucleotides long that folds into elaborate stem-loop structures.1Biologicals. Molecular Biology and Cell-free Synthesis of Poliovirus These structures assemble the ribosome and kick off cap-independent translation, letting the virus hijack the cell’s machinery even after it has sabotaged normal translation.2PubMed Central. Advances and Breakthroughs in IRES-Directed Translation and Replication of Picornaviruses
How Different IRES Types Recruit the Ribosome
Not all IRES elements work the same way. Researchers have classified viral IRES elements into several types based on their structure and the set of helper proteins they need to function. Picornavirus IRES elements alone span five recognized types, and other virus families add further variety.3PubMed Central. Structure and function of type IV IRES in picornaviruses: a systematic review The differences are not just academic; they reveal fundamentally different strategies for commandeering the ribosome.
Types That Still Use Host Initiation Factors
Type 1 (found in poliovirus and rhinovirus) and Type 2 (found in encephalomyocarditis virus) IRES elements rely on a stripped-down but still recognizable version of the normal initiation pathway. Both types work by binding directly to a host initiation factor called eIF4G, the same scaffolding protein used in cap-dependent translation, but they grab it without needing the cap-binding protein eIF4E.4PubMed Central. Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites From there, eIF4G recruits other standard factors and ultimately loads a small ribosomal subunit onto the RNA. Type 1 IRES elements also require a host protein called PCBP2, classified as an IRES trans-acting factor, or ITAF, a helper protein from the host cell that the IRES co-opts.5PubMed Central. The mechanism of translation initiation on Type 1 picornavirus IRESs
The Hepatitis C Approach
Type 3 IRES elements, best known from hepatitis C virus, take a more radical shortcut. Instead of relying on the full suite of initiation factors, the HCV IRES binds directly to the small (40S) ribosomal subunit in a single step, without any initiation factors or initiator transfer RNA being present at all.6Nature Communications. Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Ã… resolution Structural imaging has shown that when the HCV IRES docks onto the 40S subunit, it forces the subunit to change shape, closing the channel where mRNA normally threads through and locking the viral RNA into the decoding center where codons are read.7PubMed. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit The Type IV IRES found in certain picornaviruses is sometimes called “HCV-like” because it shares this compact, streamlined structure and similarly reduced dependence on initiation factors.3PubMed Central. Structure and function of type IV IRES in picornaviruses: a systematic review
The Most Extreme Shortcut
The most stripped-down IRES elements belong to a group found in insect viruses like cricket paralysis virus (CrPV). These fold into a compact shape featuring structures called pseudoknots, one of which mimics the interaction between a transfer RNA and a messenger RNA codon so convincingly that it slots directly into the ribosome’s decoding center.8PubMed Central. Initiation of Translation by Cricket Paralysis Virus IRES Requires Its Translocation in the Ribosome These IRES elements need no initiation factors whatsoever, and they can even start translation from a non-standard start codon. The CrPV IRES physically occupies the ribosome’s A, P, and E sites, the three functional positions where transfer RNAs normally cycle through.9Cell. Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes: The IRES Functions as an RNA-Based Translation Factor Translation can only begin after the ribosome physically moves the pseudoknot out of the decoding site, a step called translocation, which places the first real codon into position for a transfer RNA to recognize it.8PubMed Central. Initiation of Translation by Cricket Paralysis Virus IRES Requires Its Translocation in the Ribosome
Despite achieving the same goal, these different IRES types share almost no sequence similarity and have strikingly different structures. Evolutionary analysis suggests that conserved structural motifs within each virus family preserve the RNA shapes and protein interactions needed for IRES activity, even as the underlying sequences drift apart.10PubMed Central. Insights into Structural and Mechanistic Features of Viral IRES Elements
IRES Elements in Human Genes
IRES activity is not exclusive to viruses. Researchers have reported IRES-like sequences in the messenger RNAs of certain human genes, particularly those involved in regulating cell death and stress responses. During apoptosis, caspase enzymes chew up several of the initiation factors required for cap-dependent translation, effectively shutting down most protein production. Under these conditions, specific proteins needed for the apoptosis program are instead translated by internal ribosome entry.11Cell Death & Differentiation. Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors The anti-apoptotic protein XIAP, for instance, has an IRES in its messenger RNA that can form initiation complexes under conditions where cap-dependent translation is blocked.12Nucleic Acids Research. IRES-mediated translation of cellular messenger RNA operates in eIF2α-independent manner during stress
This type of translation has been linked to tumor cell survival. When cells face hypoxia, nutrient deprivation, or radiation, IRES-mediated translation of several genes involved in regulating apoptosis increases, potentially helping cancer cells resist chemotherapy.13PubMed Central. IRES-mediated translation of cellular messenger RNA operates in eIF2α-independent manner during stress
The Controversy Over Cellular IRES Claims
The existence of bona fide IRES elements in human genes is one of the more contentious debates in molecular biology. The standard test for IRES activity uses a “dicistronic” reporter, a synthetic RNA carrying two protein-coding sequences separated by the candidate IRES. If the second protein gets made, the logic goes, ribosomes must have entered internally. The problem is that this assay can produce false positives. If the dicistronic RNA unexpectedly gets cut or spliced into two separate pieces, the second protein can be made from its own smaller RNA through normal cap-dependent translation, mimicking IRES activity.
