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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 GFP fused with the short or long N-terminal sequence of Ilf3/NF90.Plasmids pEGFP-N1 (Control, left panels), pEGFP-N1-Ilf3/NF90 common N-terminal short sequence (Short-GFP, mid panels) and pEGFP-N1-Ilf3/NF90 common N-terminal long sequence (Long-GFP, right panels) were transfected into HeLa cells. After 24 hours, cells were fixed and co-stained with anti-α-tubulin antibody (α-Tub) and DAPI. GFP or GFP fusion proteins appear in green, α-tubulin in red and DAPI staining in blue. Arrows point to intranuclear foci.
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pone-0022296-g001: Subcellular localization of GFP fused with the short or long N-terminal sequence of Ilf3/NF90.Plasmids pEGFP-N1 (Control, left panels), pEGFP-N1-Ilf3/NF90 common N-terminal short sequence (Short-GFP, mid panels) and pEGFP-N1-Ilf3/NF90 common N-terminal long sequence (Long-GFP, right panels) were transfected into HeLa cells. After 24 hours, cells were fixed and co-stained with anti-α-tubulin antibody (α-Tub) and DAPI. GFP or GFP fusion proteins appear in green, α-tubulin in red and DAPI staining in blue. Arrows point to intranuclear foci.

Mentions: In control cells expressing GFP alone as in cells transfected with the Short-GFP plasmids, fluorescence was observed in the cytoplasm and especially in the nucleus, where GFP has been previously shown to accumulate (Figure 1, left and middle panels). In contrast, fluorescence was barely detectable in the cytoplasm of cells transfected with the Long-GFP plasmids and rather accumulated in discrete nuclear foci that may correspond to nucleoli (Figure 1, right panels). This 13-aa sequence could thus function as an efficient nucleolar localization signal (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 localization of GFP fused with the short or long N-terminal sequence of Ilf3/NF90.Plasmids pEGFP-N1 (Control, left panels), pEGFP-N1-Ilf3/NF90 common N-terminal short sequence (Short-GFP, mid panels) and pEGFP-N1-Ilf3/NF90 common N-terminal long sequence (Long-GFP, right panels) were transfected into HeLa cells. After 24 hours, cells were fixed and co-stained with anti-α-tubulin antibody (α-Tub) and DAPI. GFP or GFP fusion proteins appear in green, α-tubulin in red and DAPI staining in blue. Arrows point to intranuclear foci.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022296-g001: Subcellular localization of GFP fused with the short or long N-terminal sequence of Ilf3/NF90.Plasmids pEGFP-N1 (Control, left panels), pEGFP-N1-Ilf3/NF90 common N-terminal short sequence (Short-GFP, mid panels) and pEGFP-N1-Ilf3/NF90 common N-terminal long sequence (Long-GFP, right panels) were transfected into HeLa cells. After 24 hours, cells were fixed and co-stained with anti-α-tubulin antibody (α-Tub) and DAPI. GFP or GFP fusion proteins appear in green, α-tubulin in red and DAPI staining in blue. Arrows point to intranuclear foci.
Mentions: In control cells expressing GFP alone as in cells transfected with the Short-GFP plasmids, fluorescence was observed in the cytoplasm and especially in the nucleus, where GFP has been previously shown to accumulate (Figure 1, left and middle panels). In contrast, fluorescence was barely detectable in the cytoplasm of cells transfected with the Long-GFP plasmids and rather accumulated in discrete nuclear foci that may correspond to nucleoli (Figure 1, right panels). This 13-aa sequence could thus function as an efficient nucleolar localization signal (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