Fully processed mRNAs in the nucleus remain bound by hnRNP proteins in complexes referred to as nuclear mRNPs. Before an mRNA can be translated into its encoded protein, it must be exported from the nucleus into the cytoplasm. The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. Like the plasma membrane surrounding a cell, each nuclear mem brane consists of a water-impermeable phospholipid bi layer and multiple associated proteins. mRNPs and other macromolecules, including tRNAs and ribosomal subunits, traverse the nuclear envelope through nuclear pore complexes (NPCs). This section focuses on the export of mRNPs through NPCs and the mechanisms that allow some level of regulation of this step.

Embedded in the nuclear envelope, NPCs are cylindrical in shape with a diameter of about 30 nm. Proteins and RNPs larger than 40–60 kDa must be selectively transported across the nuclear envelope with the assistance of trans porter proteins that bind them and also interact reversibly with components in the central channel of the NPC. mRNPs are transported through the NPC by the mRNP exporter, a heterodimer consisting of a large subunit, called nuclear export factor 1 (NXF1), and a small subunit, nuclear export transporter 1 (NXT1). NXF1 binds nuclear mRNPs through associations with both RNA and proteins in the mRNP com plex. One of the most important of these proteins is REF (RNA export factor), a component of the exon-junction complexes discussed earlier, which is bound approximately 20 nucleotides 5′ to each exon-exon junction (Figure 1). The mRNP exporter also associates with SR proteins bound to exonic splicing enhancers. Thus SR proteins associated with exons function to direct both the splicing of pre-mRNAs and the export of fully processed mRNAs through NPCs to the cytoplasm. mRNPs are probably bound along their length by multiple mRNP exporters, which interact reversibly with unstructured protein domains that fill the NPC central channel.

Fig1. Remodeling of mRNPs during nuclear export. Some mRNP proteins (rectangles) dissociate from nuclear mRNP complexes before their export through an NPC. Others (ovals) are exported through the NPC with the mRNP, but dissociate from it in the cytoplasm and are shuttled back into the nucleus through an NPC. In the cytoplasm, translation initiation factor eIF4E replaces CBC bound to the 5′ cap, and PABPC1 replaces PABPN1.

Protein filaments extend from the core NPC scaffold into the nucleoplasm, forming an NPC nuclear basket (see Figure 1). Other protein filaments extend from the cytoplasmic face of the NPC into the cytoplasm. Both sets of filaments assist in mRNP export. Gle2, an adapter protein that reversibly binds both NXF1 and a protein in the nuclear basket, brings nuclear mRNPs to the NPC in preparation for export. A protein in the cytoplasmic filaments of the NPC binds an RNA helicase (Dbp5) that functions in the dissociation of NXF1/NXT1 and other hnRNP proteins from the mRNP as it reaches the cytoplasm.

In a process called mRNP remodeling, the proteins associated with an mRNA in the nuclear mRNP are exchanged for a different set of proteins as the mRNP is transported through the NPC (see Figure 1). Some nuclear mRNP proteins dissociate early in transport, remaining in the nucleus to bind to newly synthesized nascent pre-mRNA. Other nuclear mRNP proteins remain with the mRNP as it traverses the NPC and do not dissociate from the mRNP until the complex reaches the cytoplasm. Proteins in this category include the NXF1/NXT1 mRNP exporter, the nuclear cap-binding complex (CBC) bound to the 5′ cap, and PABPN1 bound to the poly(A) tail. These proteins dissociate from the mRNP on the cytoplasmic side of the NPC through the action of the Dbp5 RNA helicase that as sociates with the cytoplasmic NPC filaments, as discussed above. These proteins are then imported back into the nucleus, where they can function in the export of another mRNP. In the cytoplasm, the cap-binding translation initiation factor eIF4E replaces the CBC bound to the 5′ cap of nuclear mRNPs. In vertebrates, the nuclear poly(A)-binding protein PABPN1 is replaced with the cytoplasmic poly(A)-binding protein PABPC1 (so named to distinguish it from the nuclear PABPN1). Only a single PABP is found in budding yeast, in both the nucleus and the cytoplasm.

Phosphorylation and Dephosphorylation of SR Proteins Imposes Directionality on mRNP Export Across the Nuclear Pore Complex

Studies of S. cerevisiae indicate that the direction of mRNP export from the nucleus into the cytoplasm is controlled by the phosphorylation and dephosphorylation of mRNP adapter proteins, such as REF, that assist in the binding of the NXF1/NXT1 mRNP exporter to mRNPs. In one case, a yeast SR protein (Npl3) functions as an adapter protein that promotes the binding of the yeast mRNP exporter ( Figure 2). In its phosphorylated form, the SR protein initially binds to nascent pre-mRNA. When 3′ cleavage and polyadenylation are completed, the adapter protein is de phosphorylated by a specific nuclear protein phosphatase that is essential for mRNP export. Only the dephosphorylated adapter protein can bind the mRNP exporter, thereby coupling mRNP export to correct polyadenylation. This mechanism is one form of mRNA “quality control.” If the nascent mRNP is not correctly processed, it is not recognized by the phosphatase that dephosphorylates Npl3, and consequently, it is not bound by the mRNP exporter and is not exported from the nucleus. Instead, it is degraded by exosomes, the multiprotein complexes that degrade unprotected RNAs in the nucleus and cytoplasm.

Fig2. Reversible phosphorylation and direction of mRNP nuclear export. Step 1 : The yeast SR protein Npl3 binds nascent pre-mRNAs in its phosphorylated form. Step 2 : When poly adenylation has occurred successfully, the Glc7 nuclear phosphatase dephosphorylates Npl3, promoting the binding of the mRNP exporter, NXF1/NXT1. Step 3 : The mRNP exporter allows diffusion of the mRNP complex through the central channel of the nuclear pore complex (NPC). Step 4 : The cytoplasmic protein kinase Sky1 phosphorylates Npl3 in the cytoplasm, causing step 5 dissociation of the phosphorylated Npl3 from the mRNP exporter, probably through the action of an RNA helicase associated with NPC cytoplasmic filaments step 6. The mRNA transporter and phosphorylated Npl3 are transported back into the nucleus through NPCs. Step 7 Transported mRNA is available for translation in the cytoplasm. See E. Izaurralde, 2004, Nat. Struct. Mol. Biol. 11:210–212; see also W. Gilbert and C. Guthrie, 2004, Mol. Cell 13:201–212.

Following export to the cytoplasm, the Npl3 SR protein is phosphorylated by a specific cytoplasmic protein kinase.

This phosphorylation causes it to dissociate from the mRNP, along with the mRNP exporter. In this way, dephosphory lation of mRNP adapter proteins in the nucleus once RNA processing is complete and their phosphorylation and result ing dissociation in the cytoplasm result in a higher concentration of mRNP exporter–mRNP complexes in the nucleus, where they form, and a lower concentration of these complexes in the cytoplasm, where they dissociate. As a result, the direction of mRNP export may be driven by simple diffusion down a concentration gradient of the mRNP exporter mRNP complex across the NPC, from high in the nucleus to low in the cytoplasm.

Balbiani Rings in Insect Larval Salivary Glands Allow Direct Visualization of mRNP Export Through NPCs

The larval salivary glands of the insect Chironomus tentans provide a good model system for electron microscopic studies of the formation of hnRNPs and their export through NPCs. In these larvae, genes in large chromosomal puffs called Balbiani rings are abundantly transcribed into nascent pre-mRNAs that associate with hnRNP proteins and are processed into coiled mRNPs with a final mRNA length of about 75 kb (Figure 3a, b). These giant mRNAs encode large glue proteins that adhere the developing larva to a leaf. After processing of the pre-mRNA in Balbiani ring hnRNPs, the resulting mRNPs move through NPCs to the cytoplasm. Electron micrographs of sections of these cells show mRNPs that appear to uncoil during their passage through NPCs and then bind to ribosomes as they enter the cytoplasm. This uncoiling is probably a consequence of the remodeling of mRNPs as the result of phosphorylation of mRNP proteins by cytoplasmic kinases and the action of the RNA helicase associated with NPC cytoplasmic filaments, as discussed in the previous section. The observation that mRNPs become associated with ribosomes during transport indicates that the 5′ end leads the way through the NPC. Detailed electron microscopic studies of the transport of Balbiani ring mRNPs through nuclear pore complexes led to the model depicted in Figure 3c.