A careful study of six previously reported cellular IRES elements found that four of them contained splice sites whose activity was required for the apparent IRES function. In other words, the test RNA was being chopped into simpler pieces, and those pieces were being translated normally, not internally.14PubMed Central. Splicing mediates the activity of four putative cellular internal ribosome entry sites A more recent analysis went further, providing evidence that the vast majority of human transcript leader sequences reported to have IRES activity overlap with promoters, enhancers, or splice sites that could account for the observed results.15PubMed Central. False-positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5′ UTR annotations
This does not mean cellular IRES elements do not exist. A handful have survived increasingly stringent scrutiny, and the phenomenon of cap-independent translation during stress is real and well documented. But the number of genuine human IRES elements is almost certainly smaller than the literature suggests, and new claims now require far more rigorous controls than were standard a decade or two ago.
A Related Path to Cap-Independent Translation
IRES elements are not the only way cells bypass cap-dependent translation under stress. A chemical modification called m6A (a methyl group added to adenosine) found in the untranslated region at the front end of certain messenger RNAs can recruit the ribosome independently of the cap. A single m6A modification in the right position directly binds the initiation factor eIF3, which is enough to load the ribosome without the cap-binding factor eIF4E. After heat shock, increased m6A levels in the Hsp70 messenger RNA regulate its cap-independent translation, and cellular stress broadly redistributes m6A across the transcriptome, increasing the number of messages that carry this modification in positions where it can drive cap-independent initiation.16PubMed Central. 5′ UTR m(6)A Promotes Cap-Independent Translation
The intersection between m6A and IRES-like activity is an active research front. In triple-negative breast cancer, for example, the RNA-binding protein IGF2BP3 has been found to recognize m6A marks on the c-Met messenger RNA and recruit an alternative initiation factor to promote cap-independent translation, driving metastasis through an autophagy pathway.17PubMed Central. IGF2BP3 promotes autophagy-mediated TNBC metastasis via m6A-dependent, cap-independent c-Met translation Whether these m6A-mediated mechanisms qualify as true IRES activity or represent a distinct pathway is still being sorted out, but they clearly expand the repertoire of ways cells can make proteins when the normal cap-dependent route is compromised.
IRES in Biotechnology
Outside of basic science, IRES elements have become workhorses in genetic engineering. The most common application is the bicistronic expression vector: a synthetic DNA construct that produces a single messenger RNA encoding two different proteins, separated by an IRES. The ribosome translates the first protein by the normal cap-dependent route and then re-enters at the IRES to translate the second. This approach was first widely adopted using the IRES from encephalomyocarditis virus. One classic design pairs a gene of interest with an antibiotic-resistance gene downstream of the IRES, so that any cell surviving antibiotic selection is guaranteed to also be producing the desired protein.18PubMed. IRES bicistronic expression vectors for efficient creation of stable mammalian cell lines
Gene therapy vectors have used the same principle. A retroviral vector carrying both adenosine deaminase and a multidrug-resistance gene linked by an IRES produced a single bicistronic mRNA that expressed both proteins independently, an important feature because the two proteins function in completely different cellular compartments.19PubMed. Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector
IRES-based vectors do have a well-known limitation: the protein coded downstream of the IRES is usually expressed at a lower level than the upstream protein. This is because internal entry is less efficient than cap-dependent initiation. For applications like antibody production, where two protein chains need to be made in roughly equal amounts, this imbalance matters. An alternative strategy uses short self-cleaving peptide sequences (often called 2A peptides) that cause the ribosome to “skip” and release one protein before continuing to make the next. When researchers compared IRES-linked and 2A-linked antibody expression in mammalian cells, the 2A approach appeared to give higher total yields, but more than 40% of the product from 2A vectors was aggregated or improperly processed fusion protein. The IRES vectors produced antibodies with correct sizes and proper processing.20PubMed Central. Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells In stable mouse cell lines, similar tradeoffs appeared: 2A constructs gave higher expression of the downstream protein, but roughly 42% of the product was an unwanted fusion rather than two cleanly separated proteins.21PLOS ONE. Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines
A newer consideration is how nucleotide modifications interact with IRES function. Modified nucleotides like N1-methylpseudouridine (m1Ψ), which are now widely used in mRNA therapeutics because they reduce unwanted immune activation, can dramatically impair IRES-driven translation. Even replacing just a quarter of the uridines with m1Ψ was enough to disrupt type I IRES activity, while 2A peptide linkers tolerated the same modifications and actually benefited from them.22PubMed Central. Platform-dependent performance of P2A and IRES linkers in multicistronic gene expression This means that for the growing field of mRNA-based therapeutics, where modified nucleotides are standard, IRES elements may not be the best choice for multi-protein expression.
