IRES-mediated cap-independent translation, a path leading to hidden proteome

Abstract

Most eukaryotic mRNAs are translated in a cap-dependent fashion; however, under stress conditions, the cap-independent translation driven by internal ribosomal entry sites (IRESs) can serve as an alternative mechanism for protein production. Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trans-acting factors. Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood, the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities. A recent screen identified > 1000 putative IRESs from viral and human mRNAs, expanding the scope and mechanism for cap-independent translation. Additionally, a large number of circular RNAs lacking free ends were identified in eukaryotic cells, many of which are found to be translated through IRESs. These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA, implying a hidden proteome produced from cap-independent translation.

Keywords: IRES; ITAF; bicistronic system; circular RNA; translation.

Figure 1

Four groups of viral IRESs. Viral IRESs can be classified into four groups based on their structures and the requirement of ITAFs and IFs. Generally, IRESs with more compact structures require less auxiliary protein factors.

Figure 2

Mechanism of translation initiation driven by cellular IRES. (A) Type I cellular IRES. The cis-elements in the cellular IRESs are bound by the ITAFs. These ITAFs will directly interact with the 40S ribosomal subunit or recruit the 40S ribosomal subunit through the ‘bridge’—IFs. Finally, the 60S ribosomal subunit is recruited to start translation. (B) Type II cellular IRES. Cellular IRESs contain the short cis-elements that can base pair to the 18S rRNA. Therefore, these cellular IRESs can directly interact with 40S ribosomal subunit by base-pairing between itself and 18S rRNA and recruit 60S ribosomal subunit to start the translation.

Figure 3

Reporter systems using in IRES study. (A) Bicistronic reporter system. The most popular reporter system is used to examine IRES activity. In the plasmid, two ORFs are inserted into downstream of the transcription promoter, and the IRES is inserted between these two ORFs. During translation, the first ORF is translated through cap-dependent translation whereas the second ORF is translated by cap-independent translation. This reporter can be in vitro transcribed into RNA and used for the in vitro or in vivo translation system (left panel). There are three reasons that may introduce the false positive activity of the ‘IRES’ sequence: first, the cryptic promoter activity; second, the cryptic splicing sites; and third, translation readthrough (right panel). (B) circRNA reporter system. The circRNA reporter is inserted into a single exon containing two split GFP ORFs in a reversed order and two flanking introns with complement elements. After transcription, the circRNA will be generated through back-splicing and then translated into protein through IRES-dependent translation (left panel). The transcription readthrough produces a very long linear RNA concatemer including two or more exons with the split GFP ORFs. This RNA concatemer may be spliced into the mature linear mRNA containing a whole ORF. Therefore, the transcription readthrough will introduce the false discovery activity of IRESs (right panel).