An endogenous hybrid mRNA encodes TWE-PRIL, a functional cell surface TWEAK-APRIL fusion protein.

TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT-PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs. The encoded TWE-PRIL protein is composed of TWEAK cytoplasmic and transmembrane domains fused to the APRIL C-terminal domain. TWE-PRIL mRNA is expressed and translated in human primary T cells and monocytes, and endogenous TWE-PRIL protein was detected in primary human T lymphocytes and monocytic cell lines. TWE-PRIL is membrane anchored and presents the APRIL receptor-binding domain at the cell surface. It is a biologically active ligand, as it stimulates cycling in T- and B-lymphoma cell lines. Much like membrane-bound and secreted TNF-alpha, the different cellular localizations of TWE-PRIL and APRIL suggest that they exert distinct biological roles.

Effects of rapeseed meal-glucosinolates on thyroid metabolism and feed utilization in rainbow trout.

Two rapeseed meals (RM1 and RM2), containing glucosinolates at a concentration of 26 and 40 micromol/g, respectively, were incorporated at increasing levels (10, 20, and 30% for RM1 and 30 and 50% for RM2) in diets of juvenile rainbow trout. Disturbances in the thyroid axis appeared after 14 days of feeding (with a dietary incorporation level of 10%). The dietary supplementation with T(3) or iodine induced an increase in plasma T(3) levels, compared to that in fish fed the RM diets, and reduced the deleterious effect of RM on growth. When trout were reared in seawater, there was also a slight increase in thyroid hormone levels. TSH treatment had no effect on the thyroid hormone plasma levels. The incorporation of 30% of RM1, which induced a lower dietary content of toxic compounds than RM2, led to a rapid decrease of plasma T(4) and T(3) levels, but growth was affected only after 6 months of feeding. During these studies, the deiodinase activities responded in a complex manner to restore plasma and tissue levels of T(3).

Reliability of mRNA profiling: verification for samples with different complexities.

Normalization of mRNA profiling data remains an open issue, which turns critical when comparing divergent samples or mRNA populations with different complexities. To address this question, we generated samples with different RNA amounts and complexities by subcellular fractionation of cytoplasmic RNA into the mutually exclusive ribosome-free and polysome-bound RNA pools. For each of the 563 mRNAs analyzed, the hybridization signal corresponding to the cytoplasmic sample equals the sum of signals from the ribosome-free plus the polysome-bound targets (cytoplasmic mRNA = ribosome-free mRNA + polysome-bound mRNA). This intuitive equation was fulfilled only after data normalization following "spiking" of the samples with an exogenous RNA. This is the first demonstration that spiking allows one to correct not only for differences in reaction efficiencies but also to reflect the variations in amount and complexity between the initial mRNA populations.

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

Isolation of translationally controlled mRNAs by differential screening.

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.

Heterogeneity of eel thyrotropin beta mRNAs is due to a minisatellite in the 3' untranslated region of the gene.

The aim of this study was to determine the causes of the high heterogeneity, in the number and the length, of the thyrotropin (TSH) beta mRNA in the European eel. Northern blot analysis showed that removal of the poly(A) tail did not affect this heterogeneity. PCR amplification on reverse-transcribed pituitary RNAs (RT) showed the main source of heterogeneity to be a highly variable region in the 3' untranslated region (UTR). PCR amplification of the 3' UTR from RTs and genomic DNAs demonstrated that the high variability reflected polymorphism within the eel TSH beta gene. Isolation and sequencing of 3' UTR amplification fragments showed that the variable region comprised more or less exact repetitions of a 26-42-bp fragment. The number of repetitions varied from one allele to another. This variable region could be characterized as a minisatellite. In conclusion, instability of a minisatellite in the 3' UTR of the TSH beta gene generated the multiple and widely differing TSH beta mRNAs.

Down-regulation of TSH subunit mRNA levels by thyroid hormones in the European eel.

The effects of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) on alpha and thyroid-stimulating hormone (TSH) beta subunit mRNA pituitary levels were examined in a teleost, the European silver eel. Northern blot analysis showed that the number and length of mRNAs encoding TSH beta varied among individuals, a variability apparently not related to thyroidal status. When several bands were present, their intensities were summed for quantitative analysis. Increasing circulating thyroid hormones (THs) by implantation of T3 or T4 significantly decreased TSH beta mRNA levels. Depression of circulating THs by thiourea treatment increased alpha and TSH beta mRNA levels. In vitro studies showed that T3 and T4 decrease TSH beta mRNA levels in primary cultures of eel pituitary cells. In conclusion, in vivo and in vitro experiments indicate that T3 and T4 exert a negative feedback action on pituitary TSH beta mRNA level in the European eel and that this effect might be exerted, at least partly, through a direct action on the pituitary.