IRES and Circular RNA
Circular RNAs are a class of RNA molecules whose ends are joined into a loop, which means they have no 5′ cap and no free end for a ribosome to latch onto. For years, these molecules were assumed to be non-coding curiosities. The realization that IRES elements can drive translation from circular RNAs has opened a new field of research, and circular RNA is now being explored as a platform for protein-based therapeutics because its loop structure resists degradation and can persist longer in cells than linear mRNA.
A challenge has been that IRES-driven translation from circular RNA tends to be inefficient. Recent work tested a dual-IRES strategy, placing one IRES upstream and a second one downstream of the coding sequence within the circle. The approach significantly boosted protein output, but only when both IRES elements came from the encephalomyocarditis virus family. Other combinations failed to cooperate, suggesting that structural compatibility between the two IRES elements is critical.23PubMed Central. Enhancing Circular RNA Translation Efficiency Through Dual Internal Ribosome Entry Sites
Targeting IRES Elements as Antiviral Strategy
Because IRES elements are essential for viral replication and have no direct equivalent in normal host-cell translation, they make attractive drug targets. If a small molecule can jam the IRES, it could shut down viral protein production without disrupting the cell’s own cap-dependent translation. Several research groups are pursuing this idea.
One compound, a benzimidazole derivative called IRAB, was shown to preferentially inhibit IRES-dependent translation of foot-and-mouth disease virus. Structural analysis revealed that the drug increased the flexibility of specific stem-loops within the IRES, distorting its shape enough to impair function.24PubMed Central. Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation A different molecule, DMA-135, inhibits enterovirus 71 by working through an allosteric mechanism: it locks the IRES RNA into an altered shape that traps a host protein called AUF1 in a stable complex with the RNA, preventing translation from proceeding.25Nature Communications. IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex
More recently, computational screening of the enterovirus A71 IRES structure led to the identification of a compound called IRE-03-3 that inhibited viral proliferation in cell-based assays.26PubMed. Identification of Small-Molecule Inhibitors for Enterovirus A71 IRES by Structure-Based Virtual Screening The ability to use virtual screening against an RNA target, rather than the protein targets that dominate traditional drug discovery, represents a broader shift in how researchers think about drugging RNA structures. IRES elements, with their complex and well-characterized three-dimensional folds, are among the most tractable RNA targets for this kind of approach.
Why the Poly(A) Tail Still Matters
One common misconception is that IRES-mediated translation is completely independent of everything associated with normal translation. While it is true that IRES elements bypass the 5′ cap, other features of an mRNA can still influence how well an IRES performs. The poly(A) tail, the string of adenine nucleotides at the 3′ end of most messenger RNAs, significantly boosts IRES-driven translation. In cell-free systems, the poly(A) tail enhanced poliovirus IRES activity by more than tenfold and encephalomyocarditis virus IRES activity by roughly threefold.27RNA. Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system This means that even for viruses using internal entry, the overall architecture of the RNA message still matters. The IRES handles ribosome recruitment at the front end, but the tail end of the molecule contributes to efficiency through interactions that are still being mapped out.
Plant viruses, which face similar cap-independence challenges, have evolved yet another solution: cap-independent translation enhancers (CITEs) located in the 3′ untranslated region rather than near the start codon. These elements communicate with the ribosome through long-range RNA-RNA interactions that loop the end of the message back to the beginning. In pea enation mosaic virus, deleting the primary 3′ CITE reduced translation by more than 80%, showing how critical these back-end structures can be.28Nucleic Acids Research. Concerted action of two 3′ cap-independent translation enhancers increases the competitive strength of translated viral genomes Although CITEs are mechanistically distinct from IRES elements, they illustrate that cap-independent translation has evolved repeatedly through different structural strategies, each tailored to the constraints of a particular type of RNA genome.