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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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A representation of important domains for four evolutionarily distinct La proteins. Bars proportional to the length of  the human, Drosophila, S. pombe, and S. cerevisiae La proteins  are shown. Black lines, RRMs; two-headed arrows, proteins  NLSs. The ATP-binding domain of human La is indicated.
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Figure 7: A representation of important domains for four evolutionarily distinct La proteins. Bars proportional to the length of the human, Drosophila, S. pombe, and S. cerevisiae La proteins are shown. Black lines, RRMs; two-headed arrows, proteins NLSs. The ATP-binding domain of human La is indicated.

Mentions: The evolution of La, a multidomain, multifunctional protein most likely occurred through a number of discrete steps. It is possible that evolution of a Kapα/Kapβ1-dependent NLS allowed La to attain additional functionality, perhaps by decreasing the mass of La used as a signal for nuclear localization (see Fig. 7 for a schematic representation of the four La proteins studied here). As shown above, even a large, 113-aa domain encompassing a Kap108p-dependent NLS only weakly concentrated fragment 2 in the nucleus (Fig. 2). NLSs for α-Kap–independent β-Kaps appear to be larger than NLSs recognized by Kapα. The NLS of Nab2p for Kap104p-mediated import has been mapped to a region 50- or 110-aa long, and the hnRNP A1 NLS for Kapβ2-mediated import is at least 38-aa long (Siomi and Dreyfuss, 1995; Siomi et al., 1998; Truant et al., 1998). In addition, Kap111p/Mtr10p, which imports Npl3p, uses an NLS on the order of 130-aa long (Pemberton et al., 1997; Senger et al., 1998). In contrast, a 27-aa domain of human La (Simons et al., 1996) and a 35-aa domain of Drosophila La (Fig. 6 b) strongly concentrate fusion proteins to the nucleus via α-Kap–dependent pathways. In fact, α-Kap–dependent NLSs are often shorter than 12 aa and can even be as short as 5 aa (Dingwall and Laskey, 1991). In addition, the Kap108p-dependent NLS of Lhp1p includes the RNA-binding domain, and it is therefore possible that RNA binding and Kap108p binding of Lhp1p are mutually exclusive (many NLSs of DNA- or RNA-binding proteins have been mapped to sites overlapping or adjacent to DNA- or RNA-binding domains; LaCasse and Lefebvre, 1995). An example of this exclusivity of binding La has been described for the human protein. A monoclonal antibody, mAb SW3, which binds within the RRM of hsLa, is unable to bind to RNA-associated La (Pruijn et al., 1995). It could be envisioned that directionality for Lhp1p import could be conveyed in this manner. Newly synthesized, cytoplasmic Lhp1p could be recognized in the cytoplasm by Kap108p. Kap108p could then direct Lhp1p through the NPC by virtue of its interaction with nucleoporins (Rosenblum et al., 1997). Once in the nucleus, the Kap108p/Lhp1p complex could be dissembled in the presence of a high concentration of oligouridylate-containing RNA polymerase III transcripts in the nucleus (Senger et al., 1998). The overlap between the Lhp1p NLS and its RNA recognition motif may also affect cytoplasmic functions of La proteins. As shown in Fig. 1 b, a large proportion of cytoplasmic Lhp1p is stably bound to Kap108p. Given that this binding appears to be mediated by a large region that includes the RNA-binding domain of Lhp1p, it appears unlikely that this domain would be accessible for cytoplasmic functions in S. cerevisiae. As such, there may have been an evolutionary advantage to have the NLS of the more complex La proteins in a region not involved in direct RNA binding.


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

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

A representation of important domains for four evolutionarily distinct La proteins. Bars proportional to the length of  the human, Drosophila, S. pombe, and S. cerevisiae La proteins  are shown. Black lines, RRMs; two-headed arrows, proteins  NLSs. The ATP-binding domain of human La is indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132966&req=5

Figure 7: A representation of important domains for four evolutionarily distinct La proteins. Bars proportional to the length of the human, Drosophila, S. pombe, and S. cerevisiae La proteins are shown. Black lines, RRMs; two-headed arrows, proteins NLSs. The ATP-binding domain of human La is indicated.
Mentions: The evolution of La, a multidomain, multifunctional protein most likely occurred through a number of discrete steps. It is possible that evolution of a Kapα/Kapβ1-dependent NLS allowed La to attain additional functionality, perhaps by decreasing the mass of La used as a signal for nuclear localization (see Fig. 7 for a schematic representation of the four La proteins studied here). As shown above, even a large, 113-aa domain encompassing a Kap108p-dependent NLS only weakly concentrated fragment 2 in the nucleus (Fig. 2). NLSs for α-Kap–independent β-Kaps appear to be larger than NLSs recognized by Kapα. The NLS of Nab2p for Kap104p-mediated import has been mapped to a region 50- or 110-aa long, and the hnRNP A1 NLS for Kapβ2-mediated import is at least 38-aa long (Siomi and Dreyfuss, 1995; Siomi et al., 1998; Truant et al., 1998). In addition, Kap111p/Mtr10p, which imports Npl3p, uses an NLS on the order of 130-aa long (Pemberton et al., 1997; Senger et al., 1998). In contrast, a 27-aa domain of human La (Simons et al., 1996) and a 35-aa domain of Drosophila La (Fig. 6 b) strongly concentrate fusion proteins to the nucleus via α-Kap–dependent pathways. In fact, α-Kap–dependent NLSs are often shorter than 12 aa and can even be as short as 5 aa (Dingwall and Laskey, 1991). In addition, the Kap108p-dependent NLS of Lhp1p includes the RNA-binding domain, and it is therefore possible that RNA binding and Kap108p binding of Lhp1p are mutually exclusive (many NLSs of DNA- or RNA-binding proteins have been mapped to sites overlapping or adjacent to DNA- or RNA-binding domains; LaCasse and Lefebvre, 1995). An example of this exclusivity of binding La has been described for the human protein. A monoclonal antibody, mAb SW3, which binds within the RRM of hsLa, is unable to bind to RNA-associated La (Pruijn et al., 1995). It could be envisioned that directionality for Lhp1p import could be conveyed in this manner. Newly synthesized, cytoplasmic Lhp1p could be recognized in the cytoplasm by Kap108p. Kap108p could then direct Lhp1p through the NPC by virtue of its interaction with nucleoporins (Rosenblum et al., 1997). Once in the nucleus, the Kap108p/Lhp1p complex could be dissembled in the presence of a high concentration of oligouridylate-containing RNA polymerase III transcripts in the nucleus (Senger et al., 1998). The overlap between the Lhp1p NLS and its RNA recognition motif may also affect cytoplasmic functions of La proteins. As shown in Fig. 1 b, a large proportion of cytoplasmic Lhp1p is stably bound to Kap108p. Given that this binding appears to be mediated by a large region that includes the RNA-binding domain of Lhp1p, it appears unlikely that this domain would be accessible for cytoplasmic functions in S. cerevisiae. As such, there may have been an evolutionary advantage to have the NLS of the more complex La proteins in a region not involved in direct RNA binding.

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