EIF3 p170, a Mediator of Mimosine Effect on Protein Synthesis and Cell Cycle Progression

Abstract

l-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been suggested to affect translation of mRNAs such as p27, the CDK inhibitor. However, the mechanism of this effect is not known. Regulation of translation generally occurs at the initiation step that, in mammalian cells, is a complex process that requires multiple eukaryotic initiation factors (eIFs) and ribosome. The effects of mimosine on initiation factors or regulators consequently will influence translation initiation. P170, a putative subunit of eIF3, has been suggested to be nonessential for eIF3 function to form preinitiation complexes and it may function as a regulator for translation of a subset of mRNAs. In this article, we tested this hypothesis and investigated whether eIF3 p170 mediates mimosine effect on mRNA translation. We found that p170 translation was dramatically reduced by mimosine due to its iron-chelating function. The decreased expression of p170 by mimosine caused diminished de novo synthesis of tyrosinated ␣-tubulin and elevated translation of p27 before cell cycle arrest. These observations suggest that p170 is likely an early response gene to mimosine treatment and a mediator for mimosine effect on mRNA translation. The effect of p170 on the synthesis of tyrosinated ␣-tubulin and p27 in a reciprocal manner also suggests that p170 functions as a regulator for mRNA translation. INTRODUCTIONl-Mimosine, a plant amino acid derived from seeds of Leucaena leucocephala or Mimosa pudica, can specifically and reversibly block mammalian cells at late G1 phase (Lalande, 1990;Mosca et al., 1992). Mimosine is commonly used as a synchronizing agent for investigating cell cycle progression in mammalian cells (Wang et al., 1995;Hughes and Cook, 1996). However, the molecular mechanism of mimosine action is ill-defined. It has been reported that the cells treated with mimosine lack proteins required for initiation and elongation of DNA replication (Kalejta and Hamlin, 1997). Although mimosine does not affect mRNA synthesis in general (Tsai and Ling, 1971), a subset of mRNAs was found to relocate to the mono-ribosome pool at the expense of polyribosome fraction after mimosine treatment. On mimosine removal, they reappeared in poly-ribosome pool, indicating that the mimosine effect is reversible (Hanauske-Abel et al., 1995). A recent study showed that expression of the cyclindependent kinase inhibitor p27 was elevated at the translational level by mimosine (Wang et al., 2000). These findings suggest that mimosine may modulate the synthesis of some proteins, which in turn causes inhibition of DNA replication and cell cycle arrest. One possible mechanism for such effect is that mimosine affects the synthesis and/or the function of proteins controlling mRNA translation.The rate-limiting step in mRNA translation is the initiation step. In eukaryotes, 11 factors have been identified that participate in the translation initiation step (Hershey and Merrick, 2000). Among these factors, eIF3 is the largest and the most ...

Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis

“…RNA Extraction and Ribonuclease Protection Assay-Total RNA was extracted using the RNeasy minikit (Qiagen) as described previously (10). Nuclear RNAs were prepared by first isolating nuclei as described previously (16,17).…”

“…Previously it has been shown that mimosine functions as an iron chelator (10,35,36). Although chelating iron would increase the expression and activity of HIF-1 and c-Jun/AP-1 transcription factors (30), it also decreases the expression and activity of N-Myc and c-Myc (47,48).…”

“…We have identified several proteins that showed altered expression after mimosine treatment. Although eukaryotic translation initiation factor 3a (eIF3a 1 or eIF3 p170), tyrosinated ␣-tubulin, and ribonucleotide reductase M2 were decreased, the cyclin-dependent protein kinase inhibitor p27 was elevated by mimosine treatment (10,11). The change in the expression level of these proteins by mimosine may be an important prelude to mimosine-mediated G 1 cell cycle arrest.…”

ABCE1 is essential for S phase progression in human cells

“…Among them are p27, an essential regulator of S phase entry; RRM2, a key enzyme supplying converted deoxyribonucleotides for DNA synthesis; and RPA2 functioning in multiple DNA metabolic pathways. [66][67][68] The steady-state levels of these proteins were also not consistently altered in ABCE1-silenced cells (Fig. 6B), implying that translation of at least these few candidates may not be critically dependent on the presence of ABCE1.…”

“…In particular, eIF3A is expressed periodically during S phase and may regulate translation of several proteins important for cell cycle control and DNA replication, such as the cyclin-dependent kinase inhibitor p27, ribonucleotide reductase M2 subunit (RRM2) and replication protein RPA2. [65][66][67][68] eIF3A has been previously shown to stimulate RRM2 and to inhibit p27 and RPA2 translation, thus acting in reciprocal directions. [66][67][68] In our study, in both HEK293 and HeLa cells with depleted ABCE1, the expression of none of these proteins was consistently altered as if they were co-regulated by ABCE1 (Fig.…”