Preferential Translation of Internal Ribosome Entry Site-containing mRNAs during the Mitotic Cycle in Mammalian Cells

Abstract

A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, ϳ3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5-non-coding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis.Cells of higher organisms can regulate gene expression at the translational level in response to a wide variety of stimuli and conditions. While much is known about the control of translational initiation and elongation in response to nutritional deprivation (1, 2) and environmental stress (2), the role of translational control in mammalian cell cycle progression is not well understood. It has long been known that specific proteins are needed at specific times during the cell cycle to ensure cell cycle progression. For the most part, the expression of these cell cycle-specific proteins is thought to be regulated at the transcriptional or posttranslational level (3).In cultured mammalian cells arrested at G 2 and M phases, the rate of total protein synthesis was markedly decreased to about 25% of the rate in non-arrested, cycling cells (4). This reduction was shown to be, at least partly, due to inhibition of the initiation step of polypeptide synthesis (4 -6). Subsequently, several eukaryotic initiation factors that regulate the assembly of 40 S ribosomes at the 5Ј ends of capped mRNAs were observed to change their phosphorylation status (7-9). Therefore, it seemed likely that inhibition of translation initiation was caused by diminishing ribosome recruitment to capped mRNAs during mitosis. Viral mRNAs whose translation is initiated by an internal ribosome entry site (IRES) 1 mechanism (10), such as polioviral and hepatitis C viral mRNAs (4,(11)(12)(13)(14) or mRNAs which lack significant structures in their 5Ј-non-coding regions (5Ј-NCRs), such as the mRNAs containing the late leader of adenovirus (15), were found to be selectively translated during mitosis due to their lessened requirement for eIF4F. More recently, additional IREScontaining mRNAs, those encoding ornithine decarboxylase and kinase p58 PITSLRE , have been reported to be selectively translated during G 2 /M of the cell cycle in cultured cells (11,12). These findings raise the question whether all IREScontaining genes are preferentially translated during mitosis and whether any of these encoded products play roles in cell cycle progression. We have begun to address these questions by genomic analysis of cellular mRNAs that are associated with mitotic polysomes and are, thus, predicted to be translated during the overall translation repression in mitosis. We determined that many, but not all, IRES elements are present in mRNAs which are selectively translated...

Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B–Cdk1 activation

“…Here we also show that Int-6 silencing does not modify the amount of various mRNAs in polysomes. It is also known that capdependent translation is inhibited during mitosis (Pyronnet et al, 2001;Qin and Sarnow, 2004). Although it remains possible that Int-6 silencing affects translation of specific mRNAs, an effect of this factor on degradation of specific proteins is the most tentative explanation.…”

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

“…Polysome profiling has been carried out under a number of conditions when cap-dependent translation has been inhibited including following polioviral infection, 30,31 during mitosis, 32 hypoxia and apoptosis (our unpublished data). In each of these diverse conditions, it has been found that approximately 3% of the messages remain associated with the polysomes including c-myc mRNA.…”

Translational regulation of the cell cycle: when, where, how and why?

“…Some of these nuclear RNA-binding proteins are known as ITAFs. ITAFs are thought to recognize IRES and could contribute to translation of IRES-containing mRNAs [66][67][68]. Although the role of IRES in mitosis is controversial, it has been proposed that IRESs, through ITAFs, allow recruitment of translational machinery independently of eIF4E, which is largely inhibited in mitosis [45,46,[69][70][71][72].…”

“…For example, polysome profiling combined with microarray analysis has allowed comprehensive identification of translationally regulated mRNAs, including those that encode cell cycle players, during HeLa cell mitosis, mouse male germ cell meiosis, as well as early Drosophila embryogenesis [66,126,127]. For a majority of these transcripts, the exact mechanisms of their cell cycle-dependent translational regulation and their roles in cell cycle progression still remain to be elucidated.…”

“…During the archetypal cell cycle, cap-dependent translation peaks in G1 phase and decreases by 60-80% in mitosis, when cellular energy is mostly invested into ensuring accurate cell division [45,46,65]. Genome-wide polysome profiling showed that approximately 3 per cent of HeLa cell mRNAs, encoding mostly nuclear RNA-binding proteins, efficiently associate with actively translating polysomes in mitosis [66]. Some of these nuclear RNA-binding proteins are known as ITAFs.…”