Weighardt, F. et al. A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock. J. Cell Sci. 112, 1465-1476

Istituto di Genetica Biochimica ed Evoluzionistica, CNR Via Abbiategrasso 207, Italy.
Journal of Cell Science (Impact Factor: 5.43). 06/1999; 112 ( Pt 10)(10):1465-76.
Source: PubMed

ABSTRACT A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.

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Available from: Giuseppe Biamonti, Sep 29, 2015
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    • "SAFB1 and SAFB2 proteins share a highly conserved RNA-recognition motif (RRM) with 98% similarity in the central region, although until now their direct RNA-binding potential has remained unclear. SAFB1 has also been labelled as a novel hnRNP protein due to its similarity to the highly conserved RBD found in the hnRNP protein family [6]. Subsequent studies have implicated both SAFB proteins in alternative splicing, as overexpression of SAFB1 and SAFB2 inhibits splicing of a TRA2B variable exon [5] [7]. "
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    ABSTRACT: Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3' or 5' untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells.
    08/2015; 2015:395816. DOI:10.1155/2015/395816
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    • "If this kind of response is indeed a physiological one, we thought that it should be an environmental- or cellular-stress that triggers this specific response. It is well known that in higher organisms, heat shock induces an elaborate stress response that involves different signalling pathways [57], which, among other things, shuts down the transcription of most genes [58], [59]. In epimastigotes, which are normally cultured at 28°C, the classical heat shock response is initiated at 40°C [60]. "
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    ABSTRACT: In this work we show that under Actinomycin D (ActD) treatment, several RNA Binding Proteins (RBPs) involved in mRNA metabolism are relocalized into the nucleolus in Trypanosoma cruzi as a specific stress response. ATP depletion as well as kinase inhibition markedly reduced the nucleolar localization response, suggesting that an energy-dependent transport modulated by the phosphorylation status of the parasite might be required. Deletion analyses in one of such proteins, TcSR62, showed that a domain bearing basic amino acids located in the COOH terminal region was sufficient to promote its nucleolar relocalization. Interestingly, we showed that in addition to RBPs, poly(A)+ RNA is also accumulated into the nucleolus in response to ActD treatment. Finally, we found out that nucleolar relocalization of RBPs is also triggered by severe heat shock in a reversible way. Together, these results suggest that the nucleolus of an early divergent eukaryote is either able to sequester key factors related to mRNA metabolism in response to transcriptional stress or behaves as a RBP processing center, arguing in favour to the hypothesis that the non-traditional features of the nucleolus could be acquired early during evolution.
    PLoS ONE 05/2011; 6(5):e19920. DOI:10.1371/journal.pone.0019920 · 3.23 Impact Factor
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    • "Although HSF1 granules form at the onset of the heat shock response and rapidly disappear during the recovery period following heat shock, HAP bodies become visible after 1 hour of mild heat shock, with a maximum size reached after 3 hours of recovery. The formation of HAP bodies requires ongoing transcription (Weighardt et al. 1999) and these structures are sensitive to RNAse treatment (Chiodi et al. 2000), suggesting that RNA is a major component of nSBs. "
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    ABSTRACT: Nuclear stress bodies (nSBs) are unique subnuclear organelles which form in response to heat shock. They are initiated through a direct interaction between heat shock transcription factor 1 (HSF1) and pericentric tandem repeats of satellite III sequences and correspond to active transcription sites for noncoding satellite III transcripts. Given their unusual features, nSBs are distinct from other known transcription sites. In stressed cells, they are thought to participate in rapid, transient, and global reprogramming of gene expression through different types of mechanisms including chromatin remodeling and trapping of transcription and splicing factors. The analysis of these atypical and intriguing structures uncovers new facets of the relationship between nuclear organization and nuclear function.
    Cold Spring Harbor perspectives in biology 06/2010; 2(6):a000695. DOI:10.1101/cshperspect.a000695 · 8.68 Impact Factor
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