Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160

Abstract

Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

Figure 1.

Scp160 is localized to the cytoplasm at steady state. (A) Schematic representation of Scp160 domain structure and location of putative nuclear targeting motifs. The predicted NLS is indicated by the filled diamond and the predicted NES is indicated by the open diamond. The sequences for the predicted NLS and NES motifs as well as their positions within the open reading frame are indicated. Numbered boxes represent the 14 KH domains. (B) The Scp160-GFP protein was expressed in wild-type cells that express a red-fluorescent protein-tagged nuclear rim protein, Mlp1 (56) to mark the position of the nucleus. Fluorescent protein localization was examined by direct fluorescence microscopy. Cells were also stained with Hoechst dye to mark the position of chromatin within the nucleus. A merged fluorescence image is shown as well as the corresponding DIC image. (C) Immunoblot analysis of GFP in protein lysate from wild-type cells transformed with plasmids encoding FLAG-Scp160-GFP (right lane) or a control NLS-NES-GFP (left lane). Migration of protein standards is indicated to the left of the image. The position of the bands corresponding to FLAG-Scp160-GFP and NLS-NES-GFP is indicated.

Figure 2.

Localization of Scp160-GFP in crm1-3 and wild-type cells. The localization of Scp160-GFP was examined in crm1-3 cells following a 2 h shift to 37°C where NES-dependent nuclear export is blocked. Scp160 is localized to the cytoplasm in both wild-type and crm1-1 cells. As controls, we also localized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected, NLS-NES-GFP accumulates in the nucleus of crm1-1 cells, but ΔRGG-Nab2-GFP, which is exported via an mRNA export-dependent pathway, does not. Corresponding DIC images are shown.

Figure 3.

Localization of Scp160-GFP in rpb1-1, mex67-5 and wild-type cells. The localization of Scp160-GFP was examined in wild-type, rpb1-1 and mex67-5 cells following a 1 h shift to 37°C. Results indicates that Scp160 is localized to the cytoplasm in both rpb1-1 and mex67-5 cells. As controls, we also localized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected, ΔRGG-Nab2-GFP, which is exported in an mRNA export-dependent manner, accumulates in the nuclei of both rpb1-1 and mex67-5 cells, but NLS-NES-GFP does not.

Figure 4.

Localization of Scp160-GFP in rat7-1 and wild-type cells. The localization of Scp160-GFP was examined in rat7-1 cells following a 30 min shift to 37°C where all known transport pathways are blocked. As shown, Scp160 is localized to the cytoplasm in rat7-1 cells. As controls, we also visualized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected both control proteins accumulate in the nuclei of rat7-1 cells.

Figure 5.

Functional analysis of Scp160-NLS_mut. (A) An alignment of the predicted NLS sequence from Scp160 with functional NLS sequences from nucleoplasmin and p53 is shown. The lysine residues that were changed to alanines to create Scp160-NLS_mut are indicated by asterisks. (B) To assess the functional importance of the predicted NLS within Scp160, we tested whether an Scp160 variant with a mutant NLS (Scp160-NLS_mut) could replace wild-type Scp160 in Δscp160Δeap1 cells where Scp160 is required for viability (36). Cultures were grown to log phase and equal numbers of cells were serially diluted by orders of magnitude and spotted onto control plates lacking uracil and leucine (left) or test plates lacking leucine but containing 5-FOA (right). The top row shows cells carrying LEU2 vector (pRS315); the middle row shows cells containing a wild-type copy of Scp160; the bottom row shows cells that express Scp160-NLS_mut as the sole copy of Scp160.