Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora in termedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected fo r a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mit ochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA, This 7-kb insertion also contains a perfectly duplicated tRNA(Trp) gene, segments of several mitochondrial plasmids and n umerous GC-rich palindromic sequences that are repeated elsewhere in the mt DNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergo es 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt in tron-like sequence in TRL-78, which is missing in the TRL-64 form, is not s pliced detectably in vivo or in vitro. Our results show that TRL-78 is an u nusual tRNA-like species that could be incorporated into suppressive retrop lasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containi ng nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA vi a reverse transcription, analogous to SINE elements in animal cells.