Role and Mechanism of RNase-L Action in Senescence

本研究探讨了RNase-L在体外作为一种新的衰老介质的作用，以及在体内作为一种衰老的生化机制。RNase-L是干扰素调控的2-5A通路的核糖核酸酶组分。RNase-L通过降解病毒、核糖体和信使rna来发挥抗病毒和生长抑制作用。转染的RNase-L的组成性表达导致有丝分裂指数下降和形态学衰老。此外，人类RNASE- L基因映射到染色体1q25，该区域被认为包含复制性衰老所需的基因。基于这些发现，我们假设：1)激活RNASE-L对体外诱导衰老至关重要，并在体内发挥衰老作用；ii) RNASE-L通过转录后下调增殖刺激基因来促进衰老的发生，iii) RNASE-L通过选择性降解mrna和/或通过rRNA切割抑制mrna的翻译来抑制基因表达。为了确定RNase-L在衰老中的作用，我们将测量RNase-L活性增加或抑制的人和胚胎小鼠成纤维细胞的衰老诱导（目的1）。2- 5a通路酶的表达和活性在年轻和年老的人成纤维细胞以及年轻和年老的小鼠组织中是不同的（目的2）。这项综合调查将结合一种新的基于PCR的rRNA切割试验来检测完整细胞中的RNASE-L活性。预计RNASE-L通过基因表达的转录后调节在衰老中起作用。我们将研究已知的衰老调节基因在RNASE-L活性降低或增加的细胞中的表达（目的3）。表现出RNASE-L依赖性调控的基因代表候选底物，其mrna的半衰期将被确定。这项工作将增加我们对衰老细胞的理解，通过确定：i) RNASE-L作为衰老介质的作用；ii)衰老细胞中2-5A通路的表达和活性；以及iii) RNASE-L如何调节衰老细胞中mRNA的稳定性，从而调节基因表达。全面了解细胞衰老对于确定其与衰老和癌症的关系至关重要；这些信息可能会导致新的治疗方法。

This proposal examines the role of RNase-L as a novel mediator of senescence in vitro, and as a biochemical mechanism of aging in vivo. RNase-L is the ribonuclease component of the interferon-regulated 2-5A pathway. RNase-L functions in antiviral and growth inhibitory activities through the degradation of viral, ribosomal, and messenger RNAs. Constitutive expression of transfected RNase-L results in a decreased mitotic index and senescent morphology. Moreover, the human RNASE- L gene maps to chromosome 1q25, a region thought to harbor gene(s) required for replicative senescence. Based on these finding, we hypothesize that: i) activation of RNASE-L is critical for induction of senescence in vitro, and functions in aging in vivo; ii) RNASE-L promotes the onset of senescence through the post-transcriptional down- regulation of proliferation stimulatory genes and iii) RNASE-L suppresses gene expression through the selective degradation of mRNAs and/or the inhibition of their translation via rRNA cleavage. To determine the role of RNase-L in senescence, we will measure the induction of senescence in human and embryonic mouse fibroblasts in which RNASE-L activity is increased or inhibited (aim 1). The expression and activity of 2-5A pathway enzymes varies in young and aged human fibroblasts and in tissues from young and aged mice (aim 2). This comprehensive survey will incorporate a novel PCR based rRNA cleavage assay to detect RNASE-L activity in intact cells. RNASE-L is predicted to function in senescence via the post-transcriptional modulation of gene expression. We will examine the expression of known senescence regulated genes in cells with reduced or increased RNASE-L activity (aim 3). Genes that exhibit RNASE-L dependent regulation represent candidate substrates and the half-lives of their mRNAs will be determined. This work will increase our understanding of senescent cells by determining: i) the role of RNASE-L as a mediator of senescence; ii) the expression and activity of the 2-5A pathway in aging cells; and iii) how RNASE-L modulates mRNA stability, and thus gene expression, in senescent cells. A comprehensive understanding of cellular senescence is essential to determine its relationship to aging and cancer; such information may lead to the novel therapies.