mRNAs For Other Proteins Research Articles

Advances in therapeutic RNA-targeting

This paper reviews the advances in the past decade of different applications of modulating the level and content of mRNA by antisense oligonucleotide (AON)-based exon skipping. The primary aim of such modulation is the correction of genetic defects by alteration of the resulting protein such that the dysfunction is reduced or relieved. This application is in several clinical phase III trails, notably for Duchenne muscular dystrophy and earlier clinical trials are in preparation for other diseases, a.o. spinal muscular atrophy. An alternative aim may be to disrupt the reading frame of dysfunctional proteins when they have a dominant negative effect and their absence may ameliorate disease. A third aim is to target mRNAs for other proteins, the engineering of which might improve or prevent the disease. A final application, which is as yet under-explored but has major promise, is the functional in vivo study of protein isoforms by modulating their relative levels by AON-based skipping of alternative exons.

Stimulation of human omental adipose tissue lipolysis by growth hormone plus dexamethasone

Growth hormone [GH] administration results in a reduction in adiposity of humans that is attributed to stimulation of lipolysis. We examined the effect of direct addition of human GH, in both the absence and presence of dexamethasone [Dex], as well as that of interferon beta on lipolysis by omental adipose tissue explants from obese women incubated for 48h in primary culture. There was a significant stimulation of lipolysis by GH in the presence of Dex but not by Dex or GH alone. There was also a significant further stimulation by GH in the presence of Dex of hormone-sensitive lipase, perilipin, lipoprotein lipase and beta1 adrenergic receptor mRNA. We conclude that the direct lipolytic effect of GH is accompanied by an increase in HSL mRNA in the presence of DEX, but GH also increased the mRNAs for other proteins that could explain all or part of its lipolytic action.

Neurosteroids and Female Reproduction: Estrogen Increases 3β-HSD mRNA and Activity in Rat Hypothalamus

A central event in mammalian reproduction is the LH surge that induces ovulation and corpus luteum formation. Typically, the LH surge is initiated in ovariectomized rats by sequential treatment with estrogen and progesterone (PROG). The traditional explanation for this paradigm is that estrogen induces PROG receptors (PR) that are activated by exogenous PROG. Recent evidence suggests that whereas exogenous estrogen is necessary, exogenous PROG is not. In ovariectomized-adrenalectomized rats, estrogen treatment increases hypothalamic PROG levels before an LH surge. This estrogen-induced LH surge was blocked by an inhibitor of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD), the proximal enzyme for PROG synthesis. These data indicate that estrogen induces de novo synthesis of PROG from cholesterol in the hypothalamus, which initiates the LH surge. The mechanism(s) by which estrogen up-regulates neuro-PROG is unknown. We investigated whether estrogen increases 1) mRNA levels for several proteins involved in PROG synthesis and/or 2) activity of 3beta-HSD in the hypothalamus. In ovariectomized-adrenalectomized rats, estrogen treatment increased 3beta-HSD mRNA in the hypothalamus, as measured by relative quantitative RT-PCR. The mRNAs for other proteins involved in steroid synthesis (sterol carrier protein 2, steroidogenic acute regulatory protein, and P450 side chain cleavage) were detectable in hypothalamus but not affected by estrogen. In a biochemical assay, estrogen treatment also increased 3beta-HSD activity. These data support the hypothesis that PROG is a neurosteroid, produced locally in the hypothalamus from cholesterol, which functions in the estrogen positive-feedback mechanism driving the LH surge.

Protein synthesis in Metarhizium anisopliae growing on host cuticle

In vitro protein synthesis using poly(A + )RNA and a two-step gel system for proteins were used in this study on the entomopathogenic fungus Metarhizium anisopliae to provide an estimate of the magnitude of differential protein synthesis and secretion that may be involved in adapting to growth on insect cuticle. Shortly after being transferred to a media containing cockroach cuticle, mRNAs for certain proteins are repressed while a broad array of mRNAs for other proteins is induced. Concurrent with this, a least 42 proteins were secreted into the media in a process which was sensitive to actinomycin D. The majority of these proteins were acidic (pI range 4·2–5·6) and co-migrated with Con-A/peroxidase stained bands indicating that they might be glycoconjugates. Microsequencing of those polypeptides accumulated in large amounts revealed two NH 2 -terminal amino acid sequences from acidic proteins that were highly homologous to those of animal trypsins. The trypsin nature of the two proteins was confirmed using a combination of gelatin-SDS-polyacrylamide gel electrophoresis and enzyme overlay membranes. The NH 2 -terminal sequence of the major basic protein identified it as a know subtilisin-like proteinase (Pr1). A second basic sequence was identified as a carboxypeptidase. No other homologies were found.

Changing patterns of gene expression during sporulation in yeast.

Analysis of RNAs isolated from the yeast Saccharomyces cerevisiae reveals a dramatic series of changes in protein coding sequences during sporulation. Shortly after transfer to sporulation medium, mRNAs for certain proteins are repressed while a broad array of mRNAs for other proteins is induced. Superimposed on this general increase in transcriptional activity is the very strong induction of a particular subset of heat shock mRNAs, the same subset that is induced during the normal course of oogenesis in Drosophila. At distinct times later in sporulation, two sets of abundant mRNAs are coordinately induced. Unlike the earlier changes in the message complement, these changes are unique to sporulating cells. As asci mature, one set of sporulation-specific RNAs is selectively degraded. The second set, as well as the broad array of mRNAs induced earlier in development, is retained in a highly stable and fully translatable form.