Molecular analysis of a U3 RNA gene locus in tomato: transcription signals, the coding region, expression in transgenic tobacco plants and tandemly repeated pseudogenes

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Abstract
By screening a tomato genomic library with a tomato U3 RNA probe, we detected a U3 genomic locus whose coding region was determined by primer extension (5'' end) and direct RNA sequencing of purified U3 RNA from tomato (3'' end). Tomato U3 RNA is 216 nucleotides long, contains all the four evolutionarily highly conserved sequence blocks (Boxes A to D), has at its 5'' end a cap not precipitable with anti-m3G antibodies and can be folded into a peculiar secondary structure with two stem-loops at its 5'' end. A tagged derivative of the U3 gene was faithfully expressed in transgenic tobacco plants. In the 5'' flanking region both plant-specific UsnRNA transcription signals [the TATA-like sequence and the upstream sequence element (USE)] were present, but were positioned closer to each other and also to the cap site in the U3 gene than in the genes for the plant spliceosomal UsnRNAs studied so far. The 3'' flanking region of the tomato U3 gene lacked the consensus sequence of the putative termination signal established for the plant spliceosomal UsnRNA genes and contained a pyrimidine-rich tract (R1) followed by four tandemly repeated U3 pseudogenes (U3.1 ps to U3.4 ps) flanked by slightly altered forms (R2 to R5) of R1 and most probably generated by DNA-mediated events. Our results are in line with the conjecture that the enzyme transcribing the tomato U3 gene has different structural requirements for transcriptional activity than the enzyme transcribing plant U1, U2 and U5 genes.