Limits...
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.

Show MeSH
Subcellular distribution of exogenously-expressed L-NF90 isoforms in HeLa cells treated with DRB.Plasmids pEGFP-N1-L-NF90 (L-NF90-GFP) and mcherry-B23 (B23-mcherry) were transfected into HeLa cells. 24 hours later, cells were fixed either immediately (Control, upper panels) or after a DRB treatment during two hours (DRB 2 h, mid panels) followed by a chase of one hour (Chase 1 h, lower panels). GFP and mcherry fusion proteins appear in green and red, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3139624&req=5

pone-0022296-g007: Subcellular distribution of exogenously-expressed L-NF90 isoforms in HeLa cells treated with DRB.Plasmids pEGFP-N1-L-NF90 (L-NF90-GFP) and mcherry-B23 (B23-mcherry) were transfected into HeLa cells. 24 hours later, cells were fixed either immediately (Control, upper panels) or after a DRB treatment during two hours (DRB 2 h, mid panels) followed by a chase of one hour (Chase 1 h, lower panels). GFP and mcherry fusion proteins appear in green and red, respectively.

Mentions: When L-NF90-GFP and mcherry-B23 were co-expressed in HeLa cells, both proteins colocalized in the granular component (Figure 7, upper panels), as L-Ilf3 (Figure 5; lower panels). After treatment with DRB, the mcherry fluorescence was greatly reduced whereas the L-NF90-GFP fluorescence diffused in the nucleoplasm (Figure 7, middle panels). One hour after DRB removal, a partial restoration of nucleolar structures was observed. While B23 seemed to rapidly and almost totally localize into the reforming nucleoli, only a part of L-NF90 also relocalized with B23. The remaining L-NF90 was detected at the periphery of reforming nucleoli, around the B23 foci (Figure 7, lower panels). Identical results were obtained with L-Ilf3-GFP (data not shown).


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 exogenously-expressed L-NF90 isoforms in HeLa cells treated with DRB.Plasmids pEGFP-N1-L-NF90 (L-NF90-GFP) and mcherry-B23 (B23-mcherry) were transfected into HeLa cells. 24 hours later, cells were fixed either immediately (Control, upper panels) or after a DRB treatment during two hours (DRB 2 h, mid panels) followed by a chase of one hour (Chase 1 h, lower panels). GFP and mcherry fusion proteins appear in green and red, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022296-g007: Subcellular distribution of exogenously-expressed L-NF90 isoforms in HeLa cells treated with DRB.Plasmids pEGFP-N1-L-NF90 (L-NF90-GFP) and mcherry-B23 (B23-mcherry) were transfected into HeLa cells. 24 hours later, cells were fixed either immediately (Control, upper panels) or after a DRB treatment during two hours (DRB 2 h, mid panels) followed by a chase of one hour (Chase 1 h, lower panels). GFP and mcherry fusion proteins appear in green and red, respectively.
Mentions: When L-NF90-GFP and mcherry-B23 were co-expressed in HeLa cells, both proteins colocalized in the granular component (Figure 7, upper panels), as L-Ilf3 (Figure 5; lower panels). After treatment with DRB, the mcherry fluorescence was greatly reduced whereas the L-NF90-GFP fluorescence diffused in the nucleoplasm (Figure 7, middle panels). One hour after DRB removal, a partial restoration of nucleolar structures was observed. While B23 seemed to rapidly and almost totally localize into the reforming nucleoli, only a part of L-NF90 also relocalized with B23. The remaining L-NF90 was detected at the periphery of reforming nucleoli, around the B23 foci (Figure 7, lower panels). Identical results were obtained with L-Ilf3-GFP (data not shown).

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