Small-scale nuclear extracts for functional assays of gene-expression machineries.

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3.

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.

Human mRNA export machinery recruited to the 5' end of mRNA.

Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.

A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay.

In eukaryotes, a surveillance mechanism known as nonsense-mediated decay (NMD) degrades the mRNA when a premature-termination codon (PTC) is present. NMD requires translation to read the frame of the mRNA and detect the PTC. During pre-mRNA splicing, the exon-exon junction complex (EJC) is recruited to a region 20-24 nt upstream of the exon junction on the mature mRNA. The presence of a PTC upstream from the EJC elicits NMD. Eukaryotic initiation factor 4A (eIF4A) III is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. Here we show that siRNA against eIF4AIII, but not against eIF4AI/II, inhibits NMD. Moreover, eIF4AIII, but not eIF4AI, is specifically recruited to the EJC during splicing. The observations that eIF4AIII is loaded onto the mRNA during splicing in the nucleus, has properties related to a translation initiation factor, and functions in NMD raises the possibility that eIF4AIII substitutes for eIF4AI/II during NMD.

A new on-line resource for psycholinguistic studies.

Picture naming is a widely used technique in psycholinguistic studies. Here, we describe new on-line resources that our project has compiled and made available to researchers on the world wide web at http://crl.ucsd.edu/~aszekely/ipnp/. The website provides access to a wide range of picture stimuli and related norms in seven languages. Picture naming norms, including indices of name agreement and latency, for 520 black-and-white drawings of common objects and 275 concrete transitive and intransitive actions are presented. Norms for age-of-acquisition, word-frequency, familiarity, goodness-of-depiction, and visual complexity are included. An on-line database query system can be used to select a specific range of stimuli, based on parameters of interest for a wide range of studies on healthy and clinical populations, as well as studies of language development.

Timed picture naming in seven languages.

Timed picture naming was compared in seven languages that vary along dimensions known to affect lexical access. Analyses over items focused on factors that determine cross-language universals and cross-language disparities. With regard to universals, number of alternative names had large effects on reaction time within and across languages after target-name agreement was controlled, suggesting inhibitory effects from lexical competitors. For all the languages, word frequency and goodness of depiction had large effects, but objective picture complexity did not. Effects of word structure variables (length, syllable structure, compounding, and initial frication) varied markedly over languages. Strong cross-language correlations were found in naming latencies, frequency, and length. Other-language frequency effects were observed (e.g., Chinese frequencies predicting Spanish reaction times) even after within-language effects were controlled (e.g., Spanish frequencies predicting Spanish reaction times). These surprising cross-language correlations challenge widely held assumptions about the lexical locus of length and frequency effects, suggesting instead that they may (at least in part) reflect familiarity and accessibility at a conceptual level that is shared over languages.