Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.

Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors.

BACKGROUND: The various steps of mRNP biogenesis (transcription, processing and export) are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. RESULTS: We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC) formation. CONCLUSIONS: Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

Dbp5, Gle1-IP6 and Nup159: a working model for mRNP export.

Gene expression is a stepwise process involving distinct cellular processes including transcription, mRNA (mRNA) processing, mRNA export, and translation. As mRNAs are being synthesized, proteins associate with the RNA to form messenger ribonucleoprotein particles (mRNPs). Previous studies have demonstrated that the RNA-binding protein composition of these mRNPs is dynamic, changing as the mRNP moves through the different steps of gene expression, and playing a critical role in these events. An important step during this maturation process occurs at the cytoplasmic face of the nuclear pore complex (NPC) where the export protein Gle1 bound to inositol hexakisphosphate (IP 6) spatially activates the ATP-hydrolysis and mRNP-remodeling activity of the DEAD-box protein Dbp5. Recent work from our laboratory and others has provided important insights into the function and regulation of Dbp5. These include a more detailed explanation of the mechanism of Dbp5 RNP remodeling, the role of Gle1-IP6 in stimulating Dbp5 ATPase activity, and the identification of a novel paradigm for regulation of Dbp5 by Nup159. Based on in vitro biochemical assays, X-ray crystallography, and corresponding in vivo phenotypes, we propose here an updated model of the Dbp5 cycle during mRNP export through the NPC. This takes into account all available data and provides a platform for future studies.

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1.

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1.

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins.

The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum.

Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12 Delta. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport.

Uncovering growth-suppressive MicroRNAs in lung cancer.

PURPOSE: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer. EXPERIMENTAL DESIGN: miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal-malignant human lung tissue bank. RESULTS: miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1 and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided a mechanism for observed growth suppression. CONCLUSIONS: miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.

A genetic screen in Saccharomyces cerevisiae identifies new genes that interact with mex67-5, a temperature-sensitive allele of the gene encoding the mRNA export receptor.

The Mex67p protein, together with Mtr2p, functions as the mRNA export receptor in Saccharomyces cerevisiae by interacting with both mRNA and nuclear pore complexes. To identify genes that interact functionally with MEX67, we used transposon insertion to search for mutations that suppressed the temperature-sensitive mex67-5 allele. Four suppressors are described here. The screen revealed that mutant Mex67-5p, but not wild-type Mex67p, is a target of the nuclear protein quality control mediated by San1p, a ubiquitin-protein ligase that participates in degradation of aberrant chromatin-associated proteins. Our finding that overexpression of the SPT6 gene alleviates the growth defects of the mex67-5 strain, together with the impairment of poly(A)(+) RNA export caused by depletion of Spt6p or the related protein Iws1p/Spn1p, supports the mechanism proposed in mammalian cells for Spt6-mediated co-transcriptional loading of mRNA export factors during transcription elongation. Finally, our results also uncovered genetic connections between Mex67p and the poly(A) nuclease complex and with components of chromatin boundary elements.

Synthetic genetic array analysis in Saccharomyces cerevisiae provides evidence for an interaction between RAT8/DBP5 and genes encoding P-body components.

Coordination of the multiple steps of mRNA biogenesis helps to ensure proper regulation of gene expression. The Saccharomyces cerevisiae DEAD-box protein Rat8p/Dbp5p is an essential mRNA export factor that functions at the nuclear pore complex (NPC) where it is thought to remodel mRNA/protein complexes during mRNA export. Rat8p also functions in translation termination and has been implicated in functioning during early transcription. We conducted a synthetic genetic array analysis (SGA) using a strain harboring the temperature-sensitive rat8-2 allele. Although RAT8 had been shown to interact genetically with >15 other genes, we identified >40 additional genes whose disruption in a rat8-2 background causes synthetic lethality or dramatically reduced growth. Included were five that encode components of P-bodies, sites of cytoplasmic mRNA turnover and storage. Wild-type Rat8p localizes to NPCs and diffusely throughout the cell but rat8-2p localized to cytoplasmic granules at nonpermissive temperature that are distinct from P-bodies. In some genetic backgrounds, these granules also contain poly(A)-binding protein, Pab1p, and additional mRNA export factors. Although these foci are distinct from P-bodies, the two merge under heat-stress conditions. We suggest that these granules reflect defective mRNP remodeling during mRNA export and during cytoplasmic mRNA metabolism.

Harnessing genomics to explore the processes and evolution of mRNA export.

The export of quality-controlled mRNAs across the nuclear membrane for translation is mediated by a set of core proteins that are conserved throughout eukaryotes and have been best-characterized in Saccharomyces cerevisiae. The increased genomic complexity that arose during metazoan evolution, however, has resulted in increased systematic complexity that is reflected in the presence of additional metazoan mRNA export factors. In some cases, metazoans encode families of closely-related factors whereas in fungi, a single homolog is present. An exciting new study examines metazoan mRNA export from a global perspective through the use of a genome-wide RNA interference screen in Drosophila melanogaster. This screen identified several novel factors that contribute to mRNA export while reaffirming the strong evolutionary conservation that exists between Drosophila and yeast homologs that are essential for mRNA export. The study also showed that several factors were specifically required for the export of an intron-containing transcript but not for one lacking an intron. Taken together, this study underscores the value of genomic approaches for understanding complex biological processes.

