Localization of Nuclear-Encoded mRNAs to Mitochondria Outer Surface.

The diverse functions of mitochondria depend on hundreds of different proteins. The vast majority of these proteins is encoded in the nucleus, translated in the cytosol, and must be imported into the organelle. Import was shown to occur after complete synthesis of the protein, with the assistance of cytosolic chaperones that maintain it in an unfolded state and target it to the mitochondrial translocase of the outer membrane (TOM complex). Recent studies, however, identified many mRNAs encoding mitochondrial proteins near the outer membrane of mitochondria. Translation studies suggest that many of these mRNAs are translated locally, presumably allowing cotranslational import into mitochondria. Herein we review these data and discuss its relevance for local protein synthesis. We also suggest alternative roles for mRNA localization to mitochondria. Finally, we suggest future research directions, including revealing the significance of localization to mitochondria physiology and the molecular players that regulate it.

Absolute or relative size: What do we perceive when we look at a glass that is half full?

Given that both children and adults struggle with fractions in mathematics education, we investigated the processing of nonsymbolic fractions in a continuous form of part-of-the-whole. Continuous features of nonsymbolic numbers (e.g., the size of dots in an array) were found to influence numerosity judgment, but it should be noted that the (continuous) size of a part can be processed relative to a whole or as an absolute size. This study tested which of these size types (i.e., absolute and relative) influences comparison of parts. In two Stroop-like comparison tasks, we measured the interference of each size type on the processing of the other. In Experiment 1, stimuli were three-dimensional-like partially filled glasses of water. In both tasks, congruent trials (in which the larger absolute size was also the larger part-of-the-whole) were processed more efficiently than incongruent trials (in which the larger absolute size was the smaller part-of-the-whole). In Experiment 2, where stimuli were two-dimensional rectangles, this result was replicated under improved experimental control. We conclude that both absolute size and relative size of a part are automatically processed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos.

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.

Specific gene expression in pancreatic beta-cells: cloning and characterization of differentially expressed genes.

Identification and characterization of genes expressed preferentially in pancreatic beta-cells will clarify the mechanisms involved in the specialized properties of these cells, as well as providing new markers of the development of type 1 diabetes. Despite major efforts, relatively few beta-cell-specific genes have been characterized. We applied representational difference analysis to identify genes expressed selectively in the pancreatic beta-cell line betaTC1 compared with the pancreatic alpha-cell line alphaTC1 and isolated 26 clones expressed at higher levels in the beta-cells than in the alpha-cells. DNA sequencing revealed that 14 corresponded to known genes (that is, present in GenBank). Only four of those genes had been shown previously to be expressed at higher levels in beta-cells (insulin, islet amyloid polypeptide, neuronatin, and protein kinase A regulatory subunit [RIalpha]). The known genes include transcription factors (STAT6) and mediators of signal transduction (guanylate cyclase). The remaining 12 genes are absent from the GenBank database or are present as expressed sequence tag (EST) sequences (4 clones). Some of the genes are expressed in a highly specific pattern-expression in betaTC1 and islet cells and in relatively few of the non-beta-cell types examined; others are expressed in most cell types tested. The identification of these differentially expressed genes may aid in attaining a clearer understanding of the mechanisms involved in beta-cell function and of the possible immunogens involved in development of type 1 diabetes.

Differential expression of the protein kinase A regulatory subunit (RIalpha) in pancreatic endocrine cells.

A PCR-based subtractive cloning procedure was used to identify genes expressed at higher levels in the pancreatic beta cell line betaTC1, as compared to the pancreatic alpha cell line alphaTC1. One of the clones isolated by this procedure corresponded to the regulatory subunit (RIalpha) of protein kinase A (PKA). Using antibodies directed against RIalpha, we now demonstrate both by immunoblot and immunofluorescence that RIalpha protein is present at higher levels in cultured beta cells as compared to alpha cells. In vitro PKA assays revealed high basal PKA activity in alphaTC1 extracts, which changed little on addition of exogenous cAMP. On the other hand, extracts from beta cells showed very low basal activity of PKA, which was elevated upon addition of cAMP. A similar trend was observed in vivo using transfected luciferase constructs bearing multiple copies of a CRE element: in alphaTC1 cells, no induction by forskolin was observed, whereas in betaTC1 cells, forskolin produced a 9-fold increase in activity. Therefore, the results indicate that RIalpha of PKA is selectively expressed in pancreatic beta cells as compared to alpha cells: this selective expression is associated with major differences in the properties of the PKA signal transduction pathway. Differential expression of the regulatory subunit may play a role in determining the patterns of gene expression and signal transduction characteristic of alpha and beta cells.

Decimals are not processed automatically, not even as being smaller than one.

