Abstract
In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.
Highlights
In protein synthesis, the genetic information in mRNA is translated into an amino acid sequence with the help of ribosomes and transfer RNAs [1]
We demonstrated that RNA-binding motif protein 4 (RBM4) promoted the mRNA translation of vascular endothelial growth factor A (VEGFA) gene by binding to the G4 structure in the internal ribosome entry site (IRES)
The HSV-TK promoter upstream of the firefly luciferase (FLuc) marker gene was replaced by the VEGFA IRES-A sequence to generate a pR-IRES-F vector
Summary
The genetic information in mRNA is translated into an amino acid sequence with the help of ribosomes and transfer RNAs (tRNAs) [1]. The mRNA translation is mediated in either a cap-dependent or cap-independent manner [2,3]. IRES sequence can form a variety of RNA structures, such as stem loop, hairpin, G4 structures, and other secondary or tertiary structures These advanced RNA structures can replace the 50 -cap of mRNA and recruit ribosomes to initiate the translation process [12,13]. The 50 UTR of VEGFA is G-rich and harbors two separate IRES sites (IRES-A at 749–1038 nt and IRES-B at 91–554 nt) that facilitate VEGFA translation initiation in a cap-independent manner to respond to various cellular stresses [20,21]. RBM4 has been reported to inhibit cap-dependent translation but promote the IRES-mediated translation under cellular stress [24,26]. This study takes IRES-A of VEGFA as a target gene to explore whether RBM4 can promote the IRES-mediated mRNA translation by binding the G4 structure in the IRES element. The results revealed that RBM4 favors IRES-mediated translation as a G4 binding protein
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