Benigno C. Valdez

The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription.

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.

Silencing of RNA Helicase II/Guα Inhibits Mammalian Ribosomal RNA Production

The intricate production of ribosomal RNA is well defined in yeast, but its complexity in higher organisms is barely understood. We recently showed that down-regulation of nucleolar protein RNA helicase II/Guα (RH-II/Guα or DDX21) in Xenopus oocytes inhibited processing of 20 S rRNA to 18 S and contributed to degradation of 28 S rRNA (Yang, H., Zhou, J., Ochs, R. L., Henning, D., Jin, R., and Valdez, B. C. (2003) J. Biol. Chem. 278, 38847–38859). Since no nucleolar RNA helicase has been functionally characterized in mammalian cells, we used short interfering RNA to search for functions for RH-II/Guα and its paralogue RH-II/Guβ in rRNA production. Silencing of RH-II/Guα by more than 80% in HeLa cells resulted in an almost 80% inhibition of 18 and 28 S rRNA production. This inhibition could be reversed by exogenous expression of wild type RH-II/Guα. A helicase-deficient mutant form having ATPase activity was able to rescue the production of 28 S but not 18 S rRNA. A phenotype exhibiting inhibition of 18 S and 28 S rRNA production was also observed when the paralogue RH-II/Guβ was overexpressed. Both down-regulation of RH-II/Guα and overexpression of RH-II/Guβ slowed cell proliferation. The opposite effects of the two paralogues suggest antagonistic functions.

Phosphorothioate oligonucleotides bind in a non sequence-specific manner to the nucleolar protein C23/nucleolin

To design optimal strategies for intracellular delivery of antisense phosphorothioate oligonucleotides, it may be useful to understand their interaction with cellular macromolecules. Nuclear extracts from LOX amelanotic myeloma cells were studied for protein binding to phosphorothioate oligonucleotides using a Southwestern protocol. Multiple nuclear proteins bound to the phosphorothioate oligonucleotides but no detectable protein binding was found to phosphodiester oligonucleotides. The protein with the strongest binding signals was shown by immunoprecipitation to be nucleolar C23/nucleolin, a 110 kDa protein. With glutathione S‐transferase/nucleolin fusion protein constructs, the region of nucleolin containing the RNA recognition motifs had binding activity to phosphorothioate oligonucleotides.

c-Jun supports ribosomal RNA processing and nucleolar localization of RNA helicase DDX21.

The molecular mechanisms by which the AP-1 transcription factor c-Jun exerts its biological functions are not clearly understood. In addition to its well established role in transcriptional regulation of gene expression, several reports have suggested that c-Jun may also regulate cell behavior by non-transcriptional mechanisms. Here, we report that small interfering RNA-mediated depletion of c-Jun from mammalian cells results in inhibition of 28 S and 18 S rRNA accumulation. Moreover, we show that c-Jun depletion results in partial translocation of RNA helicase DDX21, implicated in rRNA processing, from the nucleolus to the nucleoplasm. We demonstrate that DDX21 translocation is rescued by exogenous c-Jun expression and that c-Jun depletion inhibits rRNA binding of DDX21. Furthermore, the direct interaction between c-Jun and DDX21 regulates nucleolar localization of DDX21. These results demonstrate that in addition to its transcriptional effects, c-Jun regulates rRNA processing and nucleolar compartmentalization of the rRNA processing protein DDX21. Thus, our results demonstrate a nucleolar mechanism through which c-Jun can regulate cell behavior. Moreover, these results suggest that the phenotypes observed previously in c-Jun-depleted mouse models and cell lines could be partly due to the effects of c-Jun on rRNA processing.

Down-regulation of RNA Helicase II/Gu Results in the Depletion of 18 and 28 S rRNAs in Xenopus Oocyte

Genetic manipulations have revealed the functions of RNA helicases in ribosomal RNA (rRNA) biogenesis in yeast. However, no report shows the role of an RNA helicase in rRNA formation in higher eukaryotes. This study reports the functional characterization of the frog homologue of nucleolar RNA helicase II/Gu (xGu or DDX21). Down-regulation of xGu in Xenopus laevis oocyte using an antisense oligodeoxynucleotide results in the depletion of 18 and 28 S rRNAs. The disappearance of 18 S rRNA is accompanied by an accumulation of 20 S, indicating that xGu is critical in the processing of 20 to 18 S rRNA. The degradation of 28 S rRNA into fragments smaller than 18 S is also associated with a specific decrease in the level of xGu protein. These effects are reversed in the presence of in vitro synthesized wild type xGu mRNA but not its helicase-deficient mutant form. Similar aberrant rRNA processing is observed when antibody against xGu is microinjected. The involvement of xGu in processing of rRNA is consistent with the localization of Gu protein to the granular and dense fibrillar components of PtK2 cell nucleoli by immunoelectron microscopy. Our results show that xGu is involved in the processing of 20 to 18 S rRNA and contributes to the stability of 28 S rRNA in Xenopus oocytes.

