Case Presentation Mr. A, a 34-year-old man, came to our outpatient clinic for treatment of a major depressive episode. His history was also notable for polysubstance abuse and dependence in sustained remission, with prior abuse of alcohol, LSD, and other hallucinogens and prior dependence on marijuana, cocaine, opiates, and Ecstasy (MDMA [3,4-methylenedioxymethamphetamine]). He began using alcohol at age 9, marijuana at age 12, cocaine at age 13, opiates at age 20, and Ecstasy at age 21. He had a history of depressive symptoms 6 years earlier in the context of active substance dependence and chronic back pain but had no other prior psychiatric diagnosis or treatment. One year before presentation to our clinic, he had been smoking cocaine daily, using Ecstasy several days a week, and consuming two to 10 alcoholic drinks daily but reported no depressive symptoms. After consuming cocaine, Ecstasy, oxycodone, and methadone at a party, he became aggressive and was brought to an emergency room. There, he ingested all of his remaining methadone to prevent it from being discovered. He reported no suicidal intentions surrounding this ingestion. He became unresponsive, hypoxic, and hypotensive. Mr. A was resuscitated and then stabilized in an intensive care unit over 4 days. After this overdose, Mr. A became acutely depressed. He endorsed a depressed mood, anhedonia, low energy, difficulties concentrating and remembering, feelings of hopelessness and guilt, poor self-esteem, social isolation, increased sleep, and a 20-lb weight gain over the ensuing year. He reported the disappearance of drug cravings and remained abstinent from all recreational drugs

Several enzymatic and chemical reagents were used to probe the secondary structure of Saccharomyces cerevisiae nuclear RNase P RNA in the presence and absence of its protein components. Double-stranded regions were detected with RNase V1 and single-stranded regions with RNase ONE (Escherichia coli RNase I). Nucleotides not paired at Watson-Crick positions were monitored with dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-[2-(N-methylmorpholinio)ethyl]carbodiimide p-toluenesulfonate. The results supported most aspects of the previously proposed, phylogenetically-derived RNA secondary structure, although minor refinements allowed incorporation of both the biochemical and phylogenetic data. Digestion of the RNase P protein(s) with proteinase K gave enhanced reactivities to structure probes at selected positions, indicating regions of the RNA made inaccessible by the presence of the protein subunit(s). The regions of RNA protected in the yeast nuclear holoenzyme were considerably more extensive than that seen in the Escherichia coli holoenzyme, consistent with the observation that the protein moiety generally comprises a larger percentage of the RNase P holoenzyme in eukaryotes than in eubacteria.

Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined. To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA. The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S. cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not. Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes.

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