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...

Host cellular endoplasmic reticulum α-glucosidases I and II are essential for the maturation of viral glycosylated envelope proteins that use the calnexin mediated folding pathway. Inhibition of these glycan processing enzymes leads to the misfolding and degradation of these viral glycoproteins and subsequent reduction in virion secretion. We previously reported that, CM-10-18, an imino sugar α-glucosidase inhibitor, efficiently protected the lethality of dengue virus infection of mice. In the current study, through an extensive structure-activity relationship study, we have identified three CM-10-18 derivatives that demonstrated superior in vitro antiviral activity against representative viruses from four viral families causing hemorrhagic fever. Moreover, the three novel imino sugars significantly reduced the mortality of two of the most pathogenic hemorrhagic fever viruses, Marburg virus and Ebola virus, in mice. Our study thus proves the concept that imino sugars are promising drug candidates for the management of viral hemorrhagic fever caused by variety of viruses.

RNase mitochondrial RNA processing (MRP) is an essential ribonucleoprotein endoribonuclease that functions in the degradation of specific mRNAs involved in cell cycle regulation. We have investigated where this processing event occurs and how it is regulated. As expected, results demonstrate that RNase MRP is predominantly localized in the nucleolus, where it processes ribosomal RNAs. However, after the initiation of mitosis, RNase MRP localizes throughout the entire nucleus and in a single discrete cytoplasmic spot that persists until the completion of telophase. Furthermore, this spot was asymmetrically found in daughter cells, where the RNase MRP substrate, CLB2 mRNA, localizes. Both the mitotic exit network and fourteen early anaphase release pathways are nonessential but important for the temporal changes in localization. Asymmetric localization was found to be dependent on the locasome. The evidence suggests that these spots are specialized processing bodies for the degradation of transcripts that are cell cycle regulated and daughter cell localized. We have called these TAM bodies for temporal asymmetric MRP bodies.

We recently described the discovery of oxygenated N-alkyl deoxynojirimycin (DNJ) derivative 7 (CM-10-18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising, but had an EC50 against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound 7 presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure-activity relationship studies led to analogs 2h, 2l, 3j, 3l, 3v and 4b–4c with nanomolar antiviral activity (EC50 = 0.3–0.5 μM) against DENV infection, while maintaining low cytotoxicity (CC50 > 500 μM, SI > 1000). In male Sprague-Dawley rats, compound 3l was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (F = 92 ± 4%).

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