Cytosolic rRNAs are highly dynamic and can be degraded under conditions such as apoptosis, starvation and magnesium depletion. The degradation is also related to their specific localization, as fractions of cytosolic ribosomes are localized on the surfaces of intracellular organelles, such as endoplasmic reticulum (ER) and mitochondria. Such localized translation facilitates translocation of nascent proteins into these organelles co-translationally, contributing to fast responses to cellular stresses and precise regulations of the organelle. Here, we describe a protocol to establish the in organello system to investigate rRNA degradation on mitochondrial outer membrane or ER. The protocol consists of organelle isolation, rRNA degradation on organelles and agarose gel electrophoresis to examine the remaining rRNAs.

Mitochondria have two sets of RNAs. One is encoded in mitochondrial genome, and the other that consists of imported RNAs within mitochondria and cytosolic RNAs associated with mitochondrial outer membrane is encoded in the nucleus. These mitochondrial RNAs play important roles in mitochondrion biosynthesis and signaling in and out of mitochondria. Isolation and analysis of mitochondrial RNAs can provide useful information on understanding the mitochondrial regulation of cellular processes. However, several ribonuclease activities have been found in mitochondria, which will degrade mitochondrial RNAs during the isolation process if they are not properly inactivated. Here, we describe an improved method to inactivate the ribonuclease activities prior to RNA extraction, and thus provide a reliable protocol to isolate mammalian mitochondrial RNAs for quantitative RT-PCR and other assays.