Bone marrow and bone as a source for postmortem RNA.

The susceptibility of RNA to enzymatic degradation has been considered as a tool to estimate time-since-death in forensic samples, and it has previously been demonstrated that the choice of tissue is an important factor. In this study we have extracted RNA from decaying bone and bone marrow under the hypothesis that the delayed onset of putrefaction may render them a useful source in this context. In a preliminary study, total RNA was extracted from bone and bone marrow that had been sampled from six skeletally mature rabbits at time points between zero and 31 days after death. The levels of three specific RNA transcripts could be quantified using real-time polymerase chain reaction. Bioanalyzer results show rRNA bands in bone marrow samples up to 21 days postmortem. We hereby propose bone marrow as a potential source for postmortem RNA in forensic studies.

Cartilage-hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly.

Cartilage-hair hypoplasia (CHH) is caused by mutations in the gene encoding the RNA component of RNase MRP. Currently it is unknown how these mutations affect the function of this endoribonuclease. In this study we investigated the effect of mutations in the P3 domain on protein binding and RNA folding. Our data demonstrate that a number of P3 nucleotide substitutions reduced the efficiency of its interaction with Rpp25 and Rpp20, two protein subunits binding as a heterodimer to this domain. The CHH-associated 40G>A substitution, as well as the replacement of residue 47, almost completely abrogated Rpp25 and Rpp20 binding in different assays. Also other CHH-associated P3 mutations reduced the efficiency by which the RNase MRP RNA is bound by Rpp25-Rpp20. These data demonstrate that the most important residues for binding of the Rpp25-Rpp20 dimer reside in the apical stem-loop of the P3 domain. Structural analyses by NMR not only showed that this loop may adopt a pseudo-triloop structure, but also demonstrated that the 40G>A substitution alters the folding of this part of the P3 domain. Our data are the first to provide insight into the molecular mechanism by which CHH-associated mutations affect the function of RNase MRP.