Identification of internal ribosome entry segment (IRES)-trans-acting factors for the Myc family of IRESs

Abstract

The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.

FIG. 1.

PSF, p54nrb, GRSF-1, and YB-1 bind specifically to the myc IRESs. (Ai) Biotinylated RNAs were incubated with HeLa cell extracts, and protein-RNA complexes were isolated on streptavidin magnetic beads. Proteins were separated by PAGE and identified by LC-MS-MS. (Aii) Proteins were subcloned and overexpressed in E. coli. A polyacrylamide gel of the purified proteins used in subsequent experiments is shown. (B) UV cross-linking assays were performed using radiolabeled c-, L- or N-myc IRES RNAs, which were incubated with 0.2 μg of YB-1 (i), p54nrb (ii), PSF (iii), PTB (iv), or GRSF-1 (v) in the presence/absence of unlabeled GAPDH RNA or IRES RNA. Products were separated on a 10% polyacrylamide gel. In all cases, unlabeled IRES RNA competed for binding, while no competition was observed with GAPDH RNA, even at a 10-fold (10×) molar excess.

FIG. 2.

PSF, p54nrb, GRSF-1, and YB-1 stimulate myc IRESs in vitro. (A) Schematic representation of the dicistronic reporter constructs pRF and pRIRESF, where pRIRESF contains the c-myc (pRMF), L-myc (pRLsF), N-myc (pRNF), BAG-1 (pRBF), or Apaf-1 (pRAF) 5′ UTRs inserted into the vector pRF and fused in frame with the firefly luciferase gene. SV40 prom, simian virus 40 promoter; luc, luciferase. (B and C) In vitro translation reactions in reticulocyte lysates primed with 100 ng of capped pRIRESF RNA and 50 to 200 ng of PSF, p54nrb, GRSF-1, YB-1, or PTB (see Fig. S1 in the supplemental material) show that these proteins can stimulate IRES activity from these dicistronic plasmids, albeit to different extents. The greatest effects were observed with the addition of YB-1 and p54nrb. IRES activity is expressed as a ratio of the downstream cistron to the upstream cistron (firefly/Renilla luciferase). All experiments were performed in triplicate on three independent occasions.

FIG. 3.

Reductions in the expression of p54nrb, PSF, YB-1, and GRSF-1 correlate with decreases in IRES activity in cultured cells. Cells were transfected with plasmids that reduce the expression of p54nrb, PSF, GRSF-1, or YB-1 and then transfected with the plasmids harboring the c-, L-, or N-myc (pRMF, pRLsF, pRNF, respectively); Apaf-1 (pRAF); or BAG-1 (pRBF) IRES. Cells were harvested and lysed, and a proportion of the resulting material was immunoblotted using antibodies against small interfering YB-1 (si-YB1) (A), p54nrb (B), PSF (C), or GRSF-1 (D); actin was used as a loading control (Con). In each case, the level of the protein was reduced to less than 20% of the value in the control cells. The samples were then assayed for firefly and Renilla luciferase activities, and the relative IRES activity was calculated as firefly activity relative to the transfection control β-galactosidase. All experiments were carried out in triplicate and performed on at least three independent occasions. All three myc IRESs were affected by a reduction in the levels of these four proteins, although the greatest decrease was observed when the level of YB-1 was reduced, causing the activity of all three IRESs to be reduced by 95%. In contrast, only a small reduction in Apaf-1 IRES activity was observed in cells with a decreased expression of p54nrb, and no effect was observed in BAG-1 IRES activity.

FIG. 4.

Endogenous c-Myc protein levels are reduced in cells that have decreased expression of p54nrb, GRSF-1, or YB-1. (A) Cells were transfected with a control plasmid or a plasmid that reduces the expression of YB-1 (i), p54nrb (p54) (ii), PSF (iii), or GRSF-1 (iv). Cells were harvested, and the subsequent lysates separated by SDS-PAGE and immunoblotted for the proteins indicated. c-Myc protein levels are reduced in cells that have low levels of YB-1, p54nrb, or GRSF-1 but not PSF. In contrast, BAG-1 protein levels (including the p36 isoform which can be translated by internal ribosome entry) are unaffected, and Apaf-1 protein levels are decreased only in cells with a reduction of p54nrb. (B) RNA was generated from cells transfected with plasmids which reduce the expression of p54nrb (p54), YB-1, and GRSF-1. The RNA was separated by denaturing agarose gel electrophoresis and transferred to nitrocellulose. This was then probed with radiolabeled DNA generated from GAPDH or c-myc coding regions. These Northern analyses show that the level of c-myc RNA is not reduced.

FIG. 5.

A reduction in YB-1 and p54nrb levels alters the association of c-myc mRNA with the polysomes. Cells were transfected with plasmids that reduce the expression of YB-1, p54nrb, or GRSF-1, and postnuclear extracts were separated on 10 to 55% sucrose density gradients. No overall changes in the polysome profiles were observed. RNA was generated from the individual fractions, and Northern analysis was performed using probes to c-myc and actin mRNA. When levels of YB-1 are reduced, there is a relocation of the c-myc mRNA from the polysomal region to the subpolysome particles (A). In cells with reduced expression of p54nrb, there is again a reduced polysomal location of c-myc mRNA (B), but no difference was observed in cells with a decreased expression of GRSF-1 (C). (D) To test the effect of the reduction in the levels of ITAFs on cell proliferation rates, the levels of these proteins were reduced as described above and cells were incubated with [³H]thymidine for 24 h and counted.