In mammals, polyadenylation of mRNA precursors (premRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.

The prothrombin (F2) 3 0 end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3 0 UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function. We show that the F2 USE contains a highly conserved nonameric core sequence, which promotes 3 0 end formation in a position-and sequence-dependent manner. We identify proteins that specifically interact with the USE, and demonstrate their function as trans-acting factors that promote 3 0 end formation. Interestingly, these include the splicing factors U2AF35, U2AF65 and hnRNPI. We show that these splicing factors not only modulate 3 0 end formation via the USEs contained in the F2 and the complement C2 mRNAs, but also in the biocomputationally identified BCL2L2, IVNS and ACTR mRNAs, suggesting a broader functional role. These data uncover a novel mechanism that functionally links the splicing and 3 0 end formation machineries of multiple cellular mRNAs in an USE-dependent manner.

A recessive mutant cat1-1, wild type CAT1, was isolated in Saccharomyces cerevisiae. It did not grow on glycerol nor ferment maltose even with fully constitutive, glucose resistant maltase synthesis. It prevented derepression of isocitrate lyase, fructose-1,6-diphosphatase and maltase in a constitutive but glucose sensitive maltase mutant. Derepression of malate dehydrogenase was retarded and slowed down. Sucrose fermentation and invertase synthesis was not affected. Respiration was normal. From this mutant, two reverse mutants were isolated. One was recessive, acted as a suppressor of cat1-1 and was called cat2-1, wild type CAT2; the other was dominant and allelic to CAT1 and designated CAT1-2d and cat2-1 caused an earlier derepression of enzymes studied but did not affect the repressed nor the fully derepressed enzyme levels. CAT1-2d and cat2-1 did not show any additive effects. It is proposed that carbon catabolite repression acts in two ways. The direct way represses synthesis of sensitive enzymes, during growth on repressing carbon sources whereas the other way regulates the derepression process. After alleviation of carbon catabolite repression, gene CAT1 becomes active and prevents the activity of CAT2 which functions as a repressor of sensitive enzyme synthesis. The CAT2 gene product has to be eliminated before derepression can actually occur. The time required for this causes a delay in derepression after the depletion of a repressible carbon source. cat1-1 cannot block CAT2 activity and therefore, derepression is blocked. cat2-1 is inactive and derepression can start after carbon catabolite repression has ceased. CAT1-2d permanently active as a repressor of CAT2 and eliminates the delay in derepression.

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo-and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV-VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the "Cajal bodies." However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3Ј-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions. INTRODUCTIONCell biologists had recently to recognize, much to their surprise, that certain proteins appear, often in the same cells, as "dual location proteins," i.e., as general constituents of two rather distant and different structures: On the one hand, they occur as components of cytoskeletal plaques of a specific kind of intercellular junction, and on the other hand, they are located in karyoplasmic, interchromatinic granules, even in cells devoid of any junctions. Examples include the plakophilins PKP 1-3, typical of desmosomal plaques (Mertens et al., 1996;Schmidt et al., 1997Schmidt et al., , 1999Bonné et al., 1999), the adherens junction proteins, ARVCF (Borrmann, 2000;Borrmann et al., 2000; for cDNA transfection experiments see also Mariner et al., 2000), afadin (Mandai et al., 1997) and protein 4.1 (Krauss et al., 1997;Lallena et al., 1998), and the tight junction plaque proteins ZO-1 (Gottardi et al., 1996), symplekin (Keon et al., 1996), Ash-1 (Nakamura et al., 2000), and ZONAB (Balda and Matter, 2000). Obviously, this constitutively dual location at junctions and in nuclei has to be distinguished from observations of transient nuclear accumulations of certain other junctional plaque proteins in special stages of the cell cycle or differentiation or upon expression of certain transfected cDNAs or genes (for examples see, e.g., Funayama et al., 1995;Karnovsky and Klymkowsky, 1995;Behrens et al., 1996;Huber et al. 1996;Molenaar et al., 1996;Schneider et al., 1996;Yost et al., 1996;Daniel and Reynolds, 1999; for reviews see Behrens, 2000;Hü bner et al., 2001).Such a constitutively dual localization has also been reported in many diverse cultured cells and...

With the advent of molecularly targeted agents, treatment of metastatic renal cell carcinoma (mRCC) has improved significantly. Agents targeting the vascular endothelial growth factor receptor (VEGFR) and the mammalian target of rapamycin complex 1 (mTORC1) are more effective and less toxic than previous standards of care involving cytotoxic and cytokine therapies. Unfortunately, many patients relapse following treatment with VEGFR and mTORC1 inhibitors as a result of acquired resistance mechanisms, which are thought to lead to the reestablishment of tumor vasculature. Specifically, the loss of negative feedback loops caused by inhibition of mTORC1 leads to upregulation of downstream effectors of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway and subsequent activation of hypoxia-inducible factor, an activator of angiogenesis. De novo resistance involving activated PI3K signaling has also been observed. These observations have led to the development of novel agents targeting PI3K, mTORC1/2 and PI3K/mTORC1/2, which have demonstrated antitumor activity in preclinical models of RCC. Several agents-BKM120, BEZ235 and GDC-0980-are being investigated in clinical trials in patients with metastatic/ advanced RCC, and similar agents are being tested in patients with solid tumors. The future success of mRCC treatment will likely involve a combination of agents targeting the multiple pathways involved in angiogenesis, including VEGFR, PI3K and mTORC1/2.Renal cell carcinoma (RCC) accounts for more than 2% of all human cancers, and its incidence is steadily rising by approximately 2% per year.