Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis

Abstract

The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

Fig. 1.

Expression of an Rbm3 fusion protein increases protein synthesis in N2a cells. (A) Immunoblot analysis of Rbm3 in control and c-Myc-Rbm3 cells. Cells were grown continuously at 37°C or shifted to 32°C for 24 h. β-actin was used as a loading control. (B) Incorporation of [³⁵S]Met/cysteine into control and c-Myc-Rbm3 cells. Data are the mean of four experiments ± SD. Measurements were normalized with respect to total protein as determined by Bradford assay.

Fig. 3.

Rbm3 and c-Myc-Rbm3 proteins associate with ribosomes. (A) Control or c-Myc-Rbm3 cells were grown at 37°C or shifted to 32°C for 24 h. Postmitochondrial fractions from these cells were centrifuged at 100,000 × g for 3 h. The supernatant (S100) and pellet (P100) fractions were Western blotted by using anti-Rbm3 antibodies. (B) Rbm3 coimmunoprecipitates with rat brain ribosomes. We immunoprecipitated 13 A₂₆₀ units (a) or 130 A₂₆₀ units (b) of polysome-enriched fractions of rat brain by using anti-Rbm3 antibodies. The immunoprecipitates and the supernatants of the polysomal materials were analyzed for the presence of Rbm3 and ribosomal protein L4 by Western blotting by using anti-Rbm3 and anti-L4 antibodies, respectively. IgG was used in control experiments.

Fig. 2.

Expression of the c-Myc-Rbm3 fusion protein increases monosome and polysome formation during mild hypothermia. Control (A) or c-Myc-Rbm3 (B) cells were cultured at 32°C for 24 h, and ribosomes were analyzed after centrifugation through 10–50% sucrose gradients. The top of the gradient is on the left. The positions of free small (40S) and large (60S) ribosomal subunits, monosomes (80S), polysomes, and stalled preinitiation complexes (halfmers) are indicated. An asterisk indicates the position of a complex that sediments between the top of the gradient and 40S subunits.

Fig. 4.

Rbm3 associates with translating ribosomes in an RNA-independent manner. (A) A fraction of Rbm3 copurifies with rat brain ribosomes on sucrose gradients. A polysome-enriched fraction of rat brain was centrifuged through a linear 10–50% (wt/vol) sucrose gradient. The top of the gradient is on the left. (B) Ribosomal profile of a rat brain polysome-enriched fraction after treatment with RNase A. For both A and B, the distributions of Rbm3 and ribosomal protein L4 in the fractions of the gradient were analyzed by immunoblotting using anti-Rbm3 and anti-L4 antibodies, respectively.

Fig. 5.

Rbm3 associates tightly with the large ribosomal subunit. (A) Sucrose gradient profiles of rat brain ribosomal subunits treated with 0.5 M KCl. We used 18S and 28S rRNA, isolated from the pooled 60S and 40S fractions, to confirm the purity of the 40S and 60S subunits, respectively. (B) Immunoblot analysis of 60S and 40S subunits from rat brain, reticulocyte, C6 glioma, and N2a neuroblastoma was performed by using anti-Rbm3 antibodies. (C) Purified 60S subunits were treated with 0.9 M KCl in the presence or absence of 10 mM EDTA and subjected to 24-h centrifugation at 100,000 × g to obtain a pellet containing 60S subunits and supernatant fractions. The presence of Rbm3 in these fractions was assessed by immunoblotting using anti-Rbm3 antibodies.

Fig. 6.

Expression of an Rbm3 fusion protein alters the abundance of a miRNA-containing complex. (A) RNA was extracted from fractions of a sucrose gradient profile of control cells at 32°C and stained with ethidium bromide. Lane 1 is RNA from the top of the gradient, and lane 2 is RNA from the complex that sediments between the top of the gradient and 40S subunits (indicated with an asterisk in Fig. 2 A). The positions of RNA size markers are indicated. (B) RNAs from fractions of a sucrose gradient profile of control cells at 32°C were phosphatase treated, [γ-³²P]ATP end-labeled, and separated in acrylamide–urea gels. RNAs in lanes 1–5 are from sequential fractions from the top of the gradient to the bottom. Lane 2 corresponds to the fraction represented in A, lane 2. (C) Northern blot analysis of fractions containing small RNAs from control (C) and c-Myc-Rbm3 (Rbm3) cells grown at 32°C by using a microRNA (mmu-miR-125b) probe. (D) Northern blot analysis of total RNA from control (C) and c-Myc-Rbm3 (Rbm3) cells grown at 32 or 37°C by using the mmu-miR-125b probe. The hybridization signals for C and D correspond to RNAs between the 20- and 30-nt size markers.