Post-transcriptional gene regulation (PTGR) concerns processes involved in the maturation, transport, stability and translation of coding and non-coding RNAs. RNA-binding proteins (RBPs) and ribonucleoproteins coordinate RNA processing and PTGR. The introduction of large-scale quantitative methods, such as next-generation sequencing and modern protein mass spectrometry, has renewed interest in the investigation of PTGR and the protein factors involved at a systems-biology level. Here, we present a census of 1,542 manually curated RBPs that we have analysed for their interactions with different classes of RNA, their evolutionary conservation, their abundance and their tissue-specific expression. Our analysis is a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism.

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A census of human RNA-binding proteins.

Linear arrays of spherical chromatin particles (nu bodies) about 70 angstroms in diameter have been observed in preparations of isolated eukaryotic nuclei swollen in water, centrifuged onto carbon films, and positively or negatively stained. These bodies have been found in isolated rat thymus, rat liver, and chicken erythrocyte nuclei. Favorable views also reveal connecting strands about 15 angstroms wide between adjacent particles.

https://science.sciencemag.org/content/sci/183/4122/330.full.pdf

Spheroid Chromatin Units (ν Bodies)

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

https://www.pnas.org/content/pnas/93/20/10933.full.pdf

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

Promoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.

https://www.pnas.org/content/pnas/91/20/9302.full.pdf

Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter

Electron microscopic and biochemical evidence that chromatin structure is a repeating unit

The presence of extrachromosomal nucleoli in amphibian oocytes has permitted isolation and electron microscopic observation of the genes coding for ribosomal RNA precursor molecules. Visualization of these genes is possible because many precursor molecules are simultaneously synthesized on each gene. Individual genes are separated by stretches of DNA that apparently are not transcribed at the time of synthesis of precursor rRNA in the extrachromosomal nucleoli.

https://science.sciencemag.org/content/sci/164/3882/955.full.pdf

Visualization of Nucleolar Genes

Transcriptional control regions which direct transgene expression to all tissues in transgenic animals can be useful tools for gain-of-function experiments in transgenic animals. A candidate for this purpose is the regulatory region of the human cytomegalovirus immediate early 1 gene (HCMVIE1) which is highly expressed in many lines of tissue culture cells. Here we analyzed the activity of the HCMVIE1 enhancer/promoter using a sensitive reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. Three lines of transgenic mice with an intact transgene were established. All 3 lines expressed the transgene in all 28 tissues analyzed; however, levels of expression between the three lines varied up to 100,000 fold. In addition, expression levels in the high expressing line varied over a 10,000 fold continuum, while expression levels between tissues was almost uniform in the lowest expressing line. The transgene was well expressed in the high expressing line; CAT activity in the highest expressing tissues was equivalent to levels previously reported for tissue specific CAT transgenes active only in a limited number of tissues. These data support the utilization of the HCMVIE1 enhancer/promoter as a means of expressing a transgene in all tissues, but indicate that lines with substantially different overall levels of expression may be generated, and that markedly different levels of tissue specific expression may be found when the overall level of transgene expression is high.

Barbara R. Beatty

Papers

Visualization of Nucleolar Genes

The variability in activity of the universally expressed human cytomegalovirus immediate early gene 1 enhancer/promoter in transgenic mice

Portrait of a gene.

Response to Protocol Review Scenario: The rodent species is immaterial