Altered MicroRNA expression confined to specific epithelial cell subpopulations in breast cancer.

MicroRNAs (miRNAs) are a new class of short noncoding regulatory RNAs (18-25 nucleotides) that are involved in diverse developmental and pathologic processes. Altered miRNA expression has been associated with several types of human cancer. However, most studies did not establish whether miRNA expression changes occurred within cells undergoing malignant transformation. To obtain insight into miRNA deregulation in breast cancer, we implemented an in situ hybridization (ISH) method to reveal the spatial distribution of miRNA expression in archived formalin-fixed, paraffin-embedded specimens representing normal and tumor tissue from >100 patient cases. Here, we report that expression of miR-145 and miR-205 was restricted to the myoepithelial/basal cell compartment of normal mammary ducts and lobules, whereas their accumulation was reduced or completely eliminated in matching tumor specimens. Conversely, expression of other miRNAs was detected at varying levels predominantly within luminal epithelial cells in normal tissue; expression of miR-21 was frequently increased, whereas that of let-7a was decreased in malignant cells. We also analyzed the association of miRNA expression with that of epithelial markers; prognostic indicators such as estrogen receptor, progesterone receptor, and HER2; as well as clinical outcome data. This ISH approach provides a more direct and informative assessment of how altered miRNA expression contributes to breast carcinogenesis compared with miRNA expression profiling in gross tissue biopsies. Most significantly, early manifestation of altered miR-145 expression in atypical hyperplasia and carcinoma in situ lesions suggests that this miRNA may have a potential clinical application as a novel biomarker for early detection.

The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes.

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.

The DEAD-box RNA helicase Dbp5 functions in translation termination.

In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.

Following temperature stress, export of heat shock mRNA occurs efficiently in cells with mutations in genes normally important for mRNA export.

Heat shock leads to accumulation of polyadenylated RNA in nuclei of Saccharomyces cerevisiae cells, transcriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect SSA4 mRNA (encoding Hsp70) and flow cytometry to measure the amount of Ssa4p-green fluorescent protein (GFP) produced following heat shock. Npl3p and Yra1p are mRNA-binding proteins recruited to nascent mRNAs and are essential for proper mRNA biogenesis and export. Heat shock mRNA was exported efficiently in temperature-sensitive npl3, yra1, and npl3 yra1 mutant strains. Nevertheless, Yra1p was recruited to heat shock mRNA, as were Nab2p and Npl3p. Interestingly, Yra1p was not recruited to heat shock mRNA in yra1-1 cells, suggesting that Npl3p is required for recruitment of Yra1p. The THO complex, which functions in transcription elongation and in recruitment of Yra1p, was not required for heat shock mRNA export, although normal mRNA export is impaired in growing cells lacking THO complex proteins. Taken together, these studies indicate that export following heat shock depends upon fewer factors than does mRNA export in growing cells. Furthermore, even though some mRNA-binding proteins are dispensable for efficient export of heat shock mRNA, those that are present in nuclei of heat shocked cells were recruited to heat shock mRNA.

The phylogenetic distribution of metazoan microRNAs: insights into evolutionary complexity and constraint.

How complex body plans evolved in animals such as fruit flies and vertebrates, as compared to the relatively simple jellyfish and sponges, is not known, given the similarity of developmental genetic repertoires shared by all these taxa. Here, we show that a core set of 18 microRNAs (miRNAs), non-coding RNA molecules that negatively regulate the expression of protein-coding genes, are found only in protostomes and deuterostomes and not in sponges or cnidarians. Because many of these miRNAs are expressed in specific tissues and/or organs, miRNA-mediated regulation could have played a fundamental evolutionary role in the origins of organs such as brain and heart--structures not found in cnidarians or sponges--and thus contributed greatly to the evolution of complex body plans. Furthermore, the continuous acquisition and fixation of miRNAs in various animal groups strongly correlates both with the hierarchy of metazoan relationships and with the non-random origination of metazoan morphological innovations through geologic time.

Transport of messenger RNA from the nucleus to the cytoplasm.

All movement of molecules and macromolecules between the cytoplasm and the nucleus takes place through nuclear pore complexes (NPCs), very large macromolecular complexes that are the only channels connecting these compartments. mRNA export is mediated by multiple, highly conserved protein factors that couple steps of nuclear pre-mRNA biogenesis to mRNA transport. Mature messenger ribonucleoproteins (mRNPs) diffuse from sites of transcription to NPCs, although some active genes are positioned at the nuclear periphery where they interact physically with components of NPCs. As properly processed mRNPs translocate through the pore, certain mRNP proteins are removed, probably through the enzymatic action of the DEAD-box helicase Dbp5, which binds to Nup159 and Gle1, components of the cytoplasmic filaments of the NPC. Gle1 and the phosphoinositide IP6 activate Dbp5's ATPase activity in vitro and this could provide critical spatial regulation of Dbp5 activity in vivo.

Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export.

In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export. Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription. A poly(A) tail added by the normal 3'-processing machinery is necessary but not sufficient for export. Retention depends on the exosome. In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3' processing to export. Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm. It can exit the nucleus either as cargo of exportin 1 or bound to mRNA. Pab1 is essential but several bypass suppressors have been identified. Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription. Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs. The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export.

Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export.

Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.