Nuclear and nucleolar targeting of human ribosomal protein S25: Common features shared with HIV-1 regulatory proteins

Abstract

The nuclear and nucleolar targeting properties of human ribosomal protein S25 (RPS25) were analysed by the expression of epitope-tagged RPS25 cDNAs in Cos-1 cells. The tagged RPS25 was localized to the cell nucleus, with a strong predominance in the nucleolus. At the amino terminus of RPS25, two stretches of highly basic residues juxtapose. This con®guration shares common features with the nucleolar targeting signals (NOS) of lentiviral RNA-binding transactivators, including human immunode®ciency viruses' (HIV) Rev proteins. Deletion and site-directed mutational analyses demonstrated that the ®rst NOS-like stretch is dispensable for both nuclear and nucleolar localization of RPS25, and that the nuclear targeting signal is located within the second NOS-like stretch. It has also been suggested that a set of continuous basic residues and the total number of basic residues should be required for nucleolar targeting. Signal-mediated nuclear/nucleolar targeting was further characterized by the construction and expression of a variety of chimeric constructs, utilizing three dierent backbones with RPS25 cDNA fragments. Immuno¯uorescence analyses demonstrated a 17 residue peptide of RPS25 as a potential nuclear/ nucleolar targeting signal. The identi®ed peptide signal may belong to a putative subclass of NOS, characterized by compact structure, together with lentiviral RNAbinding transactivators.

Phosphorylation-dependent Regulation of Unique Nuclear and Nucleolar Localization Signals of LIM Kinase 2 in Endothelial Cells

“…Aligning the LIMK2 NoLS with the published NoLS sequences (generally 20 -40 amino acids long) (30 -32) revealed that all these sequences retain continuous stretches of basic residues; either one continuous stretch of three to four basic residues or one to three stretches of three to four basic residues interrupted by one non-basic residue (Table 4). However, each of these basic amino acids clusters was shown to be important but not sufficient for nucleolar localization of the protein (30,31). We also found by site-directed mutagenesis that each of the basic amino acid clusters (aa 491-503) was important for nucleolar localization of the kinase domain of LIMK2.…”

The transcriptional corepressor MTG16a contains a novel nucleolar targeting sequence deranged in t (16; 21)-positive myeloid malignancies

“…We were not able to identify any known, or novel, nucleolar localization signal in the amino acid sequence crucial for nucleolar targeting (Kubota et al, 1999;Siomi et al, 1988;Tamanini et al, 2000). Thus the targeting of MTG16a to the nucleolus may implicate additional mechanisms, like specific protein folding of the N-terminal region, or the intervention of other nucleolar proteins, as is the case for the nucleolar targeting of nucleolin, which also lacks a nucleolar signal (Schmidt-Zachmann and Nigg, 1993).…”

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions

“…The resultant chimeric construct was entitled pGL3SA2. The third 3'-deletion fragment was acquired via polymerase chain reaction (PCR) with Pfu DNA polymerase (Stratagene, La Jolla, CA, USA), under the reaction cycles that had been described previously (Kubota et al, 1999a). The sense primer for the ampli®cation was described as CT3UTRS in a previous report (Kubota et al, 1999b).…”

“…The previouslydescribed CT3UTA was utilized as an anti-sense primer herein. Ampli®cation was performed under established conditions (Kubota et al, 1999a). The ampli®ed fragment was digested by XbaI and EcoRI, puri®ed, and cloned between the corresponding sites in pGL3L(+) to yield pGL3SS2.…”