A novel experimental approach for the selective isolation and characterization of human RNase MRP.

RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity.

siRNA in ovarian cancer - Delivery strategies and targets for therapy.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, and the sixth leading cause of cancer related death in women overall. Despite improved surgical techniques and advances in chemotherapy, mortality hardly decreased over the last twenty years. The major problem is that (micro)metastases persevere in the abdominal cavity, causing incurable tumor recurrence. Therefore, there is an imminent need for new therapeutic strategies. Oligonucleotide (ON) based therapies such as RNA interference (RNAi) provide the possibility to specifically address disease-related pathways. However, small interfering RNA (siRNA) molecules are unable to enter cells without a drug delivery system. Therefore, nanocarriers have been developed to aid intracellular delivery of siRNA. EOC is, in most cases, confined to the abdominal cavity, providing the possibility for peritoneal drug delivery. As a consequence, EOC should be an ideal candidate for ON therapies as intraperitoneal delivery reduces sequestration of drug formulations in other organs. In this review, we will discuss delivery strategies and siRNA targets that have been tested in EOC. Delivery strategies cover the full range of delivery approaches from polymers to exotic delivery strategies like microbubble based nanoparticles. For siRNA targets, those that aim at re-sensitizing the tumor cells to chemotherapy can be discriminated from those that reduce growth and metastasis of the tumor cells. Despite preclinical successes and the advantage that intraperitoneal delivery holds over systemic delivery, no strategy has made it into the clinic yet. We postulate that confirmatory studies that combine the most promising delivery approaches with the most promising targets are required to reach a consensus on those formulations that should be pursued for further (pre-)clinical research.

RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences.

The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.