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L-Ilf3 and L-NF90 traffic to the nucleolus granular component: alternatively-spliced exon 3 encodes a nucleolar localization motif.

Viranaicken W, Gasmi L, Chaumet A, Durieux C, Georget V, Denoulet P, Larcher JC - PLoS ONE (2011)

Bottom Line: Their heterogeneity results from posttranscriptional and posttranslational modifications.The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli.The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations.

View Article: PubMed Central - PubMed

Affiliation: UPMC Univ Paris 06, UMR 7622, Laboratoire de Biologie du Développement, Paris, France.

ABSTRACT
Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins.

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Related in: MedlinePlus

Subcellular distribution of Ilf3 and NF90 in P19 cells.After subcellular fractionation, proteins from identical percentages of each fraction were submitted to SDS-PAGE, blotted onto nitrocellulose and immunodetected with the serum Ab78 raised against Ilf3 and NF90 (S.E.: short exposure time; L.E.: long exposure time), anti-UBF serum (UBF) or anti-α-tubulin antibody (α-tub.). Molecular weight markers (kDa) are indicated at the right.
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pone-0022296-g002: Subcellular distribution of Ilf3 and NF90 in P19 cells.After subcellular fractionation, proteins from identical percentages of each fraction were submitted to SDS-PAGE, blotted onto nitrocellulose and immunodetected with the serum Ab78 raised against Ilf3 and NF90 (S.E.: short exposure time; L.E.: long exposure time), anti-UBF serum (UBF) or anti-α-tubulin antibody (α-tub.). Molecular weight markers (kDa) are indicated at the right.

Mentions: To confirm the role of the 13-aa motif in nucleolar targeting and to avoid any bias associated with overexpression, the presence of endogenous L- and S-Ilf3/NF90 isoforms was examined in subcellular fractions prepared from P19 cells. Identical results were obtained with HeLa cells (data not shown). Western blotting of different fractions using a polyclonal antibody raised against common regions of the two proteins [1] revealed that the L- and S-Ilf3 isoforms were present in both cytoplasmic and nuclear fractions (Figure 2). In contrast, only the L-NF90 isoforms were found in the nuclear fraction whereas all the NF90 isoforms were recovered in the cytoplasm. Furthermore, when purified nuclei were fractionated into nucleoplasmic and nucleolar fractions, only the L-Ilf3 and L-NF90 isoforms were found associated with the nucleoli. The identity of the different fractions was checked by immunodetecting UBF, as a nucleolar marker, and α-tubulin, as a cytosolic marker. These results strengthen the idea that the N-terminal 13-aa sequence present in the L-Ilf3 and L-NF90 isoforms acts as a potent NoLS.


L-Ilf3 and L-NF90 traffic to the nucleolus granular component: alternatively-spliced exon 3 encodes a nucleolar localization motif.

Viranaicken W, Gasmi L, Chaumet A, Durieux C, Georget V, Denoulet P, Larcher JC - PLoS ONE (2011)

Subcellular distribution of Ilf3 and NF90 in P19 cells.After subcellular fractionation, proteins from identical percentages of each fraction were submitted to SDS-PAGE, blotted onto nitrocellulose and immunodetected with the serum Ab78 raised against Ilf3 and NF90 (S.E.: short exposure time; L.E.: long exposure time), anti-UBF serum (UBF) or anti-α-tubulin antibody (α-tub.). Molecular weight markers (kDa) are indicated at the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139624&req=5

pone-0022296-g002: Subcellular distribution of Ilf3 and NF90 in P19 cells.After subcellular fractionation, proteins from identical percentages of each fraction were submitted to SDS-PAGE, blotted onto nitrocellulose and immunodetected with the serum Ab78 raised against Ilf3 and NF90 (S.E.: short exposure time; L.E.: long exposure time), anti-UBF serum (UBF) or anti-α-tubulin antibody (α-tub.). Molecular weight markers (kDa) are indicated at the right.
Mentions: To confirm the role of the 13-aa motif in nucleolar targeting and to avoid any bias associated with overexpression, the presence of endogenous L- and S-Ilf3/NF90 isoforms was examined in subcellular fractions prepared from P19 cells. Identical results were obtained with HeLa cells (data not shown). Western blotting of different fractions using a polyclonal antibody raised against common regions of the two proteins [1] revealed that the L- and S-Ilf3 isoforms were present in both cytoplasmic and nuclear fractions (Figure 2). In contrast, only the L-NF90 isoforms were found in the nuclear fraction whereas all the NF90 isoforms were recovered in the cytoplasm. Furthermore, when purified nuclei were fractionated into nucleoplasmic and nucleolar fractions, only the L-Ilf3 and L-NF90 isoforms were found associated with the nucleoli. The identity of the different fractions was checked by immunodetecting UBF, as a nucleolar marker, and α-tubulin, as a cytosolic marker. These results strengthen the idea that the N-terminal 13-aa sequence present in the L-Ilf3 and L-NF90 isoforms acts as a potent NoLS.

Bottom Line: Their heterogeneity results from posttranscriptional and posttranslational modifications.The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli.The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations.

View Article: PubMed Central - PubMed

Affiliation: UPMC Univ Paris 06, UMR 7622, Laboratoire de Biologie du Développement, Paris, France.

ABSTRACT
Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins.

Show MeSH
Related in: MedlinePlus