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Nuclear import and the evolution of a multifunctional RNA-binding protein.

Rosenblum JS, Pemberton LF, Bonifaci N, Blobel G - J. Cell Biol. (1998)

Bottom Line: Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La.As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins.Our results are consistent with an intermingling of the nuclear import and evolution of La.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021, USA.

ABSTRACT
La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

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La proteins localize to the nucleus in S. cerevisiae, but only Lhp1p, the endogenous S. cerevisiae La, needs Kap108p to do so.  (a) A phylogenetic tree of the ten cloned La proteins is shown, branches I–IV are indicated. The phylogenetic tree was generated by  aligning the NH2-terminal 250 aa of each La protein using ClustalW 1.6 followed by phylogenetic analysis using Phylip (the aligned sequences are available at http://129.85.13.212/LAS.aln). This region contains the La domain and an RRM for each protein. Species abbreviations and references for cDNA sequencing are as follows: Mm, Mus musculus (Topfer et al., 1993); Rn, Rattus norvegicus (Semsei et  al., 1993); Bs, Bos taurus (Chan et al., 1989); Hs, Homo sapiens (Chambers et al., 1988); Xla and Xlb, Xenopus laevis (Scherly et al.,  1993); Dm, Drosophila melanogaster (Bai et al., 1994); Aa, Aedes albopictus (Pardigon and Strauss, 1996); Sp, Schizosaccharomyces  pombe (Van Horn et al., 1997); Sc, Saccharomyces cerevisiae (Yoo and Wolin, 1994). (b) La proteins from the four main branches of the  phylogenetic tree in Fig. 3 a were expressed as GFP fusions in S. cerevisiae. Each protein (rows) was expressed in both wild-type (left  column) and Δkap108 (right column) cells. Fusion proteins were visualized as described above.
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Figure 3: La proteins localize to the nucleus in S. cerevisiae, but only Lhp1p, the endogenous S. cerevisiae La, needs Kap108p to do so. (a) A phylogenetic tree of the ten cloned La proteins is shown, branches I–IV are indicated. The phylogenetic tree was generated by aligning the NH2-terminal 250 aa of each La protein using ClustalW 1.6 followed by phylogenetic analysis using Phylip (the aligned sequences are available at http://129.85.13.212/LAS.aln). This region contains the La domain and an RRM for each protein. Species abbreviations and references for cDNA sequencing are as follows: Mm, Mus musculus (Topfer et al., 1993); Rn, Rattus norvegicus (Semsei et al., 1993); Bs, Bos taurus (Chan et al., 1989); Hs, Homo sapiens (Chambers et al., 1988); Xla and Xlb, Xenopus laevis (Scherly et al., 1993); Dm, Drosophila melanogaster (Bai et al., 1994); Aa, Aedes albopictus (Pardigon and Strauss, 1996); Sp, Schizosaccharomyces pombe (Van Horn et al., 1997); Sc, Saccharomyces cerevisiae (Yoo and Wolin, 1994). (b) La proteins from the four main branches of the phylogenetic tree in Fig. 3 a were expressed as GFP fusions in S. cerevisiae. Each protein (rows) was expressed in both wild-type (left column) and Δkap108 (right column) cells. Fusion proteins were visualized as described above.

Mentions: As the Kap108p-dependent NLS of Lhp1p did not coincide with the NLS of human La mapped using the Xenopus assay, we undertook an analysis of intracellular targeting of heterologous La proteins in S. cerevisiae. In the course of evolution, four major branches of La proteins have arisen (Fig. 3 a). The clearest diversion in primary structure among these branches occurs between branches I and II and branches III and IV. In diverging from branches I and II, the branch III and IV proteins grew in primary sequence by ∼30%. For example, spLa is 298-aa long whereas dmLa is 390-aa long. The added sequence is most apparent in the COOH-terminal half of the larger proteins. scLa and spLa are 36% identical, spLa and dmLa are 28% identical, and dmLa and hsLa are 34% identical. GFP fusion constructs were generated for each of these proteins. Each construct was transformed into wild-type cells and cells deleted for KAP108. As shown in Fig. 3 b (left column), human, Drosophila, and S. pombe La proteins were targeted to the nucleus when expressed in S. cerevisiae as fusions with GFP. Significantly, all of the fusion proteins were entirely restricted to the nucleus in wild-type cells, indicating that they were substrates of an S. cerevisiae Kap. Surprisingly, this Kap is Kap108p only for Lhp1p, the endogenous S. cerevisiae La protein (Fig. 3 b, right column). Even Sla1, the S. pombe La protein that is 36% identical to Lhp1p, is targeted to the nucleus in the absence of Kap108p. This targeting of Sla1 occurs even though it, like Lhp1p, diverges markedly from the COOH-terminal domain shared by the La proteins of higher eukaryotes (Van Horn et al., 1997). The NLS of the human La resides in precisely this COOH-terminal domain (Simons et al., 1996).


