The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target

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Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5′ UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.</div

Cell lines used in this study and the method of engineering.

Cell lines used in this study and the method of engineering.</p

EIF3A HLH mutation causes transcript-specific defects in initiation factor recruitment.

A, B. Representative sucrose gradient profile of in vitro translation reactions stalled with cycloheximide or GMPPNP and programmed with full length mRNA. Fractions were tracked by absorbance at 254 nm as shown, with the top of the gradient on the left. F: free RNA/top of gradient, 40S: 40S subunit, M: 60S/middle of gradient, 80S: 80S ribosome, E: end of gradient. Traces for exogenous WT EIF3A (CT, black) and for mutant HLH EIF3A (HLH, red) are shown. C, D. Representative western blot analysis of initiation factor distribution in translation reactions stalled with cycloheximide. Protein quantification normalized to RPS19 loading control and relative amounts indicated below corresponding bands. EIF4A1 was normalized to both RPS19 in the respective 40S fraction and to EIF2α, which gave equivalent ratios. E, F. Representative western blot analysis of initiation factor distribution in translation reactions stalled with GMPPNP. Protein quantification normalized as described in (C, D) and relative amounts indicated below corresponding bands. Boxes indicate fractions with decreased levels of initiation factors of interest.</p

EIF3A HLH motif selectively interacts with mRNA 5′ UTR elements.

A. In vitro translation of HCV IRES and MYC 5′ UTR Renilla mRNAs in CT and HLH* extracts. The schematic on the right shows the features and design of the mRNAs used. Globin HBB 5′ UTR Firefly mRNA was used as a control. ***P In vitro translation of HCV IRES and MYC 5′ UTR luciferase mRNAs in the presence of EIF4A1 inhibitor RocA. *P P P In vitro translation reactions programmed with full length GAPDH and MYC mRNAs inhibited with both GMPPNP and RocA and fractionated on 10–25% sucrose gradients, as in Fig 2. Traces for exogenous WT EIF3A (CT, black) and for mutant HLH EIF3A (HLH, red) are shown. D and E. Western blotting of the sucrose gradient fractions, with boxes indicating fractions of interest for EIF4A1 levels. Protein quantification normalized to RPS19 loading control and relative amounts indicated below corresponding bands. Asterisk indicates background signal in gel, which does not interfere with initiation factor distribution analysis as there is no signal to obscure (see S4 Fig panel C in S1 File). F. EIF3A HLH* motif effects on RocA-mediated repression. The schematic on the right represents the ATF4 uORF variant 5′ UTRs (WT, ΔuORF1, ΔuORF2) fused to the Renilla luciferase ORF for transfection into CRISPRi CT and HLH* cells. Live cell transfection was performed instead of in vitro translation in order to observe the effect of RocA stress. Relative Luciferase Units (RLU) percentage was normalized to internal globin HBB 5′ UTR Firefly mRNA control signal. *PPP<0.0001, Student’s t-test. Data represent mean ± S.D., n = 3 replicates.</p

S1 Raw images -

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5′ UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.</div

EIF3A HLH mutation sensitizes Burkitt’s lymphoma cells through loss of MYC.

A. Western blot analysis of HLH* sensitive proteins in Ramos shRNA lentiviral cell lines. Levels of protein normalized to ACTB control given below gels. Asterisk notes that no detectable PRL3 was found in Ramos cells. B. CT and HLH* Ramos cells cultured in the presence of increasing concentrations of RocA. Data represent mean ± S.D., n = 3 replicates. Some error bars are not visible due to being smaller than the data marker. C. Doxorubicin treatment of Ramos CT (R-CT) and HLH* (R-HLH) cell lines. Data represent mean ± S.D., n = 3 replicates. Some error bars are not visible due to being smaller than the data marker.</p

Model of EIF3A dynamic interactions with EIF4A1 and variable mRNAs.

A. Schematic of EIF4A1 and eIF3 interacting with the mRNA at the entry and exit mRNA sites of the 40S ribosomal subunit, respectively. B. Schematic representing the displacement of EIF3 HLH* from the 40S subunit that leads to general defects in initiation factor recruitment and mRNA translation. C. Schematic representing the displacement of EIF3 HLH* from the 40S subunit in the presence of select transcripts, such as MYC, and the concomitant displacement of EIF4A1 at the entry site.</p

S1 File -

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5′ UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.</div