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Characterization and prediction of protein nucleolar localization sequences.

ABSTRACT: Although the nucleolar localization of proteins is often believed to be mediated primarily by non-specific retention to core nucleolar components, many examples of short nucleolar targeting sequences have been reported in recent years. In this article, 46 human nucleolar localization sequences (NoLSs) were collated from the literature and subjected to statistical analysis. Of the residues in these NoLSs 48% are basic, whereas 99% of the residues are predicted to be solvent-accessible with 42% in ?-helix and 57% in coil. The sequence and predicted protein secondary structure of the 46 NoLSs were used to train an artificial neural network to identify NoLSs. At a true positive rate of 54%, the predictor's overall false positive rate (FPR) is estimated to be 1.52%, which can be broken down to FPRs of 0.26% for randomly chosen cytoplasmic sequences, 0.80% for randomly chosen nucleoplasmic sequences and 12% for nuclear localization signals. The predictor was used to predict NoLSs in the complete human proteome and 10 of the highest scoring previously unknown NoLSs were experimentally confirmed. NoLSs are a prevalent type of targeting motif that is distinct from nuclear localization signals and that can be computationally predicted.

SUBMITTER: Scott MS

PROVIDER: S-EPMC2995072 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Characterization and prediction of protein nucleolar localization sequences.

Scott Michelle S MS Boisvert François-Michel FM McDowall Mark D MD Lamond Angus I AI Barton Geoffrey J GJ

Nucleic acids research 20100726 21

Although the nucleolar localization of proteins is often believed to be mediated primarily by non-specific retention to core nucleolar components, many examples of short nucleolar targeting sequences have been reported in recent years. In this article, 46 human nucleolar localization sequences (NoLSs) were collated from the literature and subjected to statistical analysis. Of the residues in these NoLSs 48% are basic, whereas 99% of the residues are predicted to be solvent-accessible with 42% in ...[more]

PMID: 20663773

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Project description:Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis, we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL), from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleoli of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleoli of transfected cells. Multiple-point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed; several NoLS-mutated LEF5 proteins were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production, and occlusion body production.IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleoli of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

Dataset Information

Characterization and prediction of protein nucleolar localization sequences.

Publications

Characterization and prediction of protein nucleolar localization sequences.

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