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Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins modulates splicing

Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Nucleic Acids Research (Impact Factor: 9.11). 05/2009; 37(11):3501-13. DOI: 10.1093/nar/gkp218
Source: PubMed

ABSTRACT The biological functions of poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) are not well understood. However, it is known that hnRNPs are involved in the regulation of alternative splicing for many genes, including the Ddc gene in Drosophila. Therefore, we first confirmed that poly(ADP-ribose) (pADPr) interacts with two Drosophila hnRNPs, Squid/hrp40 and Hrb98DE/hrp38, and that this function is regulated by Poly(ADP-ribose) Polymerase 1 (PARP1) and Poly(ADP-ribose) Glycohydrolase (PARG) in vivo. These findings then provided a basis for analyzing the role of pADPr binding to these two hnRNPs in terms of alternative splicing regulation. Our results showed that Parg null mutation does cause poly(ADP-ribosyl)ation of Squid and hrp38 protein, as well as their dissociation from active chromatin. Our data also indicated that pADPr binding to hnRNPs inhibits the RNA-binding ability of hnRNPs. Following that, we demonstrated that poly(ADP-ribosyl)ation of Squid and hrp38 proteins inhibits splicing of the intron in the Hsr omega-RC transcript, but enhances splicing of the intron in the Ddc pre-mRNA. Taken together, these findings suggest that poly(ADP-ribosyl)ation regulates the interaction between hnRNPs and RNA and thus modulates the splicing pathways.

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    • "The hsrω is a developmentally active, heat shock (HS) inducible, noncoding gene in Drosophila (reviewed in Lakhotia, 2011) producing multiple lncRNAs (http://flybase.org). Its most extensively studied nucleus limited hsrω-n1 (hsrω-RB) and spliced hsrω-n2 (hsrω-RG) lncRNAs, together referred to as hsrω-n (Mallik and Lakhotia, 2011), interact with variety of RNA processing proteins including Hrb87F/Hrp36, Hrb57A/Bancal, Hrb98DE/Hrp38, Squid/Hrp40, PEP, Rumpelstiltskin/Hrp59, NonA, and Sxl (Prasanth et al., 2000; Jolly and Lakhotia, 2006; Ji and Tulin, 2009; Onorati et al., 2011; Singh and Lakhotia, 2012) to organize the omega speckles. "
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    ABSTRACT: The nucleus limited long-noncoding hsrω-n transcripts, hnRNPs, and some other RNA processing proteins organize nucleoplasmic omega speckles in Drosophila. Unlike other nuclear speckles, omega speckles rapidly disappear following cell stress, while hnRNPs and other associated proteins move away from chromosome sites, nucleoplasm, and the disappearing speckles to get uniquely sequestered at hsrω locus. Omega speckles reappear and hnRNPs get redistributed to normal locations during recovery from stress. With a view to understand the dynamics of omega speckles and their associated proteins, we used live imaging of GFP tagged hnRNPs (Hrb87F, Hrb98DE, or Squid) in unstressed and stressed Drosophila cells. Omega speckles display size-dependent mobility in nucleoplasmic domains with significant colocalization with nuclear matrix Tpr/Megator and SAFB proteins, which also accumulate at hsrω gene site after stress. Instead of moving towards the nuclear periphery located hsrω locus following heat shock or colchicine treatment, omega speckles rapidly disappear within nucleoplasm while chromosomal and nucleoplasmic hnRNPs move, stochastically or, more likely, by nuclear matrix-mediated transport to hsrω locus in non-particulate form. Continuing transcription of hsrω during cell stress is essential for sequestering incoming hnRNPs at the site. While recovering from stress, the sequestered hnRNPs are released as omega speckles in ISWI-dependent manner. Photobleaching studies reveal hnRNPs to freely move between nucleoplasm, omega speckles, chromosome regions, and hsrω gene site although their residence periods at chromosomes and hsrω locus are longer. A model for regulation of exchange of hnRNPs between nuclear compartments by hsrω-n transcripts is presented.
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    • "In fact, pADPr is already known to interfere with domain essential to protein-protein interaction and has the ability to act as a scaffold molecule. For example, pADPr will most likely modulate activities of proteins within SG, as seen with the ability of pADPr to shift activity of topoisomerase I towards ASF/SF2 (Yung et al., 2004; Malanga et al., 2008) or the postulated ability of pADPr to modulate the activity of hnRNPs, thereby affecting mRNA splicing (Ji and Tulin, 2009). Moreover, Leung et al. (Leung et al., 2011) demonstrate that pADPr modulates the assembly and maintenance of an mRNP-enriched structure. "
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    ABSTRACT: Poly(ADP-ribose) (pADPr) is heterogenic molecule synthesized from NAD by poly(ADP-ribose) polymerases (PARPs). Multiple cellular functions are affected by pADPr through its network of associated proteins ranging from genome integrity surveillance, cell cycle progression, DNA repair to apoptosis. Using quantitative proteomics, we established a temporal map of pADPr-associated complexes upon genotoxic stress. Results suggested a strong pADPr-association of multiple proteins involved in stress granule formation, notably G3BP, in latter phases of alkylation-stress-induced cells. Further investigation with dynamic imaging clearly demonstrated a pADPr-dependent initiation of stress granule assembly originating from the nucleus. The co-transfection of G3BP with poly(ADP-ribose) glycohydrolase PARG indicates that pADPr is involved in modulating the nuclear shuttling of G3BP. Moreover, a peptide pADPr blot assay of G3BP revealed that pADPr binds to the glycine-arginine rich domain of G3BP. Thereafter, we established a comprehensive G3BP interactome in presence of pADPr. Our findings establish a novel function for pADPr in the formation of G3BP-induced stress granules upon genotoxic stress.
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    • "Alternatively, the sizeable pADPr modification might cause steric hindrance to prohibit effective miRNA silencing. Recently, it has been shown that in vitro addition of pADPr inhibits the RNA-binding ability of a Drosophila heterogeneous nuclear ribonucleoprotein (Ji and Tulin, 2009). Given that pADPr also exhibited binding affinity to RNA-binding proteins (Chang et al., 2009; Gagne et al., 2008), one function of pADPr could be to regulate the binding of RNAs to RNA-binding proteins. "
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