Article

Interaction between the human nuclear cap-binding protein complex and hnRNP F

European Molecular Biology Laboratory, Heidelberg, Germany.
Molecular and Cellular Biology (Impact Factor: 5.04). 06/1997; 17(5):2587-97.
Source: PubMed

ABSTRACT hnRNP F was identified in a screen for proteins that interact with human CBP80 and CBP20, the components of the nuclear cap-binding complex (CBC). In vitro interaction studies showed that hnRNP F can bind to both CBP20 and CBP80 individually. hnRNP F and CBC bind independently to RNA, but hnRNP F binds preferentially to CBC-RNA complexes rather than to naked RNA. The hnRNP H protein, which is 78% identical to hnRNP F and also interacts with both CBP80 and CBP20 in vitro, does not discriminate between naked RNA and CBC-RNA complexes, showing that this effect is specific. Depletion of hnRNP F from HeLa cell nuclear extract decreases the efficiency of pre-mRNA splicing, a defect which can be partially compensated by addition of recombinant hnRNP F. Thus, hnRNP F is required for efficient pre-mRNA splicing in vitro and may participate in the effect of CBC on pre-mRNA splicing.

Download full-text

Full-text

Available from: Elisa Izaurralde, Sep 09, 2014
0 Followers
 · 
78 Views
  • Source
    • "The molecular machinery regulating cotranscriptional RNA metabolism is still not well understood, particularly in plants. Work in yeast and humans has shown that the CBC, composed of the two conserved subunits CBC80 and CBC20, associates with the CTD of RNA polymerase II and is required for many aspects of RNA splicing, RNA stability, and export and has been proposed to affect transcriptional elongation via a checkpoint mechanism (Gamberi et al., 1997; Shatkin and Manley, 2000). Our analysis reveals that FRI is a direct interactor of the CBC, with suppression of FRI activity by both cbp20 and cbp80 providing in vivo endorsement of the importance of the CBC for FRI function. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A major determinant of flowering time in natural Arabidopsis (Arabidopsis thaliana) variants is FRIGIDA (FRI). FRI up-regulates expression of the floral repressor FLOWERING LOCUS C (FLC), thereby conferring a vernalization requirement and a winter annual habit. FRI encodes a novel nuclear protein with no conserved domains except for two coiled-coil regions. A mutation in the large subunit of the nuclear cap-binding complex (CBC) suppresses FRI activity, so we have explored the connection between FRI and the nuclear CBC in order to gain further insight into FRI biochemical activity. Mutations in the small subunit of the CBC (CBP20) also suppress FRI up-regulation of FLC. CBP20 interacted directly with FRI in yeast and in planta, and this association of FRI with the 5' cap was reinforced by an RNA ligase-mediated rapid amplification of cDNA ends assay that showed FRI decreased the proportion of FLC transcripts lacking a 5' cap. Loss of CBP20 resulted in very low FLC mRNA levels and an increased proportion of unspliced FLC transcripts. FRI compensated for CBP20 loss, partially restoring FLC levels and normalizing the unspliced-spliced transcript ratio. Our data suggest that FRI up-regulates FLC expression through a cotranscriptional mechanism involving direct physical interaction with the nuclear CBC with concomitant effects on FLC transcription and splicing.
    Plant physiology 06/2009; 150(3):1611-8. DOI:10.1104/pp.109.137448 · 7.39 Impact Factor
  • Source
    • "Primer sequences are given in Table S2. EMSA was performed as described (Gamberi et al., 1997), except that incubations were performed at room temperature and the gels were run in 0.53 TBE at 4 C. For supershift experiments, a-Bic-C was affinity purified against immobilized MBP-Bic-C, concentrated by ultrafiltration, and titrated empirically. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bicaudal-C (Bic-C) encodes an RNA-binding protein required maternally for patterning the Drosophila embryo. We identified a set of mRNAs that associate with Bic-C in ovarian ribonucleoprotein complexes. These mRNAs are enriched for mRNAs that function in oogenesis and in cytoskeletal regulation, and include Bic-C RNA itself. Bic-C binds specific segments of the Bic-C 5' untranslated region and negatively regulates its own expression by binding directly to NOT3/5, a component of the CCR4 core deadenylase complex, thereby promoting deadenylation. Bic-C overexpression induces premature cytoplasmic-streaming, a posterior-group phenotype, defects in Oskar and Kinesin heavy chain:betaGal localization as well as dorsal-appendage defects. These phenotypes are largely reciprocal to those of Bic-C mutants, and they affect cellular processes that Bic-C-associated mRNAs are known, or predicted, to regulate. We conclude that Bic-C regulates expression of specific germline mRNAs by controlling their poly(A)-tail length.
    Developmental Cell 12/2007; 13(5):691-704. DOI:10.1016/j.devcel.2007.10.002 · 10.37 Impact Factor
  • Source
    • "Most members of the family of hnRNP proteins are known for their nuclear localization, nuclear-cytoplasmic shuttling, and their interaction with RNA or other RNA binding proteins, and are predicted to be functionally involved in diverse aspect of RNA metabolism [5] [6] [7]. Many of the hnRNP members contain so-called RGG-boxes 0006-291X/$ -see front matter Ó 2006 Elsevier Inc. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm.
    Biochemical and Biophysical Research Communications 08/2006; 346(2):517-25. DOI:10.1016/j.bbrc.2006.05.152 · 2.28 Impact Factor
Show more