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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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Subnuclear distribution of exogenously-expressed S-Ilf3, L-Ilf3 and L-NF90 isoforms in HeLa cells.Plasmids pEGFP-N1 (Control row), pEGFP-N1-S-Ilf3 (S-Ilf3 row), pEGFP-N1-L-Ilf3 (L-Ilf3 row) or pEGFP-N1-L-NF90 (L-NF90 row) were transfected into HeLa cells. After 24 hours, cells were co-stained with human anti-fibrillarin serum (Fibrillarin) and monoclonal anti-B23 antibody (B23). After confocal microscopy acquisition, focal planes were chosen to obtain optimal fibrillarin and B23 signals. DAPI (not shown here) was used to define the nuclear limits (white drawings). GFP and GFP fusion proteins appear in green, fibrillarin in red and B23 in blue.
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pone-0022296-g006: Subnuclear distribution of exogenously-expressed S-Ilf3, L-Ilf3 and L-NF90 isoforms in HeLa cells.Plasmids pEGFP-N1 (Control row), pEGFP-N1-S-Ilf3 (S-Ilf3 row), pEGFP-N1-L-Ilf3 (L-Ilf3 row) or pEGFP-N1-L-NF90 (L-NF90 row) were transfected into HeLa cells. After 24 hours, cells were co-stained with human anti-fibrillarin serum (Fibrillarin) and monoclonal anti-B23 antibody (B23). After confocal microscopy acquisition, focal planes were chosen to obtain optimal fibrillarin and B23 signals. DAPI (not shown here) was used to define the nuclear limits (white drawings). GFP and GFP fusion proteins appear in green, fibrillarin in red and B23 in blue.

Mentions: To better define the subnucleolar localization of long isoforms, the GFP sequence was fused in frame after the S-Ilf3, L-Ilf3 or L-NF90 sequences. Constructs were transfected into HeLa cells and after 24 hours cells were processed for indirect double immunofluorescence with human anti-fibrillarin serum and anti-B23 monoclonal antibodies. The intranuclear distribution of GFP and markers was then observed by confocal microscopy (Figure 6).


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)

Subnuclear distribution of exogenously-expressed S-Ilf3, L-Ilf3 and L-NF90 isoforms in HeLa cells.Plasmids pEGFP-N1 (Control row), pEGFP-N1-S-Ilf3 (S-Ilf3 row), pEGFP-N1-L-Ilf3 (L-Ilf3 row) or pEGFP-N1-L-NF90 (L-NF90 row) were transfected into HeLa cells. After 24 hours, cells were co-stained with human anti-fibrillarin serum (Fibrillarin) and monoclonal anti-B23 antibody (B23). After confocal microscopy acquisition, focal planes were chosen to obtain optimal fibrillarin and B23 signals. DAPI (not shown here) was used to define the nuclear limits (white drawings). GFP and GFP fusion proteins appear in green, fibrillarin in red and B23 in blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022296-g006: Subnuclear distribution of exogenously-expressed S-Ilf3, L-Ilf3 and L-NF90 isoforms in HeLa cells.Plasmids pEGFP-N1 (Control row), pEGFP-N1-S-Ilf3 (S-Ilf3 row), pEGFP-N1-L-Ilf3 (L-Ilf3 row) or pEGFP-N1-L-NF90 (L-NF90 row) were transfected into HeLa cells. After 24 hours, cells were co-stained with human anti-fibrillarin serum (Fibrillarin) and monoclonal anti-B23 antibody (B23). After confocal microscopy acquisition, focal planes were chosen to obtain optimal fibrillarin and B23 signals. DAPI (not shown here) was used to define the nuclear limits (white drawings). GFP and GFP fusion proteins appear in green, fibrillarin in red and B23 in blue.
Mentions: To better define the subnucleolar localization of long isoforms, the GFP sequence was fused in frame after the S-Ilf3, L-Ilf3 or L-NF90 sequences. Constructs were transfected into HeLa cells and after 24 hours cells were processed for indirect double immunofluorescence with human anti-fibrillarin serum and anti-B23 monoclonal antibodies. The intranuclear distribution of GFP and markers was then observed by confocal microscopy (Figure 6).

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