Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4.

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48-UN complex and its assembly.

ORC interacts with THSC/TREX-2 and its subunits promote Nxf1 association with mRNP and mRNA export in Drosophila.

The origin recognition complex (ORC) of eukaryotes associates with the replication origins and initiates the pre-replication complex assembly. In the literature, there are several reports of interaction of ORC with different RNAs. Here, we demonstrate for the first time a direct interaction of ORC with the THSC/TREX-2 mRNA nuclear export complex. The THSC/TREX-2 was purified from the Drosophila embryonic extract and found to bind with a fraction of the ORC. This interaction occurred via several subunits and was essential for Drosophila viability. Also, ORC was associated with mRNP, which was facilitated by TREX-2. ORC subunits interacted with the Nxf1 receptor mediating the bulk mRNA export. The knockdown of Orc5 led to a drop in the Nxf1 association with mRNP, while Orc3 knockdown increased the level of mRNP-bound Nxf1. The knockdown of Orc5, Orc3 and several other ORC subunits led to an accumulation of mRNA in the nucleus, suggesting that ORC participates in the regulation of the mRNP export.

Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae.

The nuclear export of macromolecules is facilitated by the nuclear pore complexes (NPCs), embedded in the nuclear envelope and consists of multi-protein complexes. MEX67 is one of the nuclear export factor responsible for the transport of the majority of cellular mRNAs from the nucleus to the cytoplasm. The mechanism of mRNA transport through NPCs is unclear due to the unavailability of structures and the known interacting partners of MEX67. The mex67 gene was cloned in pQE30A and was expressed in Escherichia coli. A strategy has been developed to purify the insoluble MEX67 using a nickel affinity column with chelating Sepharose fast flow media, after solubilizing with sodium lauroyl sarcosinate (Sarkosyl). The IMAC purified recombinant MEX67 was further purified using SEC to apparent homogeneity (∼8 mg/L). Following SEC, MEX67 was stable and observed to be a 67 kDa monomeric protein as determined by PAGE and the size exclusion chromatography. The availability of large quantities of the protein will help in its biochemical and biophysical characterization, which may lead to the identification of new interaction partners of MEX67 or MEX67 complex.

Purification of nuclear poly(A)-binding protein Nab2 reveals association with the yeast transcriptome and a messenger ribonucleoprotein core structure.

Nascent mRNAs produced by transcription in the nucleus are subsequently processed and packaged into mRNA ribonucleoprotein particles (messenger ribonucleoproteins (mRNPs)) before export to the cytoplasm. Here, we have used the poly(A)-binding protein Nab2 to isolate mRNPs from yeast under conditions that preserve mRNA integrity. Upon Nab2-tandem affinity purification, several mRNA export factors were co-enriched (Yra1, Mex67, THO-TREX) that were present in mRNPs of different size and mRNA length. High-throughput sequencing of the co-precipitated RNAs indicated that Nab2 is associated with the bulk of yeast transcripts with no specificity for different mRNA classes. Electron microscopy revealed that many of the mRNPs have a characteristic elongated structure. Our data suggest that mRNPs, although associated with different mRNAs, have a unifying core structure.

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.

XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.

During splicing and polyadenylation, factors that stimulate export from the nucleus are recruited to nascent mRNAs. X-inactive specific transcript (XIST) RNA is unusual among capped, spliced, polyadenylated transcripts in that it accumulates exclusively in the nucleus. It is well established that, at steady state levels, XIST RNA is primarily nuclear. However, it was unknown whether XIST RNA spends its entire lifetime in the nucleus (nuclear retention) or passes briefly through the cytoplasm during maturation, like many other functional RNAs. In this study, we present the first evidence that XIST RNA exhibits nuclear retention. We report that a green fluorescent protein (GFP)-XIST fusion RNA is detected in the nucleus and not the cytoplasm, and GFP is not translated. XIST RNA does not shuttle in a heterokaryon assay or move between chromosomes in the same nucleus when expressed at wild-type levels. These results indicate that XIST RNA's nuclear localization is mediated by nuclear retention rather than export followed by import. We present evidence that the export factor TAP/NXF1 binds poorly to XIST RNA in comparison to exported mRNAs, suggesting that reduced TAP/NFX1 binding may contribute to nuclear retention of XIST RNA.