Fig3. Formation of heterogeneous ribonucleoprotein particles (hnRNPs) and export of mRNPs from the nucleus. (a) Model of a single chromatin transcription loop and assembly of Balbiani ring (BR) mRNP in Chironomus tentans. Nascent RNA transcripts produced from the template DNA rapidly associate with proteins, forming hnRNPs. The gradual increase in the size of the hnRNPs reflects the increasing length of RNA transcripts at greater distances from the transcription start site. The model was reconstructed from electron micrographs of serial thin sections of salivary gland cells. (b) Schematic diagram of the biogenesis of hnRNPs. Fol lowing processing of the pre-mRNA, the resulting ribonucleoprotein particle is referred to as an mRNP. (c) Model for the transport of BR mRNPs through the nuclear pore complex (NPC) based on electron microscopic studies. Note that the curved mRNPs appear to uncoil as they pass through NPCs. As the mRNA enters the cytoplasm, it rapidly associates with ribosomes, indicating that the 5′ end passes through the NPC first. Parts (b) and (c), see B. Daneholt, 1997, Cell 88:585. See also B. Daneholt, 2001, Proc. Natl. Acad. Sci. USA 98:7012. [Part (a) republished with permission from Elsevier, from Erricson, C. et al., “The ultra structure of upstream and downstream regions of an active Balbiani ring gene,” Cell, 1989, 56(4): 631–9; courtesy of B. Daneholt. Permission conveyed through the Copyright Clearance Center, Inc.]

Pre-mRNAs in Spliceosomes Are Not Exported from the Nucleus

It is critical that only fully processed mature mRNAs be ex ported from the nucleus because translation of incompletely processed pre-mRNAs containing introns would produce defective proteins that might interfere with the functioning of the cell. To prevent this, pre-mRNAs associated with snRNPs in spliceosomes are usually prevented from being transported to the cytoplasm.

In one type of experiment demonstrating this restriction, a gene encoding a pre-mRNA with a single intron that is normally spliced out was mutated to introduce deviations from the consensus splice-site sequences. Mutation of either the 5′ or the 3′ invariant splice-site bases at the ends of the intron resulted in pre-mRNAs that were bound by snRNPs to form spliceosomes; however, RNA splicing was blocked, and the pre-mRNA was retained in the nucleus. In contrast, mutation of both the 5′ and 3′ splice sites in the same pre-mRNA resulted in export of the unspliced pre-mRNA, although less efficiently than for the spliced mRNA, prob ably because of the absence of an exon-junction complex. When both splice sites were mutated, the pre-mRNAs were not efficiently bound by snRNPs, and consequently, their export was not blocked.

Studies in yeast have shown that a protein component of the NPC nuclear basket is required to retain pre-mRNAs associated with snRNPs in the nucleus. If either this protein or the nuclear basket protein to which it binds is deleted, unspliced pre-mRNAs are exported. Consequently, these proteins prevent hnRNPs associated with snRNPs from traversing the NPC.

Many cases of thalassemia, an inherited disease that results in abnormally low levels of globin proteins, are due to mutations in globin-gene splice sites that decrease the efficiency of splicing but do not prevent association of the pre-mRNA with snRNPs. The resulting unspliced globin pre-mRNAs are retained in the nuclei of erythroid progenitors and are rapidly degraded.

HIV Rev Protein Regulates the Transport of Unspliced Viral mRNAs

 As discussed earlier, transport of mRNPs containing mature, functional mRNAs through NPCs from the nucleus to the cytoplasm entails a complex mechanism that is crucial to gene expression (see Figures 1, 2, and 3). Regulation of this transport theoretically could provide another means of gene control, although it appears to be relatively rare. Indeed, the only known examples of regulated mRNA export occur during the cellular response to conditions (e.g., heat shock) that cause protein denaturation or during viral infection, when virus-induced alterations in nuclear export of mRNPs maximize viral replication. Here we describe the regulation of mRNP export mediated by a protein encoded by human immunodeficiency virus (HIV).

HIV, which is a retrovirus, integrates a DNA copy of its RNA genome into the host-cell DNA. The integrated viral DNA, or provirus, contains a single transcription unit, which is transcribed into a single primary transcript by cellular RNA polymerase II. The HIV transcript can be spliced in alternative ways to yield three classes of mRNAs: a 9-kb unspliced mRNA; 4-kb mRNAs formed by removal of one intron; and 2-kb mRNAs formed by removal of two or more introns (Figure 4). After their synthesis in the host-cell nucleus, all three classes of HIV mRNAs are transported to the cytoplasm and translated into viral proteins; some of the 9-kb unspliced RNA is used as the viral genome in progeny virions that bud from the cell surface.

