While in the nucleus, most mRNA precursors undergo a series of processing events prior to moving into the cytoplasm via the nuclear pore. These maturation events include cotranscriptional capping at the 5Ј end (Lewis et al. 1995), splicing (Adams et al. 1996, and 3Ј-end cleavage followed by polyadenylation (Manley and Takagaki 1996;Wahle and Keller 1996). Proper execution of these steps is required to produce an export-competent mRNA species.The poly(A) tail has been proposed to play a role in nearly every aspect of mRNA metabolism. These include maintaining the proper stability of transcripts (Beelman and Parker 1995), promoting export from the nucleus to the cytoplasm (Huang and Carmichael 1996), and recruiting mRNAs to the translation machinery (Sachs et al. 1997). Studies have proposed that it is the actual process of polyadenylation and not just the presence of the poly(A) tail itself, which is essential for mRNA export. When 3Ј ends were artificially formed by cis-acting ribozyme cleavage in vivo, the resulting mRNA was not exported efficiently (Eckner et al. 1991;Liu and Smith 1994;Huang and Carmichael 1996).A detailed picture of the biochemical requirements for mammalian mRNA 3Ј-end formation has started to emerge (Manley and Takagaki 1996;Ruegsegger et al. 1996;Wahle and Keller 1996). Six protein factors are required for this processing step. One set of factors is required for the proper recognition and cleavage of the premRNA and consists of the cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II (CF I m and CF II m ), and for most pre-mRNAs, poly(A) polymerase (PAP). The second set of biochemically defined factors participates in poly(A) addition and includes CPSF, PAP, and the nuclear poly(A)-binding protein II (PAB II). The polypeptide constituents of many of these factors are known, but their mechanisms of action have not yet been defined.The development of a cell-free system for the accurate and efficient polyadenylation of yeast mRNAs indicated mechanistic similarities between mRNA 3Ј-end formation in Sacchacomyces cerevisiae and metazoans (Butler and Platt 1988). In yeast four separable protein factors in 3 These authors contributed equally to this study.
Eukaryotic mRNA processing and export is mediated by various heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated on arginine residues. In the yeast, Saccharomyces cerevisiae, the predominant enzyme responsible for arginine methylation is Hmt1p. Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export. Here, we employ an in vivo nuclear export assay to show that arginine methylation is important for the nuclear export of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus in cells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export. The export of a novel hnRNP-like protein, Hrb1p, which does not bind poly(A) + RNA, however, is not affected by the lack of methylation. Furthermore, we find a genetic relationship between Hmt1p and cap-binding protein 80 (CBP80). Together, these findings establish that one biological role for arginine methylation is in facilitating the export of certain hnRNPs out of the nucleus. While in the nucleus, mRNA precursors, referred to as pre-mRNAs or heterogeneous nuclear RNAs (hnRNAs), undergo a series of processing events before traveling to the cytoplasm. These maturation events include capping at the 5Ј end, splicing, and 3Ј-end cleavage followed by polyadenylation. From the time they leave the transcription complex, hnRNAs are associated with proteins, some of which have been proposed to be mediators of RNA export (for review, see Lee and Silver 1997). The set of proteins that bind hnRNAs, with the exception of small nuclear RNPs, are referred to as heterogeneous nuclear ribonucleoproteins (hnRNPs).Among the most abundant proteins in the nucleus (Kiledjian et al. 1994), there are over 20 mammalian hnRNPs, proposed to function in nearly every step of mRNA maturation including export (Piñ ol-Roma 1997). One of the best-studied hnRNPs, hnRNP A1, travels back and forth between the nucleus and the cytoplasm in a process termed nucleocytoplasmic shuttling (Piñ olRoma and Dreyfuss 1992). In addition, an hnRNP A1-like protein in the insect Chironomus tentans can be seen by immunoelectron microscopy to be associated with pre-mRNA traveling through the nuclear pore to the cytoplasm (Visa et al. 1996a).The discovery of a nuclear export signal (NES) in some hnRNPs has suggested further that these hnRNPs could play an active role in RNA export. A 38-amino-acid sequence within hnRNP A1 termed M9 has been found to be necessary and sufficient for export of the protein to the cytoplasm (Michael et al. 1995). Microinjection experiments have shown that saturating amounts of the M9 domain block mRNA export in Xenopus oocytes . A model for mRNA export has been proposed in which the export signals on the shuttling hnRNPs are directly responsible for the translocation of bound mRNAs into the cytoplasm (Fischer et al. 1996;Nigg 1997). This model is best supported by studies of the HIV Rev protein. Through its leucine-rich NES, Rev facilitates the nuclear export of partially spliced and unspliced viral RNA...
A number of RNA-binding proteins are associated with mRNAs in both the nucleus and the cytoplasm. One of these, Npl3p, is a heterogeneous nuclear ribonucleoprotein-like protein with some similarity to SR proteins and is essential for growth in the yeast S. cerevisiae. Temperature-sensitive alleles have defects in the export of mRNA out of the nucleus (1). In this report, we define a genetic relationship between NPL3 and the nonessential genes encoding the subunits of the cap-binding complex (CBP80 and CBP20). Deletion of CBP80 or CBP20 in combination with certain temperature-sensitive npl3 mutant alleles fail to grow and thus display a synthetic lethal relationship. Further evidence of an interaction between Npl3p and the cap-binding complex was revealed by co-immunoprecipitation experiments; Cbp80p and Cbp20p specifically co-precipitate with Npl3p. However, the interaction of Npl3p with Cbp80p depends on both the presence of Cbp20p and RNA. In addition, we show that Cbp80p is capable of shuttling between the nucleus and the cytoplasm in a manner dependent on the ongoing synthesis of RNA. Taken together, these data support a model whereby mRNAs are co-transcriptionally packaged by proteins including Npl3p and capbinding complex for export out of the nucleus.While in the nucleus, mRNA precursors, referred to as pre-mRNAs or heterogeneous nuclear RNAs undergo a series of processing events before entering the cytoplasm. These maturation events include co-transcriptional capping at the 5Ј-end, splicing, and cleavage and polyadenylation at the 3Ј-end. The proper execution of these steps affects the export of mRNA (reviewed in Refs. 2-4). Thus, the process of mRNA export commences long before the RNA actually reaches the nuclear membrane.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.