Epitaxial deposition of calcium oxalate on uric acid rich stone matrix is induced by a 29 kDa protein.

BACKGROUND: Association of macromolecules particularly the role of proteins in urolithiasis has been studied for last few centuries, but still a complete profile of stone matrix proteins that mediate co-precipitation of uric acid and calcium oxalate has not been characterized. We isolated and characterize proteins from uric acid rich stone matrix, which have oxalate binding activity. METHODS: Matrix proteins were isolated from uric acid rich stone matrix using EDTA as a demineralizing agent. The radiolabelled solubilized proteins were fractionated with increasing ionic concentration by DEAE cellulose column chromatography to identify the oxalate binding protein. It was purified using Sephadex G-200 column chromatography. Amino acid composition was determined and monoclonal antibody was produced against the oxalate binding uric acid rich stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers urine, their oxalate binding activity assayed and cross reactivity with the produced monoclonal antibody were checked using ELISA and Western blotting. RESULTS: Matrix on DEAE column chromatography elution yielded 3 protein peaks and they were named as fraction I, II and III among which fraction I had higher oxalate binding activity which was further purified with Sephadex G-200 column which yielded 2 protein peaks designated as Ia and Ib. Fraction Ib with molecular weight 29 kDa exhibited the maximum oxalate binding activity. Forty percent of this 29 kDa protein is comprised of basic amino acids. Monoclonal antibody (IgG1) was produced against the 29 kDa stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers, 4 protein peaks were obtained named as fraction I to IV. Among them, fraction IV having molecular weight of approximately 29 kDa cross reacted up to 85.6% with 29 kDa stone matrix protein. Moreover, urinary 29 kDa protein exhibited oxalate binding activity of 94.16 +/- 6.08 pmol/mg protein at pH 5.5. CONCLUSION: The 29 kDa protein isolated from uric acid rich stone matrix and urine are one and the same, thereby insinuating that 29 kDa protein might play a major role in epitaxial deposition of calcium oxalate over uric acid core, consequently favoring the lithogenic events like uric acid and calcium oxalate nucleation, aggregation and retention.

Nuclear transport factor 2 represents a novel cross-reactive fungal allergen.

BACKGROUND: Ubiquitously occuring moulds are important allergenic sources known to elicit IgE-mediated allergic diseases and to share cross-reactive allergens. Limited information is available about the molecular structures involved in cross-reactivity. We aimed to clone and characterize cross-reactive mould allergens. METHODS: Phage surface-displayed Alternaria alternata and Cladosporium herbarum cDNA libraries were screened using sera from Aspergillus fumigatus-sensitized patients. Inserts encoding putative allergens were sequenced, and recombinant proteins used to demonstrate cross-reactivity by inhibition experiments and skin test. Three-dimensional homology models of cloned putative nuclear transport factor 2 (NTF2) were constructed based on known NTF2 structure to corroborate the functional and structural properties of the novel allergens. RESULTS: After six rounds of affinity selection, the libraries were enriched for clones displaying allergens. Sequencing of inserts showed that some clones derived from Alternaria alternata and Cladosporium herbarum contain open reading frames predicting proteins of 124 and 125 amino acids corresponding to NTF2. The recombinant proteins were able to bind and cross-inhibit IgE binding and to elicit type I skin reactions in mould-sensitized individuals, demonstrating the allergenicity of the proteins. CONCLUSIONS: NTF2 represents a novel cross-reactive fungal allergen as demonstrated by sequence homology, three-dimensional modelling, inhibition experiments and skin test reactivity.