Fig4. Transport of HIV mRNAs from the nucleus to the cytoplasm. The HIV genome, which contains several coding regions, is transcribed into a single 9-kb primary transcript. Several 4-kb mRNAs result from the splicing out of any one of several introns (dashed lines), and several 2-kb mRNAs result from the splicing out of two or more alternative introns. After transport to the cytoplasm, these various RNA species are translated into different viral proteins. Rev protein, encoded by a 2-kb mRNA, interacts with the Rev-response element (RRE) in the unspliced and singly spliced mRNAs, stimulating their transport to the cytoplasm. See B. R. Cullen and M. H. Malim, 1991, Trends Biochem. Sci. 16:346.

Since the 9-kb and 4-kb HIV mRNAs contain splice sites, they can be viewed as incompletely spliced mRNAs. As dis cussed earlier, association of such incompletely spliced mRNAs with snRNPs in spliceosomes normally blocks their export from the nucleus. Thus HIV, as well as other retroviruses, must have some mechanism for overcoming this block, permitting export of the longer viral mRNAs. Some retroviruses have evolved an RNA sequence within their genome called the constitutive transport element (CTE), which binds to the NXF1/NXT1 mRNP exporter with high affinity. This strong interaction with the mRNP exporter allows export of unspliced retroviral RNA into the cytoplasm. HIV solved the problem differently.

Studies with HIV mutants showed that transport of un spliced 9-kb and singly spliced 4-kb viral mRNAs from the nucleus to the cytoplasm requires the virus-encoded Rev protein. Subsequent biochemical experiments demonstrated that Rev binds to a specific Rev-response element (RRE) that is present in HIV RNA. In cells infected with HIV mutants lacking the RRE, unspliced and singly spliced viral mRNAs remain in the nucleus, demonstrating that the RRE is re quired for Rev-mediated stimulation of nuclear export. Early in an infection, before any Rev protein is synthesized, only multiply spliced 2-kb mRNAs that do not retain any splice sites can be exported. One of these alternatively spliced 2-kb mRNAs encodes Rev, which contains a leucine-rich nuclear export signal that interacts with the transporter exportin 1 rather than with the NXF1/NXT1 mRNP exporter. Translation of Rev in the cytoplasm, followed by its import into the nucleus, results in export of the larger unspliced and singly spliced HIV mRNAs through the NPC.

اخر الاخبار

اخبار العتبة العباسية المقدسة

مجموعة مشاتل الكفيل ترفد مشاتلها بأكثر من 28 طنًّا من النباتات

شعبة التوجيه الديني النسوي تُحيي ذكرى شهادة الإمام الصادق (عليه السلام)

قسم الشؤون الدينية يحيي ذكرى شهادة الإمام الصادق (عليه السلام)

قسم الشؤون الفكرية يصدر العدد 17 من سلسلة السيرة للرسائل والأطاريح الجامعية

المزيد

الآخبار الصحية

هواية تعزز صحة ونشاط الدماغ بشكل ملحوظ

كيف يمكن للصيام أن يطيل العمر؟!

مواد في منزلك قد تشوه الأجنة.. دراسة تكشف مفاجأة مقلقة

بديل طبيعي للفلورايد يحمي أسنانك من التسوس

المزيد

الآخبار التكنلوجية

كارثة بيئية في العراق.. نفوق 1000 طن من الأسماك ؟!

الأرض "تومض" ليلا!.. كوكبنا ازداد سطوعا بنسبة 16% في غضون 8 سنوات

أحد أخطر البراكين العملاقة على وجه الأرض يمتلئ بالصهارة!

علماء يطورون تقنية جديدة تُسرّع التئام الجروح عبر تنشيط خلايا الجلد

المزيد

مواضيع ذات صلة

Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions

A Diverse Set of Proteins with Conserved RNA Binding Domains Associate with Pre-mRNAs

The 5′ Cap Is Added to Nascent RNAs Shortly After Transcription Initiation

Regulation of Pre-mRNA Processing

Transcription Initiation by Pol I and Pol III Is Analogous to That by Pol II

The Mediator Complex Forms a Molecular Bridge Between Activation Domains and Pol II

Pioneer Transcription Factors Initiate the Process of Gene Activation During Cellular Differentiation

Genetic Material can be Altered & Rearranged

Translation and protein production

Epigenetic Regulation of Transcription

Chromatin-Remodeling Complexes Help Activate or Repress Transcription

Activators Can Direct Histone Acetylation at Specific Genes