Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been implicated in numerous diseases ranging from neurological disorders to cancer. Here we show that NELF interacts with the nuclear cap binding complex (CBC), a multifunctional factor that plays important roles in several mRNA processing steps, and the two factors together participate in the 3' end processing of replication-dependent histone mRNAs, most likely through association with the histone stem-loop binding protein (SLBP). Strikingly, absence of NELF and CBC causes aberrant production of polyadenylated histone mRNAs. Moreover, NELF is physically associated with histone gene loci and forms distinct intranuclear foci that we call NELF bodies, which often overlap with Cajal bodies and cleavage bodies. Our results point to a surprising role of NELF in the 3' end processing of histone mRNAs and also suggest that NELF is a new factor that coordinates different mRNA processing steps during transcription.

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (K i ‫؍‬ 15 M). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.Porphyrins consist of a tetrapyrrole ring structure and are most widely and efficiently used in energy metabolism. Heme (Fe-protoporphyrin IX), a porphyrin derivative, is a prosthetic molecule for several hemoproteins, and plays an essential role in various biological processes such as oxygen transport, respiration, and signal transduction. The biosynthesis of heme is a multistep process that starts with the condensation of glycineand succinyl-CoA to form 5-aminolevulinate (1). Heme is ultimately formed in the mitochondrial matrix space following translocation of the heme precursor, co-proporphyrinogen III, which is generated in cytosol, into mitochondria (1). Heme is then utilized in the mitochondrial matrix for the synthesis of a variety of heme proteins such as the cytochromes. Thus, it is necessary for heme biosynthesis and synthesis of the mitochondrial heme proteins that heme and porphyrin precursors must be transported into mitochondria from cytosol. However, the mechanism by which this mitochondrial accumulation occurs is unclear.Some metalloporphyrin derivatives are fluorescence-or phosphorescence-emitting molecules. These molecules undergo a shift from a low energy ground state to a high energy excited state upon exposure to UV light. Fluorescent or phosphorescent light is emitted as the molecules decay from their high energy state back down to the ground state. PdTCPP 3 (Fig. 1A) emits long-lived phosphorescence in respons...

Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-␤-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.

TFII-I was initially identified as the general transcription factor that binds to initiator (Inr) elements in vitro . Subsequent studies have shown that TFII-I activates transcription of various genes either through Inr elements or through other upstream elements in vivo . Since, however, most studies so far on TFII-I have been limited to over-expression and reporter gene assays, we reevaluated the role of TFII-I in vivo by using stable knockdown with siRNA and by examining the expression of endogenous genes. Contrary to the widely accepted view, here we show that TFII-I is not important for cell viability in general but rather inhibits the growth of MCF-7 human breast cancer cells. MCF-7 cells are known to proliferate in an estrogen-dependent manner. Through analysis of TFII-I's cell-type specific growth inhibitory effect, we show evidence that TFII-I down-regulates a subset of estrogen-responsive genes, only those containing Inr elements, by recruiting estrogen receptor (ER) α α α α and corepressors to these promoters. Thus, this study has revealed an unexpected new role of TFII-I as a negative regulator of transcription and cell proliferation.

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