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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 Kap108p-dependent NLS of Lhp1p overlaps with  the RNA recognition motif of Lhp1p. (a) A diagram of the constructs localized by virtue of fusion with GFP. Three relevant features of Lhp1p, the location of the La domain, RRM, and corresponding region of the human La NLS are noted above the bar  representing full-length Lhp1p. (b) Approximately equal amounts  of total protein from cells expressing the reporter constructs were  separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. Polyclonal anti-GFP antibodies were used to visualize the  fusion proteins. (c) GFP fusion proteins were visualized in live  wild-type and Δkap108 cells. Coincident fluorescence and Nomarski optics were used to localize GFP relative to the cell periphery.  Fusion proteins were considered nuclear (Nuc) if they were able  to concentrate in the nucleus, otherwise they were considered cytoplasmic (Cyt).
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Figure 2: The Kap108p-dependent NLS of Lhp1p overlaps with the RNA recognition motif of Lhp1p. (a) A diagram of the constructs localized by virtue of fusion with GFP. Three relevant features of Lhp1p, the location of the La domain, RRM, and corresponding region of the human La NLS are noted above the bar representing full-length Lhp1p. (b) Approximately equal amounts of total protein from cells expressing the reporter constructs were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. Polyclonal anti-GFP antibodies were used to visualize the fusion proteins. (c) GFP fusion proteins were visualized in live wild-type and Δkap108 cells. Coincident fluorescence and Nomarski optics were used to localize GFP relative to the cell periphery. Fusion proteins were considered nuclear (Nuc) if they were able to concentrate in the nucleus, otherwise they were considered cytoplasmic (Cyt).

Mentions: To further characterize the nuclear import of Lhp1p via Kap108p we next delimited a subdomain of Lhp1p that was capable of conferring nuclear localization via Kap108p. To do so, we constructed plasmids for the in vivo expression of Lhp1p and three fragments of Lhp1p, each fused to GFP. A schematic of these constructs is shown in Fig. 2 a. These fragments overlap by at least 25 amino acids to minimize the likelihood that an NLS would be missed because it had been split between two fragments. That these fragments were properly expressed as full-length products was confirmed by Western blotting with anti-GFP antibodies (Fig. 2 b). Consistent with our previous observations with Lhp1–PrA, in wild-type cells, Lhp1–GFP is restricted to the nucleus, whereas in the KAP108 deletion, Lhp1–GFP is unable to target the nucleus (Fig. 2 c, first row) (Rosenblum et al., 1997). As such, the GFP tag, like the PrA tag we have previously used, did not affect the nuclear targeting of Lhp1p. Fragment 1, aa 1–136, contains the La domain which is highly conserved throughout the La family (Van Horn et al., 1997). As a GFP fusion, fragment 1 was unable to accumulate in nuclei of wild-type cells (Fig. 2 c, second row). Fragment 2 spans aa 112–224, whereas the RRM spans aa 124–209. This fragment, which was not as highly expressed as the other fragments (Fig. 2 b), did accumulate in nuclei of wild-type cells (Fig. 2 c, third row). In contrast, when fragment 2 was expressed in cells deleted for KAP108, no accumulation of GFP was seen in nuclei (Fig. 2 c, third row). Fragment 3 is composed of the COOH terminus of Lhp1p, aa 188–275. This fragment is composed of just under 50% of charged residues, and encompasses a region that corresponds to the NLS of human La that has recently been defined by microinjection of in vitro translated hsLa mutants into Xenopus laevis oocytes followed by dissection (Simons et al., 1996). Interestingly, this region was also able to direct nuclear accumulation, yet this accumulation was insensitive to the presence of Kap108p (Fig. 2 c, fourth row). That fragments 2 and 3 contained discrete NLSs was suggested by their different dependence on Kap108p for nuclear localization. To demonstrate that each fragment had its own NLS, a construct was made that encoded the portion of fragment 3 that did not overlap with fragment 2. This shorter fragment demonstrated localization identical to that of fragment 3, in both wild-type and KAP108-deleted cells (data not shown). As full-length Lhp1p is unable to accumulate in the nucleus in the absence of Kap108p (Fig. 2 c, first row), it appears that fragment 3 contains not the endogenous NLS of Lhp1p, but a secondary NLS unmasked by its expression out of context of Lhp1p.


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

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

The Kap108p-dependent NLS of Lhp1p overlaps with  the RNA recognition motif of Lhp1p. (a) A diagram of the constructs localized by virtue of fusion with GFP. Three relevant features of Lhp1p, the location of the La domain, RRM, and corresponding region of the human La NLS are noted above the bar  representing full-length Lhp1p. (b) Approximately equal amounts  of total protein from cells expressing the reporter constructs were  separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. Polyclonal anti-GFP antibodies were used to visualize the  fusion proteins. (c) GFP fusion proteins were visualized in live  wild-type and Δkap108 cells. Coincident fluorescence and Nomarski optics were used to localize GFP relative to the cell periphery.  Fusion proteins were considered nuclear (Nuc) if they were able  to concentrate in the nucleus, otherwise they were considered cytoplasmic (Cyt).
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Related In: Results  -  Collection

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Figure 2: The Kap108p-dependent NLS of Lhp1p overlaps with the RNA recognition motif of Lhp1p. (a) A diagram of the constructs localized by virtue of fusion with GFP. Three relevant features of Lhp1p, the location of the La domain, RRM, and corresponding region of the human La NLS are noted above the bar representing full-length Lhp1p. (b) Approximately equal amounts of total protein from cells expressing the reporter constructs were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. Polyclonal anti-GFP antibodies were used to visualize the fusion proteins. (c) GFP fusion proteins were visualized in live wild-type and Δkap108 cells. Coincident fluorescence and Nomarski optics were used to localize GFP relative to the cell periphery. Fusion proteins were considered nuclear (Nuc) if they were able to concentrate in the nucleus, otherwise they were considered cytoplasmic (Cyt).
Mentions: To further characterize the nuclear import of Lhp1p via Kap108p we next delimited a subdomain of Lhp1p that was capable of conferring nuclear localization via Kap108p. To do so, we constructed plasmids for the in vivo expression of Lhp1p and three fragments of Lhp1p, each fused to GFP. A schematic of these constructs is shown in Fig. 2 a. These fragments overlap by at least 25 amino acids to minimize the likelihood that an NLS would be missed because it had been split between two fragments. That these fragments were properly expressed as full-length products was confirmed by Western blotting with anti-GFP antibodies (Fig. 2 b). Consistent with our previous observations with Lhp1–PrA, in wild-type cells, Lhp1–GFP is restricted to the nucleus, whereas in the KAP108 deletion, Lhp1–GFP is unable to target the nucleus (Fig. 2 c, first row) (Rosenblum et al., 1997). As such, the GFP tag, like the PrA tag we have previously used, did not affect the nuclear targeting of Lhp1p. Fragment 1, aa 1–136, contains the La domain which is highly conserved throughout the La family (Van Horn et al., 1997). As a GFP fusion, fragment 1 was unable to accumulate in nuclei of wild-type cells (Fig. 2 c, second row). Fragment 2 spans aa 112–224, whereas the RRM spans aa 124–209. This fragment, which was not as highly expressed as the other fragments (Fig. 2 b), did accumulate in nuclei of wild-type cells (Fig. 2 c, third row). In contrast, when fragment 2 was expressed in cells deleted for KAP108, no accumulation of GFP was seen in nuclei (Fig. 2 c, third row). Fragment 3 is composed of the COOH terminus of Lhp1p, aa 188–275. This fragment is composed of just under 50% of charged residues, and encompasses a region that corresponds to the NLS of human La that has recently been defined by microinjection of in vitro translated hsLa mutants into Xenopus laevis oocytes followed by dissection (Simons et al., 1996). Interestingly, this region was also able to direct nuclear accumulation, yet this accumulation was insensitive to the presence of Kap108p (Fig. 2 c, fourth row). That fragments 2 and 3 contained discrete NLSs was suggested by their different dependence on Kap108p for nuclear localization. To demonstrate that each fragment had its own NLS, a construct was made that encoded the portion of fragment 3 that did not overlap with fragment 2. This shorter fragment demonstrated localization identical to that of fragment 3, in both wild-type and KAP108-deleted cells (data not shown). As full-length Lhp1p is unable to accumulate in the nucleus in the absence of Kap108p (Fig. 2 c, first row), it appears that fragment 3 contains not the endogenous NLS of Lhp1p, but a secondary NLS unmasked by its expression out of context of Lhp1p.

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