Internal ribosome entry site

An internal ribosome entry site, abbreviated IRES, is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis. Usually, in eukaryotes, translation can be initiated only at the 5' end of the mRNA molecule, since 5' cap recognition is required for the assembly of the initiation complex.

History
These sequences were first discovered in 1988 in poliovirus RNA and encephalomyocarditis virus RNA in the labs of Nahum Sonenberg and Eckard Wimmer, respectively. They are described as distinct regions of RNA molecules that are able to attract the eukaryotic ribosome to the mRNA molecule and, therefore, allow translation initiation to occur. This process became known as the internal initiation of translation. It has been hypothesized that IRES elements have a distinct secondary or even tertiary structure, but similar structural features at the levels of either primary or secondary structure that are common to all IRES segments have not been reported to date.

Location
It is common that IRESes are located in the 5'UTR of RNA viruses and allow translation of the RNAs in a cap-independent manner. However, mRNAs of viruses from Dicistroviridae family possess two ORFs (open reading frames), translation of the both being directed by two distinct IRESes. It was later suggested that some mammalian mRNAs also have IRES. Several cellular IRES elements are thought to be located in eukaryotic mRNAs encoding genes involved in stress survival, and other processes critical to survival. As of September 2009, there are 60 animal and 8 plant viruses reported to contain IRES segments and 115 mRNA sequences containing them as well.

Activation
IRES are often used by viruses as a means to ensure that viral translation is active during periods of time when host translation is inhibited. These mechanisms of host translation inhibition are varied, and can be initiated by both virus and host, depending on the type of virus in question. However, in the case of most picornaviruses, this is accomplished by the viral protease cleaving eIF-4G so that it cannot interact with eIF-4E. Interaction between these two initiation factors is necessary for mRNA 5'cap to 3'poly-A-tail loop formation, which is usually a necessary event for initiation of translation. The virus may even use the eIF-4G to aid in initiation of IRES-mediated translation.

The cell may also use IRES to increase translation of certain proteins during mitosis and programmed cell death. In mitosis, the cell dephosphorylates eIF-4E so that it has little affinity for the 5'cap. As a result, the pre-initiation mRNA loop is not formed, and the translational machinery is diverted to IRES within the mRNA. Many proteins involved in mitosis are encoded by IRES mRNA. In programmed cell death, cleavage of eIF-4G, such as performed by viruses, decreases translation. Lack of essential proteins contributes to the death of the cell, as does translation of IRES mRNA sequences coding proteins involved in controlling cell death.

Mechanism
The mechanism of viral IRES function to date is better characterized than the mechanism of cellular IRES function, for which no clear mechanism have been proposed yet. Hepatitis C Virus-related IRESs directly bind 40S ribosomal subunit in such a way that their initiator codons are located in ribosomal P-site without mRNA scanning. These IRESs do not require Eukaryotic initiation factors eIF1, 1A, 4A, 4B, and 4E. Picornavirus IRES do not attract 40S directly, but rather through high-affinity eIF4G-binding site. In addition, many viral IRES (as well as cellular IRES) require additional proteins to mediate their function, known as IRES trans-acting factors (ITAFs). The role of ITAFs in IRES function is currently the subject of intense research.

Testing
Testing a particular RNA sequence for IRES activity relies on a bicistronic reporter construct. When an IRES segment is located between two reporter open reading frames in a eukaryotic mRNA molecule (a bicistronic mRNA), it can drive translation of the downstream protein coding region independently of the 5'-cap structure bound to the 5' end of the mRNA molecule. In such a setup both proteins are produced in the cell. The first reporter protein located in the first cistron is synthesized by the cap-dependent initiation approach while translation initiation of the second protein is directed by the IRES segment located in the intercistronic spacer region between the two reporter protein coding regions. However, there are several caveats to be aware of when interpreting data produced using bicistronic reporter constructs. For example, there are several known cases of mis-reported IRES segments that were later recognized as promoter-containing regions. More recently, splice acceptor sites within several presumed IRES segments have been shown to be responsible for apparent IRES function in bicistronic reporter assays.