RNase MRP Cleaves the CLB2 mRNA To Promote Cell Cycle Progression: Novel Method of mRNA Degradation

Abstract

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5 untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 533 exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5-UTR to allow rapid 5 to 3 degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.RNase mitochondrial RNA processing (RNase MRP) is a site-specific endoribonuclease ribonucleoprotein (8). RNase MRP was initially isolated from mammalian mitochondria, where its activity is consistent with its playing a role in primer formation in the initiation of mitochondrial DNA replication. Cellular fractionation and immunolocalization experiments revealed that the majority of the complex is localized to the nucleolus (31), where a role in processing of rRNAs has been identified (9,16,36). Mutations in the RNA component of the human RNase MRP have been shown to be the cause of the genetic disease cartilage hair hypoplasia (32), which is characterized by short stature, brittle and sparse hair, and immunodeficiency (11,26).The protein and RNA components of RNase MRP are highly conserved in both structure and sequence in all eukaryotes (1,17,23,42,44). Biochemically, the enzymes from yeasts to humans have been found to be similar in both substrate specificity and activity (41). In addition, the RNA component of RNase MRP is structurally related the RNA component of RNase P, both enzymes contain identical protein components, and both can cleave common substrates (6, 7).The gene for the Saccharomyces cerevisiae MRP RNA is called NME1 for nuclear mitochondrial endonuclease 1 (35). In addition, at least nine yeast proteins associated with the MRP RNA in vivo have been identified. Eight of these proteins are shared with the ribonucleoprotein endoribonuclease RNase P (6, 10, 12, 23, 42). One protein encoded by the SNM1 gene encodes an RNA binding protein that is associated only with the RNase MRP RNA and not the RNase P RNA (37).All of the components of RNase MRP are essential for the viability of S. cerevisiae. Mut...

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