Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides.

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N-acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers.

Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.

An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs. OBJECTIVE: We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1. METHODS: We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD. RESULTS: The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD. CONCLUSIONS: This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.

Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle.

BACKGROUND: The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays critical roles at multiple steps in post-transcriptional regulation of gene expression. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing, and weakness in the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-specific pathology is unknown. Given the general function of PABPN1 in RNA metabolism, intrinsic characteristics of skeletal muscle may make this tissue susceptible to the effects of mutant PABPN1. METHODS: To begin to understand the muscle specificity of OPMD, we investigated the steady-state levels of PABPN1 in different tissues of humans and mice. Additionally, we analyzed the levels of PABPN1 during muscle regeneration after injury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay in skeletal muscle compared to kidney. RESULTS: Here, we show that the steady-state levels of both PABPN1 mRNA and protein are drastically lower in mouse and human skeletal muscle, particularly those impacted in OPMD, compared to other tissues. In contrast, PABPN1 levels are increased during muscle regeneration, suggesting a greater requirement for PABPN1 function during tissue repair. Further analysis indicates that modulation of PABPN1 expression is likely due to post-transcriptional mechanisms acting at the level of mRNA stability. CONCLUSIONS: Our results demonstrate that PABPN1 steady-state levels and likely control of expression differ significantly in skeletal muscle as compared to other tissues, which could have important implications for understanding the muscle-specific nature of OPMD.

PABPN1: molecular function and muscle disease.

The polyadenosine RNA binding protein polyadenylate-binding nuclear protein 1 (PABPN1) plays key roles in post-transcriptional processing of RNA. Although PABPN1 is ubiquitously expressed and presumably contributes to control of gene expression in all tissues, mutation of the PABPN1 gene causes the disease oculopharyngeal muscular dystrophy (OPMD), in which a limited set of skeletal muscles are affected. A major goal in the field of OPMD research is to understand why mutation of a ubiquitously expressed gene leads to a muscle-specific disease. PABPN1 plays a well-documented role in controlling the poly(A) tail length of RNA transcripts but new functions are emerging through studies that exploit a variety of unbiased screens as well as model organisms. This review addresses (a) the molecular function of PABPN1 incorporating recent findings that reveal novel cellular functions for PABPN1 and (b) the approaches that are being used to understand the molecular defects that stem from expression of mutant PABPN1. The long-term goal in this field of research is to understand the key molecular functions of PABPN1 in muscle as well as the mechanisms that underlie the pathological consequences of mutant PABPN1. Armed with this information, researchers can seek to develop therapeutic approaches to enhance the quality of life for patients afflicted with OPMD.

Expression of interferon-γ, interferon-α and related genes in individuals with Down syndrome and periodontitis.

BACKGROUND: Recently, attenuation of anti-inflammatory and increase of pro-inflammatory mediators was demonstrated in individuals with Down syndrome (DS) in comparison with euploid patients during periodontal disease (PD), suggesting a shift to a more aggressive inflammation in DS. AIM: To determine the influence of DS in the modulation of interferons (IFNs) signaling pathway in PD. MATERIALS AND METHODS: Clinical periodontal assessment was performed and gingival tissue samples obtained from a total of 51 subjects, including 19 DS individuals with PD, 20 euploid individuals with PD and 12 euploid individuals without PD. Expression levels of interferon-gamma (IFNG) and interferon-alpha (IFNA), and their receptors IFNGR1, IFNGR2, IFNAR1 and IFNAR2, the signaling intermediates Janus kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1) were determined using real time quantitative polymerase chain reaction (qPCR). RESULTS: Clinical signs of periodontal disease were markedly more severe in DS and euploid patients with PD in comparison to euploid and periodontally healthy patients. There was no difference on mRNA levels of IFNA, IFNG, INFGR2, IFNAR1 and IFNAR2 between DS and euploid individuals, even though some of these genes are located on chromosome 21. STAT1 and IRF1 mRNA levels were significantly lower in DS patients in comparison with euploid individuals with PD. In euploid individuals, PD was associated with an increased expression of IFNGR1, IFNGR2, IFNAR1, STAT1 and IRF1. CONCLUSIONS: Reduced expression of STAT1 and IRF1 genes indicate an impaired activation of IFNs signaling in individuals with DS and PD. Expression of IFNA, IFNG and IFN receptors was not altered in DS patients, indicating that indirect mechanisms are involved in the reduced activation of IFN signaling.

RNA-binding proteins and gene regulation in myogenesis.

Skeletal muscle development, repair and function are dependent on highly coordinated expression of many genes. RNA-binding proteins are crucial determinants of gene expression in the health and disease of various tissues, including skeletal muscle. A variety of RNA-binding proteins are associated with a transcript during its life cycle and define the lifetime, cellular localization, processing and rate at which that transcript is translated and ultimately degraded. The focus of this review is to highlight the roles of the best-characterized RNA-binding proteins in muscle, including HuR, KSRP, CUGBP1, PABPN1, Lin-28 and TTP. Recent studies indicate key functions for these RNA-binding proteins in different aspects of muscle physiology. Understanding the role of specific RNA-binding proteins in skeletal muscle will provide insights not only into basic mechanisms regulating gene expression in muscle, but also into the etiology and pathology of muscle disease.

Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans.

Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.

The mitogen-activated protein kinase Slt2 regulates nuclear retention of non-heat shock mRNAs during heat shock-induced stress.

Cellular adaptation to environmental stress conditions requires rapid and specific changes in gene expression. During heat shock, most polyadenylated mRNAs are retained in the nucleus, whereas the export of heat shock-induced mRNAs is allowed. Although essential mRNA export factors are known, the precise mechanism for regulating transport is not fully understood. Here we find that during heat shock in Saccharomyces cerevisiae, the mRNA-binding protein Nab2 is phosphorylated on threonine 178 and serine 180 by the mitogen-activated protein (MAP) kinase Slt2/Mpk1. Slt2 is required for nuclear poly(A(+)) mRNA accumulation upon heat shock, and thermotolerance is decreased in a nup42 nab2-T178A/S180A mutant. Coincident with phosphorylation, Nab2 and Yra1 colocalize in nuclear foci with Mlp1, a protein involved in mRNA retention. Nab2 nuclear focus formation and Nab2 phosphorylation are independent, suggesting that heat shock induces multiple cellular alterations that impinge upon transport efficiency. Under normal conditions, we find that the mRNA export receptor Mex67 and Nab2 directly interact. However, upon heat shock stress, Mex67 does not localize to the Mlp1 nuclear foci, and its association with Nab2 complexes is reduced. These results reveal a novel mechanism by which the MAP kinase Slt2 and Mlp1 control mRNA export factors during heat shock stress.

Chemokine expression and control of muscle cell migration during myogenesis.

Adult regenerative myogenesis is vital for restoring normal tissue structure after muscle injury. Muscle regeneration is dependent on progenitor satellite cells, which proliferate in response to injury, and their progeny differentiate and undergo cell-cell fusion to form regenerating myofibers. Myogenic progenitor cells must be precisely regulated and positioned for proper cell fusion to occur. Chemokines are secreted proteins that share both leukocyte chemoattractant and cytokine-like behavior and affect the physiology of a number of cell types. We investigated the steady-state mRNA levels of 84 chemokines, chemokine receptors and signaling molecules, to obtain a comprehensive view of chemokine expression by muscle cells during myogenesis in vitro. A large number of chemokines and chemokine receptors were expressed by primary mouse muscle cells, especially during times of extensive cell-cell fusion. Furthermore, muscle cells exhibited different migratory behavior throughout myogenesis in vitro. One receptor-ligand pair, CXCR4-SDF-1alpha (CXCL12), regulated migration of both proliferating and terminally differentiated muscle cells, and was necessary for proper fusion of muscle cells. Given the large number of chemokines and chemokine receptors directly expressed by muscle cells, these proteins might have a greater role in myogenesis than previously appreciated.

Recognition of polyadenosine RNA by the zinc finger domain of nuclear poly(A) RNA-binding protein 2 (Nab2) is required for correct mRNA 3'-end formation.

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. We also demonstrate genetic interactions between mutant nab2 alleles and mutant alleles of the mRNA 3'-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length.

Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis.

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein.

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2, 3, and 3 short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain.

Expression and purification of human respiratory syncytial virus recombinant fusion protein.

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. coli and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies.

Synthetic lethality between eIF5A and Ypt1 reveals a connection between translation and the secretory pathway in yeast.

The putative translation initiation factor 5A (eIF5A) is a small protein, highly conserved and essential in all organisms from archaea to mammals. Although the involvement of eIF5A in translation initiation has been questioned, new evidence reestablished the connection between eIF5A and this cellular process. In order to better understand the function of elF5A, a screen for synthetic lethal gene using the tif51A-3 mutant was carried out and a new mutation (G80D) was found in the essential gene YPT1, encoding a protein involved in vesicular trafficking. The precursor form of the vacuolar protein CPY is accumulated in the ypt1-G80D mutant at the nonpermissive temperature, but this defect in vesicular trafficking did not occur in the tif51A mutants tested. Overexpression of eIF5A suppresses the growth defect of a series of ypt1 mutants, but this suppression does not restore correct CPY sorting. On the other hand, overexpression of YPT1 does not suppress the growth defect of tif51A mutants. Further, it was revealed that eIF-5A is present in both soluble and membrane fractions, and its membrane association is ribosome-dependent. Finally, we demonstrated that the ypt1 and other secretion pathway mutants are sensitive to paromomycin. These results confirm the link between translation and vesicular trafficking and reinforce the implication of eIF5A in protein synthesis.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis.

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins--eIF5A(K56A) and eIF5A(Q22H,L93F)--and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA processing/export and mRNA stability.

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex.

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy (Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. (2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.