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Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2.

ABSTRACT: MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target ?-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, suggesting that MeCP2 can be retained in the nucleus by its affinity for DNA. Consistent with these findings, we demonstrate that disease progression in a mouse model of RTT is unaffected by an inactivating mutation in the NLS of MeCP2. Taken together, our work reveals an unexpected redundancy between functional domains of MeCP2 in targeting this protein to the nucleus, potentially explaining why NLS-inactivating mutations are rarely associated with disease.

SUBMITTER: Lyst MJ

PROVIDER: S-EPMC6130050 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2.

Lyst Matthew J MJ Ekiert Robert R Guy Jacky J Selfridge Jim J Koerner Martha V MV Merusi Cara C De Sousa Dina D Bird Adrian A

Cell reports 20180801 9

MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target β-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, sugge ...[more]

PMID: 30157418

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Project description:BackgroundThe nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system.ResultsIn this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families.ConclusionThe results of this study reveal that Planctomycetaceae separates slightly from the members of non-planctomycete bacteria but still has substantial differences from fungi, based on the NLS-like motifs and NLS-bearing protein analysis.

Project description:BackgroundPorcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication.ResultsThe PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2.ConclusionsRecovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.

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Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2.

Publications

Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2.

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