No abstract

The human protein C23 (nucleolin) is a major nucleolar protein. Its interactions with other proteins were studied with the two-hybrid system which identified nucleolar protein B23 (nucleoph'osmin) as being associated with C23. Both proteins were co-immunoprecipitated from HeLa cell nuclear extract by either monoclonal antLC23 or monoclonal anti-B23. Binding studies utilizing deletion mutants indicated that the binding of C23 and B23 involves specific motifs. In addition to an approximately 46-amino-acidbinding domain in B23 (amino acids 194-239), amino acids 540-628 of C23 were required for binding; this region of C23 is required for the nucleolar localization. In addition, nucleolar protein p120 was also found to be co-immunoprecipitated with B23. A fragment of p120 containing a functional nucleolar localization signal bound to the truncated binding domain of B23, as did C23. These results suggest that the interaction of C23 and B23 may represent a nucleolar-targeting mechanism in which B23 acts as a nucleolar-localization signal-binding protein.Keywords: C23 ; nucleolin; B23 ; nucleophosmin ; protein-protein interaction.The nucleolus is the site where preribosomes are formed. Recent studies showed that the nucleolus may also be important for poly(A)-rich RNA transport and processing [l]. Over the years, a number of nucleolar proteins have been discovered and characterized in our laboratory, including protein C23 (nucleolin) [2], protein B23 (nucleophosmin) [2], and protein p120 [3].Although a great deal of knowledge on the chemistry of these proteins has been accumulated, the role of these proteins in nucleolar structure and function is still unknown. In this regard, we consider that understanding the interactions between certain nucleolar proteins would be useful. Thus, the interaction of nucleolar protein C23 with other proteins was studied.C23 is a highly conserved major nucleolar protein found in multiple species including human, rodent, insect, frog and chicken cells Abbreviations. NOR, nucleolar organizer regions ; RRM, RNA recognition motif; GST, glutathione S-transferase; NoLS, nucleolar-localization signal.College of Medicine, Houston, TX 77030 has also been confirmed by co-immunoprecipitation of the two proteins from HeLa cell nuclear extracts. Further studies utilizing deletion mutants of C23 or B23 revealed that the binding of the two proteins requires specific amino acid motifs and such binding may be important for the nucleolar localization of C23.

Treacher Collins syndrome (TCS) is characterized by defects in craniofacial development, which results from mutations in the TCOF1 gene. TCOF1 encodes the nucleolar phosphoprotein treacle, which interacts with upstream binding factor (UBF) and affects transcription of the ribosomal DNA gene. The present study shows participation of treacle in the 2'-O-methylation of pre-rRNA. Antisense-mediated down-regulation of treacle expression in Xenopus laevis oocytes reduced 2'-O-methylation of pre-rRNA. Analysis of RNA isolated from wild-type and Tcof1+/- heterozygous mice embryos from strains that exhibit a lethal phenotype showed significant reduction in 2'-O-methylation at nucleotide C463 of 18S rRNA. The level of pseudouridylation of U1642 of 18S rRNA from the same RNA samples was not affected suggesting specificity. There is no significant difference in rRNA methylation between wild-type and heterozygous embryos of DBA x BALB/c mice, which have no obvious craniofacial phenotype. The function of treacle in pre-rRNA methylation is most likely mediated by its direct physical interaction with NOP56, a component of the ribonucleoprotein methylation complex. Although treacle co-localizes with UBF throughout mitosis, it co-localizes with NOP56 and fibrillarin, a putative methyl transferase, only during telophase when rDNA gene transcription and pre-rRNA methylation are known to commence. These observations suggest that treacle might link RNA polymerase I-catalyzed transcription and post-transcriptional modification of pre-rRNA. We hypothesize that haploinsufficiency of treacle in TCS patients results in inhibition of production of properly modified mature rRNA in addition to inhibition of rDNA gene transcription, which consequently affects proliferation and proper differentiation of specific embryonic cells during development.

Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfered with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells.