Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export

Vinciguerra, Patrizia; Iglesias, Nahid; Camblong, Jurgi; Zenklusen, Daniel; Stutz, Françoise

doi:10.1038/sj.emboj.7600527

Cited by 128 publications

(153 citation statements)

References 37 publications

(63 reference statements)

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“…4). We also identified Mlp1p and Mlp2p, proteins associated with the NPC and the nuclear periphery, whose interaction with Yra1p had previously been demonstrated 23 , and 24 proteins that had not been seen in any previous affinity purification using tagged Yra1p, including NPC components, splicing factors, a protein (Esc1p) found at the nuclear periphery and resembling in structure Mlp1p and Mlp2p (ref. 24), an E3 ubiquitin ligase (Tom1p) that functions during mRNA maturation 25 , and several mRNA-associated proteins.…”

Section: Rnp Complexes Do Not Rearrange During Isolationmentioning

confidence: 76%

Comprehensive analysis of diverse ribonucleoprotein complexes

et al. 2007

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The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.During and after eukaryotic transcription, RNA is packaged into RNP complexes, then modified, spliced, folded and exported into the cytoplasm through nuclear pore complexes (NPCs). There are two main forms of RNPs. Each contains a different type of RNA: mRNPs contain mRNA, and rRNPs contain ribosomal RNA (rRNA). Each also uses discrete processing factors, which associate with RNPs in a dynamic fashion to define the order of transcript maturation. The function and composition of many of these RNP complexes, however, is poorly understood.In recent years, there have been considerable efforts to study the composition of mRNPs and rRNPs in greater detail. A favored approach has been to purify these complexes from cell lysates [1][2][3][4] . But the limitations of presently available purification techniques pose a major hurdle to the study of RNA processing and export. Here we describe a method that aims to overcome these limitations and capture the dynamic interactome of maturing RNP complexes.We used this method to analyze complexes along both the ribosome and mRNA biogenesis pathways. RESULTSA strategy for isolation of RNP complexes Our strategy comprises seven steps that we optimized for efficient isolation of intact RNP complexes ( Fig. 1 and Supplementary Methods online). Collected cells are rapidly frozen in liquid nitrogen and broken open in the solid phase by milling 5 , using a planetary ball mill that produces particles of B1-2 mm in diameter (small enough for rapid and efficient RNP extraction). Because cell breakage occurs in the frozen state, we avoid damage to RNP complexes by released nucleases and proteases, as well as redistribution of proteins within the extract. The resulting frozen grindate is rapidly thawed into an RNP-compatible buffer (see Supplementary Methods), to produce an extract that is then immediately clarified by filtration. We isolate the tagged complexes using antibody-conjugated magnetic beads, rather than more commonly used antibody-conjugated resins such as Sepharose. The magnetic beads are small (2.8 mm) and so have a large surface area-to-volume ratio that increases the speed of binding. Moreover, the beads are impermeable, such that the antibodies are densely conjugated to their exposed surface; hence, there is no theoretical upper limit to the size of isolated complexes (unlike with permeable resins such as Sepharose, which are limited by their pore size). Incubation times...

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Section: Rnp Complexes Do Not Rearrange During Isolationmentioning

confidence: 76%

Comprehensive analysis of diverse ribonucleoprotein complexes

et al. 2007

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“…Notably, the C-terminal domain of Mlp1 (CT-Mlp1) binds to the hnRNPs, Nab2 and Npl3, and expression of CT-Mlp1 or the C-terminal domain of human Tpr causes nuclear accumulation of poly(A) RNA (38,46), linking the Mlp1/Tpr proteins with mRNA export. The Mlp1/2 proteins also bind to the mRNA export adaptor, Yra1 (39). Significantly, Mlp2 binds more strongly to the Yra1-8 export mutant protein than to wild-type Yra1, suggesting that Mlp2 recruits Yra1 for docking of mRNA-protein complexes, but retains malformed complexes that contain the Yra1-8 mutant protein (39).…”

