No abstract

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100 -300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly ( Moreover the exon junction complex, which is deposited during splicing, is removed during the so-called pioneering round of translation (for reviews, see Refs. 1 and 2). Finally a particular mRNA becomes embroidered with nuclear RNA-binding proteins, and the specific ensemble may determine cytoplasmic events such as RNA localization, translation, and stability (for a review, see Ref.3). Cytoplasmic mRNPs may become destined for local translation. In support of this possibility, RNAs have been found in large mRNP granules, which are transported along cytoskeletal structures and anchored at their final destination. Messenger RNA localization has mainly been examined in polarized oocytes and neurons, and it has been proposed that local postsynaptic protein synthesis is required for synaptic plasticity (4). Previous studies have identified neuronal Staufen (5) and FMRP granules (6, 7), containing mRNAs, small and large ribosomal subunits, translation initiation factors including eIF4E and eIF2␣, and RNA-binding proteins (Refs. 8 -11; for a review, see Ref. 12). The protein composition of neuronal mRNP granules is to some degree overlapping with stress granules and processing bodies (P-bodies). The hallmark of stress granules is the presence of stalled 48 S initiation complexes and stress-dependent RNA-binding factors such as G3BP (13,14), whereas P-bodies contain components of the 5Ј-3Ј mRNA decay machinery and factors involved in nonsense-mediated decay (15).The zip code-binding proteins IMP1, -2, and -3 (human), ZBP1 (chicken), Vg1-RBP/Vera (Xenopus), and coding region determinant-binding protein (mouse) are members of the From the ‡Department

a b s t r a c tMetabolic flux analysis (MFA) is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA) has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA) to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37°C to 32°C maintained a constant IgG volumespecific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32°C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity.