Refractoriness of hepatitis C virus internal ribosome entry site to processing by Dicer in vivo.

BACKGROUND: Hepatitis C virus (HCV) is a positive-strand RNA virus harboring a highly structured internal ribosome entry site (IRES) in the 5' nontranslated region of its genome. Important for initiating translation of viral RNAs into proteins, the HCV IRES is composed of RNA structures reminiscent of microRNA precursors that may be targeted by the host RNA silencing machinery. RESULTS: We report that HCV IRES can be recognized and processed into small RNAs by the human ribonuclease Dicer in vitro. Furthermore, we identify domains II, III and VI of HCV IRES as potential substrates for Dicer in vitro. However, maintenance of the functional integrity of the HCV IRES in response to Dicer overexpression suggests that the structure of the HCV IRES abrogates its processing by Dicer in vivo. CONCLUSION: Our results suggest that the HCV IRES may have evolved to adopt a structure or a cellular context that is refractory to Dicer processing, which may contribute to viral escape of the host RNA silencing machinery.

A central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1.

Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.

Powerful beneficial effects of macrophage colony-stimulating factor on beta-amyloid deposition and cognitive impairment in Alzheimer's disease.

Alzheimer's disease is a major cause of dementia in humans. The appearance of cognitive decline is linked to the overproduction of a short peptide called beta-amyloid (Abeta) in both soluble and aggregate forms. Here, we show that injecting macrophage colony-stimulating factor (M-CSF) to Swedish beta-amyloid precursor protein (APP(Swe))/PS1 transgenic mice, a well-documented model for Alzheimer's disease, on a weekly basis prior to the appearance of learning and memory deficits prevented cognitive loss. M-CSF also increased the number of microglia in the parenchyma and decreased the number of Abeta deposits. Senile plaques were smaller and less dense in the brain of M-CSF-treated mice compared to littermate controls treated with vehicle solution. Interestingly, a higher ratio of microglia internalized Abeta in the brain of M-CSF-treated animals and the phagocytosed peptides were located in the late endosomes and lysosomes. Less Abeta(40) and Abeta(42) monomers were also detected in the extracellular protein enriched fractions of M-CSF-treated transgenic mice when compared with vehicle controls. Finally, treating APP(Swe)/PS1 mice that were already demonstrating installed Abeta pathology stabilized the cognitive decline. Together these results provide compelling evidence that systemic M-CSF administration is a powerful treatment to stimulate bone marrow-derived microglia, degrade Abeta and prevent or improve the cognitive decline associated with Abeta burden in a mouse model of Alzheimer's disease.

MicroRNA-298 and microRNA-328 regulate expression of mouse beta-amyloid precursor protein-converting enzyme 1.

MicroRNAs (miRNAs) are key regulatory RNAs known to repress mRNA translation through recognition of specific binding sites located mainly in their 3'-untranslated region (UTR). Loss of specific miRNA control of gene expression is thus expected to underlie serious genetic diseases. Intriguingly, previous post-mortem analyses showed higher beta-amyloid precursor protein-converting enzyme (BACE) protein, but not mRNA, levels in the brain of patients that suffered from Alzheimer disease (AD). Here we also observed a loss of correlation between BACE1 mRNA and protein levels in the hippocampus of a mouse model of AD. Consistent with an impairment of miRNA-mediated regulation of BACE1 expression, these findings prompted us to investigate the regulatory role of the BACE1 3'-UTR element and the possible involvement of specific miRNAs in cultured neuronal (N2a) and fibroblastic (NIH 3T3) cells. Through various experimental approaches, we validated computational predictions and demonstrated that miR-298 and miR-328 recognize specific binding sites in the 3'-UTR of BACE1 mRNA and exert regulatory effects on BACE1 protein expression in cultured neuronal cells. Our results may provide the molecular basis underlying BACE1 deregulation in AD and offer new perspectives on the etiology of this neurological disorder.

A polymerase chain reaction-based cloning strategy applicable to functional microRNA studies.

MicroRNAs (miRNAs) are key regulatory RNAs that act in concert to coordinately control messenger RNA translation through imperfect recognition of multiple specific binding sites (BSs) located in their 3' untranslated region. Here, we present a polymerase chain reaction-based cloning strategy that allows the rapid and efficient generation of regulatory elements harboring up to 10 miRNA BSs. Amenable for the study of regulatory elements of any multiplicity, such as those recognized by miRNAs and transcription factors, this methodology will facilitate functional miRNA/miRNA BS studies and accelerate discoveries mainly in the field of gene regulation.

Regulatory RNAs: future perspectives in diagnosis, prognosis, and individualized therapy.

With potentially up to 1000 microRNAs (miRNAs) present in the human genome, altogether regulating the expression of thousands of genes, one can anticipate that miRNAs will play a significant role in health and disease. Deregulated protein expression induced by a dysfunctional miRNA-based regulatory system is thus expected to lead to the development of serious, if not lethal, genetic diseases. A relationship among miRNAs, Dicer, and cancer has recently been suggested. Further investigations will help establish specific causal links between dysfunctional miRNAs and diseases. miRNAs of foreign origin, e.g., viruses, may also be used as specific markers of viral infections. In these cases, miRNA expression profiles could represent a powerful diagnostic tool. Regulatory RNAs may also have therapeutic applications, by which disease-causing genes or viral miRNAs could be neutralized, or functional miRNAs be restored. Will bedside miRNA expression profiling eventually assist physicians in providing patients with accurate diagnosis, personalized therapy, and treatment outcome?