Common fractions have been found to be processed intentionally but not automatically, which led to the conclusion that they are not represented holistically in long-term memory. However, decimals are more similar to natural numbers in their form and thus might be better candidates to be holistically represented by educated adults. To test this hypothesis, we investigated the automatic processing of decimals by college students in 4 experiments. When decimals were presented in a familiar form (e.g., 0.3, 0.05) the length of the stimuli (i.e., the number of digits) dominated performance rather than the decimal value. When controlling for the number of digits and their location within the digit string, using the place-value task, decimals were not processed automatically in either a numerical comparison task or a physical comparison task. Under the same conditions, natural numbers were processed automatically. We conclude that decimals are not represented holistically. Results of mixed pairs of a decimal and a natural number suggest that, unlike common fractions, decimals are not automatically perceived as smaller than natural numbers. We conclude that decimal place-values (e.g., tenths, hundredths) are not represented well enough to be automatically activated, and we discuss possible explanations.

The place-value of a digit in multi-digit numbers is processed automatically.

The automatic processing of the place-value of digits in a multi-digit number was investigated in 4 experiments. Experiment 1 and two control experiments employed a numerical comparison task in which the place-value of a non-zero digit was varied in a string composed of zeros. Experiment 2 employed a physical comparison task in which strings of digits varied in their physical sizes. In both types of tasks, the place-value of the non-zero digit in the string was irrelevant to the task performed. Interference of the place-value information was found in both tasks. When the non-zero digit occupied a lower place-value, it was recognized slower as a larger digit or as written in a larger font size. We concluded that place-value in a multi-digit number is processed automatically. These results support the notion of a decomposed representation of multi-digit numbers in memory.

When meaningful components interrupt the processing of the whole: the case of fractions.

Numerical fractions are composed of a numerator and a denominator that are natural numbers. These components influence processing of the fraction. This study was conducted to test whether eliminating the fractional components would result in the processing of fractions as unique numerical entities. Participants that learned to relate fractional values to arbitrary figures in a training task showed automatic processing of the numerical values of the new figures. The processing of fractions written in regular form improved following training, but did not show automatic processing. The results suggest that eliminating the influence of the fractional components allowed individual fractions to be represented in long-term memory.

Mental arithmetic activates analogic representations of internally generated sums.

The internal representation of numbers generated during calculation has received little attention. Much of the mathematics learning literature focuses on symbolic retrieval of math facts; in contrast, we critically test the hypothesis that internally generated numbers are represented analogically, using an approximate number system. In an fMRI study, the spontaneous processing of arithmetical expressions was tested. Participants passively viewed a sequence of double-digit addition expressions that summed to the same number. Adaptation was found in number-related regions in a fronto-parietal network. Following adaptation, arrays of dots were introduced, differing in their numerical distance from the sum of the addition expressions. Activation in voxels that showed adaptation to a repeated sum was also sensitive to the distance of the dot quantity from the sum. We conclude that participants exhibited adaptation to an internally generated number, that adapted representations were analogic in nature, and that these analogic representations may undergird arithmetic calculation.

A generalized fraction: an entity smaller than one on the mental number line.

The representation of fractions in long-term memory (LTM) was investigated by examining the automatic processing of such numbers in a physical comparison task, and their intentional processing in a numerical comparison task. The size congruity effect (SiCE) served as a marker of automatic processing and consequently as an indicator of the access to the primitives of numerical representation in LTM. Mixed pairs composed of a natural number and a fraction showed both a SiCE and a distance effect. The SiCE for mixed pairs was stable across relative sizes of natural numbers compared to the fraction digits (Experiment 4). However, comparing pairs of fractions revealed a strong influence of fractional components: An inverse SiCE was found for pairs of unit fractions (Experiment 1), while no SiCE was found for pairs of non-unit fractions (Experiments 2-3). This leads to the conclusions that: (1) there are no unique representations of distinct fraction values in LTM, and (2) there is a representation of a "generalized fraction" as an "entity smaller than one" that emerges from the notational structure common to all fractions.

GRFbeta, a novel regulator of calcium signaling, is expressed in pancreatic beta cells and brain.

By screening for genes expressed differentially in pancreatic beta cells, we have isolated a cDNA encoding GRFbeta, a novel 178-amino acid protein whose N terminus is identical to that of GRF1, a calcium-dependent guanine nucleotide exchange factor, and whose C terminus is unrelated to known proteins. We show that both GRF1 and GRFbeta are expressed selectively in beta cell lines, pancreatic islet cells and brain. Treatment of beta cell lines (betaTC1 and HIT) with calcium ionophore led to a significant elevation in activity of the Ras signal transduction pathway, as determined by phosphorylation of extracellular signal-related kinase (ERK). Transfection of beta cells with a plasmid encoding a dominant negative variant of GRF1 led to 70% reduction in ERK phosphorylation, consistent with a role for GRF1 in calcium-dependent Ras signaling in these cells. To examine the possible function of GRFbeta, cultured cells were transfected with a GRFbeta expression vector. This led to a significant reduction in both GRF1-dependent ERK phosphorylation and AP1-dependent reporter gene activity. The results suggest that GRF1 plays a role in mediating calcium-dependent signal transduction in beta cells and that GRFbeta represents a novel dominant negative modulator of Ras signaling.