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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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The extreme COOH termini of La proteins are key elements of their evolution. (a) The final 35 amino acids of ten La  proteins (for abbreviations see Fig. 3 legend). White on black  background, basic residues; gray on shaded background, acidic  residues; gray, other residues. (b) The final 35 amino acids of  S. pombe and D. melanogaster were each expressed as fusions  with GFP in S. cerevisiae. The fusion constructs were visualized as  previously described.
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Figure 6: The extreme COOH termini of La proteins are key elements of their evolution. (a) The final 35 amino acids of ten La proteins (for abbreviations see Fig. 3 legend). White on black background, basic residues; gray on shaded background, acidic residues; gray, other residues. (b) The final 35 amino acids of S. pombe and D. melanogaster were each expressed as fusions with GFP in S. cerevisiae. The fusion constructs were visualized as previously described.

Mentions: As we had detected a division in the route to the nucleus taken by La proteins of different species, we sought a possible evolutionary explanation. We had mapped the Lhp1p NLS to the central region, overlapping its RRM, whereas the human La NLS resides at the COOH terminus. In Fig. 6 a, we have listed the COOH-terminal 35 amino acids of each of the La proteins cloned to date, highlighting basic residues which are critical for the interaction with Kapα (Conti et al., 1998). Although this stretch from each protein contains several basic residues, scLa and spLa only have one basic residue on the COOH-terminal end of this region, whereas bipartite NLSs generally have three basic residues in their COOH-terminal cluster (Makkerh et al., 1996; Conti et al., 1998). The La proteins from the multicellular eukaryotes have clear bipartite NLSs at their COOH termini and are ∼100 amino acids larger than those from the yeasts (Fig. 6 a, right column). To asses the ability of this region to act as an NLS in the branch II and branch III proteins, we constructed reporter constructs of the last 35 aa of both spLa and dmLa. When expressed in S. cerevisiae, the last 35 aa of dmLa concentrated in the nucleus, with no observable GFP signal in the cytoplasm (Fig. 6 b). This suggests that the last 35 aa of dmLa can mediate its nuclear import, presumably by Kap60p/ Kap95p in S. cerevisiae as a bipartite consensus region begins at Lys-358, 35 aa from the COOH terminus of dmLa. In contrast, the last 35 aa of spLa did not concentrate in the nucleus, possibly due to the insufficiency of the single COOH-terminal basic residue (Fig. 6 b). However, a larger COOH-terminal construct of Sla1, beginning with Met-232 could concentrate in the nucleus (data not shown). This region of Sla1 contains a putative bipartite NLS beginning at Lys-255, which is 44 aa from the COOH terminus.


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

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

The extreme COOH termini of La proteins are key elements of their evolution. (a) The final 35 amino acids of ten La  proteins (for abbreviations see Fig. 3 legend). White on black  background, basic residues; gray on shaded background, acidic  residues; gray, other residues. (b) The final 35 amino acids of  S. pombe and D. melanogaster were each expressed as fusions  with GFP in S. cerevisiae. The fusion constructs were visualized as  previously described.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132966&req=5

Figure 6: The extreme COOH termini of La proteins are key elements of their evolution. (a) The final 35 amino acids of ten La proteins (for abbreviations see Fig. 3 legend). White on black background, basic residues; gray on shaded background, acidic residues; gray, other residues. (b) The final 35 amino acids of S. pombe and D. melanogaster were each expressed as fusions with GFP in S. cerevisiae. The fusion constructs were visualized as previously described.
Mentions: As we had detected a division in the route to the nucleus taken by La proteins of different species, we sought a possible evolutionary explanation. We had mapped the Lhp1p NLS to the central region, overlapping its RRM, whereas the human La NLS resides at the COOH terminus. In Fig. 6 a, we have listed the COOH-terminal 35 amino acids of each of the La proteins cloned to date, highlighting basic residues which are critical for the interaction with Kapα (Conti et al., 1998). Although this stretch from each protein contains several basic residues, scLa and spLa only have one basic residue on the COOH-terminal end of this region, whereas bipartite NLSs generally have three basic residues in their COOH-terminal cluster (Makkerh et al., 1996; Conti et al., 1998). The La proteins from the multicellular eukaryotes have clear bipartite NLSs at their COOH termini and are ∼100 amino acids larger than those from the yeasts (Fig. 6 a, right column). To asses the ability of this region to act as an NLS in the branch II and branch III proteins, we constructed reporter constructs of the last 35 aa of both spLa and dmLa. When expressed in S. cerevisiae, the last 35 aa of dmLa concentrated in the nucleus, with no observable GFP signal in the cytoplasm (Fig. 6 b). This suggests that the last 35 aa of dmLa can mediate its nuclear import, presumably by Kap60p/ Kap95p in S. cerevisiae as a bipartite consensus region begins at Lys-358, 35 aa from the COOH terminus of dmLa. In contrast, the last 35 aa of spLa did not concentrate in the nucleus, possibly due to the insufficiency of the single COOH-terminal basic residue (Fig. 6 b). However, a larger COOH-terminal construct of Sla1, beginning with Met-232 could concentrate in the nucleus (data not shown). This region of Sla1 contains a putative bipartite NLS beginning at Lys-255, which is 44 aa from the COOH terminus.

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.

Show MeSH