Immunodominant RNA recognition motifs of human nucleolin/C23

Nucleolin/C23 is a nucleolar phosphoprotein implicated in the synthesis, processing and transport of ribosomal RNA and gene transcription. Auto-antibodies to human nucleolin/C23 have been reported in patients with systemic lupus erythematosus and other systemic autoimmune disorders. To identify immunodominant regions in nucleolin/C23, deletion fragments of nucleolin/C23 were fused in frame with the glutathione S-transferase gene. Seven monoclonal anti-nucleolin/C23 antibodies were used to determine the immunoreactivity of the bacterially expressed fusion proteins. Two sets of immunogenic regions at amino acids 314-389 and 387-461 were identified; each contained overlapping discontinuous epitopes and a centrally located RNA recognition motif. An auto-immune serum from a patient with systemic lupus erythematosus patient was found to contain antibodies against human nucleolin/C23 which recognized amino acids 387-461 of nucleolin/C23.

Functional interaction between RNA helicase II/Guα and ribosomal protein L4

RNA helicase II/Guα is a multifunctional nucleolar protein involved in ribosomal RNA processing in Xenopus laevis oocytes and mammalian cells. Downregulation of Guα using small interfering RNA (siRNA) in HeLa cells resulted in 80% inhibition of both 18S and 28S rRNA production. The mechanisms underlying this effect remain unclear. Here we show that in mammalian cells, Guα physically interacts with ribosomal protein L4 (RPL4), a component of 60S ribosome large subunit. The ATPase activity of Guα is important for this interaction and is also necessary for the function of Guα in the production of both 18S and 28S rRNAs. Knocking down RPL4 expression using siRNA in mouse LAP3 cells inhibits the production of 47/45S, 32S, 28S, and 18S rRNAs. This inhibition is reversed by exogenous expression of wild‐type human RPL4 protein but not the mutant form lacking Guα‐interacting motif. These observations have suggested that the function of Guα in rRNA processing is at least partially dependent on its ability to interact with RPL4.

Cloning and characterization of a new silver-stainable protein SSP29, a member of the LRR family

Silver‐stainable proteins (SSPs) are aspartic acid‐rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxynucleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA‐derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine‐rich repeats (LRR) near the NH2‐terminal region and acidic clusters at the COOH‐terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen‐mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag‐NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.

The effect of antisense p120 construct on p120 expression and cell proliferation in human breast cancer MCF-7 cells

Malignant transformation of NIH3T3 cells was observed by transfection with the pSVX vector containing a sense human p120 cDNA construct (pSVX120). Subsequent transfection of these transformed cells with a dexamethasone inducible antisense p120 construct (pMSG021) markedly reduced the expression of human p120 and the growth rate of these transformed cells (Perklaky et al., Cancer Res., (1992) 52, 428-436). In the present study, a human breast cancer cell line (MCF-7) which expresses the p120 protein was transfected by electroporation with a pSVX plasmid-construct containing the antisense p120 cDNA (pSVX021). Clones containing the pSVX021 construct were selected and analyzed for expression of p120 mRNA, protein and growth characteristics. The expression of the p120 protein was inhibited by 44% in the antisense-transfected MCF-7pSVX021 cells; a 56% inhibition of cell-growth and a reduced colony formation in soft agarose were also observed. The growth of MCF-7 cells transfected with the p120 antisense construct was reduced by 93% in nude mice.

Genomic structure of newly identified paralogue of RNA helicase II/Gu: detection of pseudogenes and multiple alternatively spliced mRNAs

RNA helicase II/Gu (RH-II/Gu or DDX21) is a DEAD-box enzyme that localizes to the nucleoli and may be involved in ribosomal RNA synthesis or processing. It has two paralogues, RH-II/Gualpha and RH-II/Gubeta, both genes of which are on chromosome 10. Their similar genomic structures suggest the two genes arose by gene duplication. Both genes are expressed at higher levels in some normal human tissues compared to matching tumor tissues. Pseudogenes for RH-II/Gubeta exist on chromosomes 2, 3 and 4. No pseudogene was identified for RH-II/Gualpha. Both exon inclusion and exon skipping were found to post-transcriptionally regulate RH-II/Gubeta gene expression. No alternative splicing was identified for RH-II/Gualpha. Overall, the results suggest that the two paralogues of RH-II/Gu arose by gene duplication but the resulting genes are differentially regulated.