Richard J. Jackson

The mechanism of eukaryotic translation initiation and principles of its regulation

Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control of gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions in ribosomal initiation complexes, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity (which affects almost all scanning-dependent initiation) and through sequence-specific RNA-binding proteins and microRNAs (which affect individual mRNAs).

Ultrasensitive assays for endogenous antimicrobial polypeptides.

We developed two sensitive methods for identifying antimicrobial molecules in leukocytes and other tissues. One method uses a gel overlay technique and was designed to identify antimicrobial polypeptides in samples subjected to polyacrylamide gel electrophoresis. The other, a radial diffusion assay, allows multiple fractions obtained by chromatographic procedures to be tested for antimicrobial activity conveniently. When we used E. coli ML-35p or Salmonella typhimurium 14028S as test organisms in the radial diffusion assay, we routinely detected 5-10 ng of rabbit defensin NP-1 in 5 microliters of sample. With minor modifications, both methods can be applied to other organisms, including Gram-positive bacteria, several Candida species and Cryptococcus neoformans.

Phosphorylation of initiation factor eIF-2 and the control of reticulocyte protein synthesis

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.

How Do MicroRNAs Regulate Gene Expression

Several thousand human genes, amounting to about one-third of the whole genome, are potential targets for regulation by the several hundred microRNAs (miRNAs) encoded in the genome. The regulation occurs posttranscriptionally and involves the ~21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. The target sites are almost invariably in the 3′-untranslated region of the messenger RNA (mRNA), often in multiple copies. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. However, recent studies have questioned these suppositions. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. Repressed target mRNAs as well as miRNAs themselves accumulate in cytoplasmic foci known as P-bodies, where many enzymes involved in mRNA degradation are concentrated. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression. The human genome encodes several hundred short (~21 residue) microRNAs (miRNAs), which are predicted to collectively regulate several thousand distinct genes, amounting to about one-third of all human genes. MicroRNAs generally interact with their target mRNAs through imperfect or incomplete complementary base-pairing to sites in the 3′-untranslated region of the messenger RNA (mRNA), which are usually present in multiple copies. These interactions result in a decrease in synthesis of the protein encoded by the mRNA, but until very recently, little was known about the mechanism(s) of this repression. An miRNA can decrease the intracellular concentration of its target mRNAs by accelerating the normal process of mRNA degradation and can inhibit the translation or decoding of the target mRNA, with the relative importance of these two mechanisms differing between different miRNA-mRNA pairs. The mechanism of inhibition of translation remains controversial, with some data pointing to inhibition of the translation initiation step, and other results indicative of inhibition at some later stage during the actual decoding of the mRNA sequence. This article reviews these recent data, discusses the controversies that remain unresolved, and makes suggestions for future research.

[4] Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA

Publisher Summary The nuclease-treated rabbit reticulocyte lysate supplemented with heterologous tRNA is the most widely used translation system for eukaryotic mRNAs. This chapter describes the procedures to make the lysate, how to make it dependent on added mRNA, and provide some suggestions for methods of preparing gel-filtered nuclease-treated lysates. The ideal translation system is a reticulocyte lysate from which the endogenous mRNA is removed by fractionation or selective destruction without impairing the intrinsic high activity of the translation machinery. The resulting nuclease-treated lysate has a very low activity, unless eukaryotic mRNAs are added. The reticulocytes are highly specialized cells, and the translation machinery shows little specialization or preference with regard to initiation of protein synthesis on different eukaryotic mRNAs. It is only with respect to the complement of tRNA species that the reticulocyte lysate shows special properties, which may impair its ability to translate heterologous mRNA.

The Impact of the Built Environment on Health: An Emerging Field

The drive from my office to my suburban Atlanta home is all too familiar: it begins with a scary 7-lane thoroughfare, infamous for its strip malls, lack of sidewalks, and high pedestrian fatality rates; progresses to a jumble of connecting interstate highways packed with rush-hour traffic despite 12 or more roadway lanes; and ends with clusters of new, low-density, single-family residential developments lacking public parks, playgrounds, libraries, nearby stores or cafes, sidewalks, bicycle trails, and public transit. Adults and children in my neighborhood travel by private automobile to virtually all of their destinations, because they have no practical transportation alternatives.

Epidemic Yersinia enterocolitica Infection Due to Contaminated Chocolate Milk

In September and October, 1976, an outbreak of illness due to chocolate milk contaminated with Yersinia enterocolitica resulted in hospitalization of 36 children, 16 of whom had appendectomies. Infection with Y. enterocolitica serotype 0:8 was demonstrated in 38 ill persons. Sixty-one per cent of the persons who were infected had a titer greater than 1:160 OH agglutinins to serotype 8 yersinia, whereas 48 per cent of the hospitalized children had a fourfold change in agglutinin titer. An epidemiologic investigation demonstrated that illness was associated with drinking of chocolate milk purchased in school cafeterias, and Y. enterocolitica 0:8 was subsequently isolated from the milk. The investigation suggested that the bacterium was introduced at the dairy during the mixing by hand of chocolate syrup with previously pasteurized milk.

