Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. A new pathway to the nucleus has been characterized, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p binds peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways (Pemberton, 1997).
MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA (Senger, 1998).
The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. RS domain-bearing proteins do not utilize any of the known nuclear import receptors and a novel nuclear import receptor specific for SR proteins has been identified. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain-bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin ß/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family (Kataoka, 1999).
Mammalian SR proteins are currently thought to function in mRNA export as well as splicing. They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides. Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear. Npl3 is a shuttling protein in budding yeast that is a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1. Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p. The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo. It is proposed that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export (Gilbert, 2001).
Telomerase is a ribonucleoprotein particle (RNP) involved in chromosome end replication, but its biogenesis is poorly understood. The RNA component of yeast telomerase (Tlc1) is synthesized as a polyadenylated precursor and then processed to a mature poly(A)- form. The karyopherin Mtr10p is required for the normal accumulation of mature Tlc1 and its proper localization to the nucleus. Neither TLC1 transcription nor the stability of poly(A)- Tlc1 is significantly affected in mtr10delta cells. Tlc1 is mostly nuclear in a wild-type background, and this localization is not affected by mutations in other telomerase components. Strikingly, in the absence of Mtr10p, Tlc1 is found dispersed throughout the entire cell. These results are compatible with two alternative models: (1) Mtr10p may import a cytoplasmic complex containing Tlc1 and perhaps other components of telomerase, and shuttling of Tlc1 from the nucleus to the cytoplasm and back may be necessary for the biogenesis of telomerase (the 'shuttling' model); (2) Mtr10p may be necessary for the nuclear import of some enzyme needed for the nuclear processing and maturation of Tlc1, and in the absence of this maturation, poly(A)+ Tlc1 is aberrantly exported to the cytoplasm (the 'processing enzyme' model) (Ferrezuelo, 2002).
Important progress in understanding messenger RNA export from the nucleus could be achieved by increasing the list of proteins that are involved in this process. Gbp2 has been identified as a novel shuttling RNA-binding protein in Saccharomyces cerevisiae. Nuclear import of Gbp2 is dependent on the receptor Mtr10 and the serine/arginine-specific protein kinase Sky1. Deletion of the genes encoding both of these proteins or disruption of two of the arginine/serine repeats each shifts the steady-state localization of Gbp2 to the cytoplasm. Interestingly, deletion of MTR10 only also causes an increase in poly(A)(+) RNA binding by Gbp2, suggesting a role of Mtr10 in the dissociation of Gbp2 from mRNA in the cytoplasm. The nuclear export of Gbp2 is always coupled to mRNA export and is dependent on continuous RNA polymerase II transcription and mRNA-export factors. Although GBP2 is not essential for normal cell growth, overexpression of this gene is toxic and causes a nuclear retention of bulk poly(A)(+) RNA. Together, these findings clearly show an involvement of Gbp2 in mRNA transport. In addition, as a non-essential protein, Gbp2 also has the interesting potential to be spatially or temporally regulated (Windgassen, 2003).
Messenger RNAs are transported to the cytoplasm bound to several shuttling mRNA-binding proteins. Hrb1, a novel component of the transported ribonucleoprotein complex in Saccharomyces cerevisiae, has been characterized. The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3. Hrb1 is nuclear at steady state and its import is mediated by the karyopherin Mtr10. Hrb1 binds to poly(A)+ RNA in vivo and its binding is significantly increased in MTR10 mutants, suggesting a role for Mtr10 in dissociating Hrb1 from the mRNAs. Interestingly, by comparing the export requirements of all three SR proteins similarities were found but also striking differences. While the export of all three proteins is dependent on the export of mRNAs in general, since no transport is observed in mutants defective in transcription (rpb1-1) or mRNA export (mex67-5), specific requirements were found for components of the THO complex, involved in transcription elongation. While both Hrb1 and Gbp2 depend on Mft1 and Hpr1 for their nuclear export, Npl3 is exported independently of both proteins. These findings suggest that Hrb1 and Gbp2, but not Npl3, might be loaded onto the growing mRNA via the THO complex components Mtf1 and Hrp1 (Hacker, 2004)
A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. This study describes the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, evidence is provided that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP (Windgassen, 2004).
Serine/arginine-rich proteins are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. The RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but is insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, a human importin-ß family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR was identified. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, these results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins (Lai, 2000).
Serine/arginine-rich proteins are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. An importin ß family protein, transportin-SR2 (TRN-SR2) specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, it has been shown that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, it was found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, it was shown that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains (Lai, 2001).
Alternative splicing of precursor mRNA is often regulated by SR proteins and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by TRN-SRs, importin ß-like nuclear transporters. A non-SR protein, RNA-binding motif protein 4 (RBM4), has been identified as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus (Lai, 2003).
SR proteins and related RS domain-containing polypeptides are an important class of splicing regulators in higher eukaryotic cells. The RS domain facilitates nuclear import of SR proteins and mediates protein-protein interactions during spliceosome assembly; both functions appear to subject to regulation by phosphorylation. Previous studies have identified two nuclear import receptors for SR proteins, transportin-SR1 and transportin-SR2. Transportin-SR1 and transportin-SR2 are the alternatively spliced products of the same gene and transportin-SR2 is the predominant transcript in most cells and tissues examined. While both receptors import typical SR proteins in a phosphorylation-dependent manner, they differentially import the RS domain-containing splicing regulators hTra2alpha and hTra2ß in different phosphorylation states. It is suggested that differential regulation of nuclear import may serve as a mechanism for homeostasis of RS domain-containing splicing factors and regulators in the nucleus and for selective cellular responses to signaling (Yun, 2003).
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