Abstract
Long noncoding RNAs (lncRNAs) are important regulators of transcription; however, their involvement in protein translation is not well known. Here we explored whether the lncRNA GAS5 is associated with translation initiation machinery and regulates translation. GAS5 was enriched with eukaryotic translation initiation factor-4E (eIF4E) in an RNA-immunoprecipitation assay using lymphoma cell lines. We identified two RNA binding motifs within eIF4E protein and the deletion of each motif inhibited the binding of GAS5 with eIF4E. To confirm the role of GAS5 in translation regulation, GAS5 siRNA and in vitro transcribed GAS5 RNA were used to knock down or overexpress GAS5, respectively. GAS5 siRNA had no effect on global protein translation but did specifically increase c-Myc protein level without an effect on c-Myc mRNA. The mechanism of this increase in c-Myc protein was enhanced association of c-Myc mRNA with the polysome without any effect on protein stability. In contrast, overexpression of in vitro transcribed GAS5 RNA suppressed c-Myc protein without affecting c-Myc mRNA. Interestingly, GAS5 was found to be bound with c-Myc mRNA, suggesting that GAS5 regulates c-Myc translation through lncRNA-mRNA interaction. Our findings have uncovered a role of GAS5 lncRNA in translation regulation through its interactions with eIF4E and c-Myc mRNA.
Highlights
Long noncoding RNAs are pervasive in the mammalian genome and are important in regulating a variety of biological functions through different molecular mechanisms [1]
Association of Growth arrest-specific 5 (GAS5) Long noncoding RNAs (lncRNAs) with eukaryotic translation initiation factor-4E (eIF4E) To explore whether GAS5 plays a role in regulating protein translation in lymphoma cells, we first detected the association of GAS5 with the translation initiation factor eIF4E since the initiation of translation is a key step in translation regulation [22]
To further confirm that GAS5 is associated with the active translation apparatus, we separated polysomes by sucrose gradient ultracentrifugation into the non-polysome fraction, where translation initiation occurs [23], and the polysome fraction, where mRNAs are associated with ribosomes and efficiently translated (Figure 1B)
Summary
Long noncoding RNAs (lncRNAs) are pervasive in the mammalian genome and are important in regulating a variety of biological functions through different molecular mechanisms [1] Their role in transcription regulation has been comprehensively studied [2]. Ribosome-profiling studies revealed that many lncRNAs have similar ribosome occupancy as the translated regions of proteincoding genes [3] yet do not encode proteins [4,5]. These reports suggest that the lncRNAs associated with the ribosome may serve a regulatory function. We investigated the association of GAS5 with the eIF4F complex ( with eIF4E) and explored its role in the control of protein translation
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