An integrated model for the nucleo-cytoplasmic transport of cytoplasmic poly(A)-binding proteins

Abstract

Cytoplasmic poly(A)-binding proteins (PABPs) regulate mRNA stability and translation. Although predominantly localized in the cytoplasm, PABP proteins also cycle through the nucleus. Recent work has established that their steady-state localization can be altered by cellular stresses such as ultraviolet (UV) radiation, and infection by several viruses, resulting in nuclear accumulation of PABPs. Here, we present further evidence that their interaction with and release from mRNA and translation complexes are important in determining their sub-cellular distribution and propose an integrated model for regulated nucleo-cytoplasmic transport of PABPs.

Keywords: PABP; PABPC1; PABPC4; RNA-binding protein; cellular stress response; nucleo-cytoplasmic transport; sub-cellular localization; translation initiation factor.

Figure 1. Heat shock results in PABP1 relocalization to stress granules but not the nucleus. HeLa cells were incubated at 44°C for the indicated time and fixed. Poly(A)-RNA (red) and PABP1 (green) were detected by FISH using an oligo dT₄₀ probe and immunofluorescence, respectively. Stress granules are visible after 1 h treatment as cytoplasmic foci containing both PABP1 and poly(A) RNA. Scale bars: 20 µm.

Figure 2. Pre-treatment with cycloheximide limits nuclear relocalization of PABP1. HeLa cells were pretreated with cycloheximide (CX, 100 μg/ml) for 30 min prior to and during treatment with (A) sodium arsenite (0.5 mM; 1 h) or (B) actinomycin D (Act D, 5 μg/ml; 12 h). Appropriate vehicle control was applied to non-treated cells. Poly(A)-RNA (red) and PABP1 (green) were detected by oligo dT₄₀ FISH and immunofluorescence. Scale bars: 20 µm.

Figure 3. A model for PABP nucleo-cytoplasmic transport. In normally growing cells most molecules of PABP1 and PABP4 are located within the cytoplasm. PABP nuclear import is dependent on interaction with α-importins. This can be retarded by PABP binding to RNA and participation in translational complexes, and may also be affected by interaction with other PABP partner proteins. In the nucleus PABP1 and PABP4 bind mRNAs alongside PABPN1. PABP1 and PABP4 do not contribute to mRNA export but rather are exported in an mRNA-dependent manner. Enhancing the levels of PABP1 available for import or blocking mRNA export results in an accumulation of PABP1 in the nucleus.

Figure 4. PABP1 and PABP4 are present in both polysomal and non-polysomal fractions. Post-nuclear HeLa extracts were sedimented through 10–50% sucrose gradients and fractionated. Proteins from the fractions were TCA precipitated prior to western blotting for PABP1 and PABP4. Cycloheximide (150 μg/ml) or EDTA (20 mM) was added to the lysis buffer to stabilize or disassemble polysomes respectively. Absorbance traces at 254nm were used to identify polysome, 80S, 60/40S and mRNP-containing fractions.

Figure 5. Nuclear export of PABP1 and PABP4 is not prevented by the CRM1 inhibitor leptomycin B. HeLa cells were treated with 5 ng/ml leptomycin B (LMB) or vehicle control for 3 h prior to fixation and immunofluorescence detection of paxillin (red) and (A) PABP1 or (B) PABP4 (green). Scale bars: 20 µm.