Nuclear import and the evolution of a multifunctional RNA-binding protein.

Rosenblum JS, Pemberton LF, Bonifaci N, Blobel G - J. Cell Biol. (1998)

La proteins localize to the nucleus in S. cerevisiae, but only Lhp1p, the endogenous S. cerevisiae La, needs Kap108p to do so.  (a) A phylogenetic tree of the ten cloned La proteins is shown, branches I–IV are indicated. The phylogenetic tree was generated by  aligning the NH2-terminal 250 aa of each La protein using ClustalW 1.6 followed by phylogenetic analysis using Phylip (the aligned sequences are available at http://129.85.13.212/LAS.aln). This region contains the La domain and an RRM for each protein. Species abbreviations and references for cDNA sequencing are as follows: Mm, Mus musculus (Topfer et al., 1993); Rn, Rattus norvegicus (Semsei et  al., 1993); Bs, Bos taurus (Chan et al., 1989); Hs, Homo sapiens (Chambers et al., 1988); Xla and Xlb, Xenopus laevis (Scherly et al.,  1993); Dm, Drosophila melanogaster (Bai et al., 1994); Aa, Aedes albopictus (Pardigon and Strauss, 1996); Sp, Schizosaccharomyces  pombe (Van Horn et al., 1997); Sc, Saccharomyces cerevisiae (Yoo and Wolin, 1994). (b) La proteins from the four main branches of the  phylogenetic tree in Fig. 3 a were expressed as GFP fusions in S. cerevisiae. Each protein (rows) was expressed in both wild-type (left  column) and Δkap108 (right column) cells. Fusion proteins were visualized as described above.
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Related In: Results  -  Collection

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Figure 3: La proteins localize to the nucleus in S. cerevisiae, but only Lhp1p, the endogenous S. cerevisiae La, needs Kap108p to do so. (a) A phylogenetic tree of the ten cloned La proteins is shown, branches I–IV are indicated. The phylogenetic tree was generated by aligning the NH2-terminal 250 aa of each La protein using ClustalW 1.6 followed by phylogenetic analysis using Phylip (the aligned sequences are available at http://129.85.13.212/LAS.aln). This region contains the La domain and an RRM for each protein. Species abbreviations and references for cDNA sequencing are as follows: Mm, Mus musculus (Topfer et al., 1993); Rn, Rattus norvegicus (Semsei et al., 1993); Bs, Bos taurus (Chan et al., 1989); Hs, Homo sapiens (Chambers et al., 1988); Xla and Xlb, Xenopus laevis (Scherly et al., 1993); Dm, Drosophila melanogaster (Bai et al., 1994); Aa, Aedes albopictus (Pardigon and Strauss, 1996); Sp, Schizosaccharomyces pombe (Van Horn et al., 1997); Sc, Saccharomyces cerevisiae (Yoo and Wolin, 1994). (b) La proteins from the four main branches of the phylogenetic tree in Fig. 3 a were expressed as GFP fusions in S. cerevisiae. Each protein (rows) was expressed in both wild-type (left column) and Δkap108 (right column) cells. Fusion proteins were visualized as described above.
Mentions: As the Kap108p-dependent NLS of Lhp1p did not coincide with the NLS of human La mapped using the Xenopus assay, we undertook an analysis of intracellular targeting of heterologous La proteins in S. cerevisiae. In the course of evolution, four major branches of La proteins have arisen (Fig. 3 a). The clearest diversion in primary structure among these branches occurs between branches I and II and branches III and IV. In diverging from branches I and II, the branch III and IV proteins grew in primary sequence by ∼30%. For example, spLa is 298-aa long whereas dmLa is 390-aa long. The added sequence is most apparent in the COOH-terminal half of the larger proteins. scLa and spLa are 36% identical, spLa and dmLa are 28% identical, and dmLa and hsLa are 34% identical. GFP fusion constructs were generated for each of these proteins. Each construct was transformed into wild-type cells and cells deleted for KAP108. As shown in Fig. 3 b (left column), human, Drosophila, and S. pombe La proteins were targeted to the nucleus when expressed in S. cerevisiae as fusions with GFP. Significantly, all of the fusion proteins were entirely restricted to the nucleus in wild-type cells, indicating that they were substrates of an S. cerevisiae Kap. Surprisingly, this Kap is Kap108p only for Lhp1p, the endogenous S. cerevisiae La protein (Fig. 3 b, right column). Even Sla1, the S. pombe La protein that is 36% identical to Lhp1p, is targeted to the nucleus in the absence of Kap108p. This targeting of Sla1 occurs even though it, like Lhp1p, diverges markedly from the COOH-terminal domain shared by the La proteins of higher eukaryotes (Van Horn et al., 1997). The NLS of the human La resides in precisely this COOH-terminal domain (Simons et al., 1996).

Bottom Line: Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La.As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins.Our results are consistent with an intermingling of the nuclear import and evolution of La.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021, USA.

ABSTRACT
La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

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