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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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Subcellular localization of human eRF1 fused with short or long N-terminal sequence of Ilf3/NF90.Plasmids pCMV-heRF1-Ilf3/NF90 short N-terminal sequence (N-heRF1, mid panels) and pCMV-heRF1-Ilf3/NF90 long N-terminal sequence (NoLS-heRF1, lower panels) were transfected into HeLa cells. After 24 hours, untransfected (Control, upper panels) or transfected cells were co-stained with anti-heRF1 antibody (heRF1), anti-α-tubulin antibody (α-Tub) and DAPI. Endogenous heRF1 or heRF1 recombinant fusion proteins appear in green, α-tubulin in red and DAPI in blue. Arrows point to intranuclear foci corresponding to nucleoli.
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pone-0022296-g004: Subcellular localization of human eRF1 fused with short or long N-terminal sequence of Ilf3/NF90.Plasmids pCMV-heRF1-Ilf3/NF90 short N-terminal sequence (N-heRF1, mid panels) and pCMV-heRF1-Ilf3/NF90 long N-terminal sequence (NoLS-heRF1, lower panels) were transfected into HeLa cells. After 24 hours, untransfected (Control, upper panels) or transfected cells were co-stained with anti-heRF1 antibody (heRF1), anti-α-tubulin antibody (α-Tub) and DAPI. Endogenous heRF1 or heRF1 recombinant fusion proteins appear in green, α-tubulin in red and DAPI in blue. Arrows point to intranuclear foci corresponding to nucleoli.

Mentions: Since GFP can enter the nucleus, we tested the ability of the 13-aa motif to target to the nucleus and nucleolus a well-known cytoplasmic protein, the human eukaryotic releasing factor 1 (heRF1). The heRF1 coding sequence was fused at the 3′ end of the sequence coding for the first 54 aa of the L-Ilf3/NF90 isoforms (NoLS-heRF1) or for the first 41 aa of the S-Ilf3/NF90 isoforms (N-heRF1). The constructs were transfected into HeLa cells and the heRF1 localization was observed by immunofluorescence microscopy (Figure 4).


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 localization of human eRF1 fused with short or long N-terminal sequence of Ilf3/NF90.Plasmids pCMV-heRF1-Ilf3/NF90 short N-terminal sequence (N-heRF1, mid panels) and pCMV-heRF1-Ilf3/NF90 long N-terminal sequence (NoLS-heRF1, lower panels) were transfected into HeLa cells. After 24 hours, untransfected (Control, upper panels) or transfected cells were co-stained with anti-heRF1 antibody (heRF1), anti-α-tubulin antibody (α-Tub) and DAPI. Endogenous heRF1 or heRF1 recombinant fusion proteins appear in green, α-tubulin in red and DAPI in blue. Arrows point to intranuclear foci corresponding to nucleoli.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022296-g004: Subcellular localization of human eRF1 fused with short or long N-terminal sequence of Ilf3/NF90.Plasmids pCMV-heRF1-Ilf3/NF90 short N-terminal sequence (N-heRF1, mid panels) and pCMV-heRF1-Ilf3/NF90 long N-terminal sequence (NoLS-heRF1, lower panels) were transfected into HeLa cells. After 24 hours, untransfected (Control, upper panels) or transfected cells were co-stained with anti-heRF1 antibody (heRF1), anti-α-tubulin antibody (α-Tub) and DAPI. Endogenous heRF1 or heRF1 recombinant fusion proteins appear in green, α-tubulin in red and DAPI in blue. Arrows point to intranuclear foci corresponding to nucleoli.
Mentions: Since GFP can enter the nucleus, we tested the ability of the 13-aa motif to target to the nucleus and nucleolus a well-known cytoplasmic protein, the human eukaryotic releasing factor 1 (heRF1). The heRF1 coding sequence was fused at the 3′ end of the sequence coding for the first 54 aa of the L-Ilf3/NF90 isoforms (NoLS-heRF1) or for the first 41 aa of the S-Ilf3/NF90 isoforms (N-heRF1). The constructs were transfected into HeLa cells and the heRF1 localization was observed by immunofluorescence microscopy (Figure 4).

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