Initiation of protein synthesis: Evidence for messenger RNA-independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit

Abstract This paper shows that reticuloeyte lysates contain 40 S/Met-tRNA f complexes which are intermediates in the initiation of protein synthesis before the involvement of messenger RNA. More than one third of the native 40 S subunits in the lysate exist as these complexes during periods of linear protein synthesis, but less than a tenth are associated with mRNA. The 40 S/Met-tRNA f complexes disappear in some situations in which initiation is inhibited (by double-stranded RNA, oxidized glutathione, or in the absence of added haemin), but persist in the presence of other inhibitors (e.g. aurintricarboxylate or poly(I)). Inhibitors of chain elongation had little effect on the amount of these complexes. The Met-tRNA f in the 40 S complexes appears to exchange readily with free Met-tRNA f ; when lysates were preincubated with sparsomycin or diphtheria toxin and then incubated with [ 35 S]Met-tRNA f , the native 40 S subunits were the only ribosomal particles labelled. This experimental system was used to examine whether 40 S/Met-tRNA f complexes could interact with mRNA; various mRNAs were added shortly after or at the same time as the [ 35 S]Met-tRNA f . This resulted in a conversion of the 40 S/Met-tRNA f complexes into 80 S complexes, which appeared to be true initiation complexes since they were capable of translating the first two codons of the added mRNA. The mRNA-dependent formation of these 80 S complexes was completely inhibited by 0.1 mM-aurintricarboxylate, but the association of Met-tRNA f with the 40 S subunits was not prevented. The 40 S/Met-tRNA f complexes also participated in initiation on endogenous mRNA, and it was shown that the Met-tRNA f in this complex was used in preference to free Met-tRNA f in this process. We propose that the first step in the initiation of protein synthesis in the reticuloeyte lysate is the formation of a 40 S/Met-tRNA f complex. In the second stage the complex binds mRNA at the correct initiation site and, after joining with a 60 S subunit, an 80 S/Met-tRNA f /mRNA initiation complex is formed.

Pancreatic cancer hENT1 expression and survival from gemcitabine in patients from the ESPAC-3 trial

BACKGROUND Human equilibrative nucleoside transporter 1 (hENT1) levels in pancreatic adenocarcinoma may predict survival in patients who receive adjuvant gemcitabine after resection. METHODS Microarrays from 434 patients randomized to chemotherapy in the ESPAC-3 trial (plus controls from ESPAC-1/3) were stained with the 10D7G2 anti-hENT1 antibody. Patients were classified as having high hENT1 expression if the mean H score for their cores was above the overall median H score (48). High and low hENT1-expressing groups were compared using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. RESULTS Three hundred eighty patients (87.6%) and 1808 cores were suitable and included in the final analysis. Median overall survival for gemcitabine-treated patients (n = 176) was 23.4 (95% confidence interval [CI] = 18.3 to 26.0) months vs 23.5 (95% CI = 19.8 to 27.3) months for 176 patients treated with 5-fluorouracil/folinic acid (χ(2) 1=0.24; P = .62). Median survival for patients treated with gemcitabine was 17.1 (95% CI = 14.3 to 23.8) months for those with low hENT1 expression vs 26.2 (95% CI = 21.2 to 31.4) months for those with high hENT1 expression (χ(2)₁= 9.87; P = .002). For the 5-fluorouracil group, median survival was 25.6 (95% CI = 20.1 to 27.9) and 21.9 (95% CI = 16.0 to 28.3) months for those with low and high hENT1 expression, respectively (χ(2)₁ = 0.83; P = .36). hENT1 levels were not predictive of survival for the 28 patients of the observation group (χ(2)₁ = 0.37; P = .54). Multivariable analysis confirmed hENT1 expression as a predictive marker in gemcitabine-treated (Wald χ(2) = 9.16; P = .003) but not 5-fluorouracil-treated (Wald χ(2) = 1.22; P = .27) patients. CONCLUSIONS Subject to prospective validation, gemcitabine should not be used for patients with low tumor hENT1 expression.

The Apaf-1 Internal Ribosome Entry Segment Attains the Correct Structural Conformation for Function via Interactions with PTB and unr

We have shown previously that polypyrimidine tract binding protein 1 (PTB) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr) is prebound. Here we show that the Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation.