Chemical shift assignments of a minimal Rna14p/Rna15p heterodimer from the yeast cleavage factor IA complex

The two yeast proteins Rna14p and Rna15p form part of the cleavage/polyadenylation factor IA (CF IA) complex that is involved in the 3′ processing of pre-mRNA. Association of the two proteins is mediated by a small C-terminal peptide from Rna14p and a region in Rna15p that corresponds to the hinge domain first identified within the human orthologue. Here I report the ¹H, ¹³C and ¹⁵N spectral assignments for a bacterially co-expressed heterodimer of Rna14p/Rna15p. Further analysis of secondary chemical shifts reveals that both peptides are predominantly α-helical within the complex.     

Rna14-Rna15 assembly mediates the RNA-binding capability of Saccharomyces cerevisiae cleavage factor IA

The Rna14–Rna15 complex is a core component of the cleavage factor IA RNA-processing complex from Saccharomyces cerevisiae. To understand the assembly and RNA-binding properties, we have isolated and characterized the Rna14–Rna15 complex using a combination of biochemical and biophysical methods. Analysis of the purified complex, using transmission electron microscopy, reveals that the two proteins assemble into a kinked rod-shaped structure and that these rods are able to further self-associate. Analytical ultracentrifugation reveals that Rna14 mediates this association and facilitates assembly of an A₂B₂ tetramer (M_r 230000), where relatively compact Rna14–Rna15 heterodimers are in rapid equilibrium with tetramers that have a more extended shape. The Rna14–Rna15 complex, unlike the individual components, binds to an RNA oligonucleotide derived from the 3′-untranslated region of the S.cerevisiae GAL7 gene. Based on these structural and thermodynamic data, we propose that CFIA assembly regulates RNA-binding activity.     

Rna14p, a component of the yeast nuclear cleavage/polyadenylation factor I, is also localised in mitochondria

RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation. These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm. By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria. In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14. We isolated thirteen new mutants. Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature - like the first mutant identified (rna14-1). However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration. The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.     

Locked tether formation by cooperative folding of Rna14p monkeytail and Rna15p hinge domains in the yeast CF IA complex

The removal of the 3' region of pre-mRNA followed by polyadenylation is a key step in mRNA maturation. In the yeast Saccharomyces cerevisiae, one component of the processing machinery is the cleavage/polyadenylation factor IA (CF IA) complex, composed of four proteins (Clp1p, Pcf11p, Rna14p, Rna15p) that recognize RNA sequences adjacent to the cleavage site and recruit additional processing factors. To gain insight into the molecular architecture of CF IA we solved the solution structure of the heterodimer composed of the interacting regions between Rna14p and Rna15p. The C-terminal monkeytail domain from Rna14p and the hinge region from Rna15p display a coupled binding and folding mechanism, where both peptides are initially disordered. Mutants with destabilized monkeytail-hinge interactions prevent association of Rna15p within CF IA. Conservation of interdomain residues reveals that the structural tethering is preserved in the homologous mammalian cleavage stimulation factor (CstF)-77 and CstF-64 proteins of the CstF complex.     

Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.     

Rna14p, a component of the yeast nuclear Cleavage / Polyadenylation Factor I, is also localised in mitochondria

RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation. These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm. By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria. In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14. We isolated thirteen new mutants. Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature – like the first mutant identified (rna14-1). However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration. The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.     

The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites.

Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton. The GTPase Cdc42 regulates actin rearrangements and signal transduction pathways in all eukaryotic cells [1], and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF). Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane [2] [3] at sites of polarized growth [4]. We show that Cdc24p labeled with green fluorescent protein (GFP-Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells. Unexpectedly, GFP-Cdc24p also localized to the nucleus and GFP-Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother-bud necks during cytokinesis. The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p. The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother-bud neck, and mating projection. Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the G(beta) subunit Ste4p. These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.     

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.     

Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells

When MCF-7 cells were incubated with 10 or 20 microM CdCl(2), p53 protein level increased after 18 h. Among serines in p53 protein immunoprecipitated from cells treated with CdCl(2), only Ser 15 was phosphorylated. No clear phosphorylation was found on Ser 6, 9, 20, 37, and 392. Accumulation of p53 protein phosphorylated at Ser 15 was also found after 18 h exposure. While phosphorylation of extracellular signal-regulated protein kinase, c-Jun NH2-terminal kinase and p38 was found in cells treated with CdCl(2), treatment with U0126, LL-Z1640-2, or SB203580 did not suppress Ser 15 phosphorylation. On the other hand, treatment with wortmannin or caffeine suppressed CdCl(2)-induced Ser 15 phosphorylation and accumulation of p53 protein. The present results showed that cadmium induces phosphorylation of p53 at Ser 15 in MCF-7 cells depending on phosphatidylinositol 3-kinase related kinases, but not on mitogen-activated protein kinases.     

Inactivation of arf-bp1 induces p53 activation and diabetic phenotypes in mice

It is well accepted that the Mdm2 ubiquitin ligase acts as a major factor in controlling p53 stability and activity in vivo. Although several E3 ligases have been reported to be involved in Mdm2-independent p53 degradation, the roles of these ligases in p53 regulation in vivo remain largely unknown. To elucidate the physiological role of the ubiquitin ligase ARF-BP1, we generated arf-bp1 mutant mice. We found that inactivation of arf-bp1 during embryonic development in mice resulted in p53 activation and embryonic lethality, but the mice with arf-bp1 deletion specifically in the pancreatic β-cells (arf-bp1(FL/Y)/RIP-cre) were viable and displayed no obvious abnormality after birth. Interestingly, these mice showed dramatic loss of β-cells as mice aged, and >50% of these mice died of severe diabetic symptoms before reaching 1 year of age. Notably, the diabetic phenotype of these mice was largely reversed by concomitant deletion of p53, and the life span of the mice was significantly extended (p53(LFL/FL)/arf-bp1(FL/Y)/RIP-cre). These findings underscore an important role of ARF-BP1 in maintaining β-cell homeostasis in aging mice and reveal that the stability of p53 is critically regulated by ARF-BP1 in vivo.     

Lead cleavage sites in the core structure of group I intron-RNA.

Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.     

p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation

The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics. In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxyl-terminal to the zinc finger DNA-binding domain. [(14)C]acetate-labeling experiments demonstrated that AR acetylation by p300 in cultured cells requires the same residues identified in vitro. Point mutation of the AR acetylation site (K632A/K633A) abrogated dihydrotestosterone-dependent transactivation of the AR in cultured cells. Mutation of the p300 CH3 region or the p300/cAMP-response element-binding protein histone acetylase domain reduced ligand-dependent AR function. The identification of the AR as a direct target of histone acetyltransferase co-activators has important implications for targeting inhibitors of AR function.     

The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosomal small nuclear ribonucleoproteins

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.