mentioning

confidence: 99%

“…The Mlp1/2 proteins also bind to the mRNA export adaptor, Yra1 (39). Significantly, Mlp2 binds more strongly to the Yra1-8 export mutant protein than to wild-type Yra1, suggesting that Mlp2 recruits Yra1 for docking of mRNA-protein complexes, but retains malformed complexes that contain the Yra1-8 mutant protein (39). The yeast MLP1 and MLP2 genes are not essential for cell viability and mlp1⌬ or mlp2⌬ single deletion mutants or mlp1⌬ mlp2⌬ double deletion mutants are viable and show no significant growth defects (41,47,48).…”

mentioning

confidence: 99%

Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export

Fasken

Stewart

Corbett

2008

Journal of Biological Chemistry

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Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal domain of Nab2 (Nab2-N; residues 1-97) interacts directly with the C-terminal globular domain of Mlp1 (CTMlp1: residues 1490 -1875). Recent structural and binding studies focused on Nab2-N have shown that Nab2-N contains a hydrophobic patch centered on Phe 73 that is critical for interaction with Mlp1. Engineered amino acid changes within this patch disrupt the Nab2/Mlp1 interaction in vitro. Given the importance of Nab2 and Mlp1 to mRNA export, we have examined the Nab2/Mlp1 interaction in greater detail and analyzed the functional consequences of disrupting the interaction in vivo. We find that the Nab2-binding domain of Mlp1 (Mlp1-NBD) maps to a 183-residue region (residues 1586 -1768) within CT-Mlp1, binds directly to Nab2 with micromolar affinity, and confers nuclear accumulation of poly(A) RNA. Furthermore, we show that cells expressing a Nab2 F73D mutant that cannot interact with Mlp1 exhibit nuclear accumulation of poly(A) RNA and that this nab2 F73D mutant genetically interacts with alleles of two essential mRNA export genes, MEX67 and YRA1. These data provide in vivo evidence for a model of mRNA export in which Nab2 is important for targeting mRNAs to the nuclear pore for export.Production of mature mRNA for translation is a complex multistep process involving an array of RNA-binding proteins (1-3). From its transcriptional inception in the nucleus, pre-mRNA that emerges from RNA polymerase II must be bound by processing factors for 5Ј-end capping, splicing, and 3Ј-end cleavage and polyadenylation to reach maturation (4). Alongside these processing events, maturing mRNA must be co-transcriptionally loaded with heterogeneous nuclear ribonucleoproteins (hnRNPs), 2 mRNA export adaptors, and an mRNA export receptor to facilitate its active export from the nucleus to the cytoplasm through nuclear pore complexes (NPCs), large proteinaceous channels that perforate the nuclear membrane and mediate nucleocytoplasmic transport (1-3, 5). There are a number of nuclear quality controls that safeguard against improperly processed mRNA being exported and translated into deleterious proteins (6, 7). In particular, this quality control machinery likely monitors the state of processed mRNA by checking for the presence or absence of mRNA processing and export factors.One of the key players in mRNA export is the conserved heterodimeric mRNA export receptor, Mex67/Mtr2 (NXF1/ NXT1 or TAP/p15 in metazoans), which plays an essential role in bulk mRNA export in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster...

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“…Deletion of Mlp1 results in the leakage of unspliced transcripts into the cytoplasm (Galy et al, 2004). Mlp1 interacts with Nab2 Grant et al, 2008;Green et al, 2003;Vinciguerra et al, 2005), and the splice site-binding protein SF1 and the branch point-binding protein also interact with Mlp1 in an RNA-dependent manner (Galy et al, 2004). At the NPC, through sequestering unspliced transcripts or facilitating the export of mature mRNA, Mlps function as 'gatekeepers' to assure that only mature mRNAs are exported from the nucleus.…”

Section: Nuclear Retention Of Aberrant Mrnamentioning

confidence: 99%