The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma - Cloning and identification of a transcribed tRNA-like element that controls its expression

U4 small nuclear RNA is essential for tra ns-splicing. Here we report the c loning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, i t carries an Sm-like site, and has the potential for extensive intermolecul ar base pairing with the U6 RNA. Sequence analysis of the U4 locus indicate s the presence of a tRNA-Iike element 86 base pairs upstream of the gene th at is divergently transcribed to yield a stable small tRNA-like RNA. Two ad ditional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this e lement. By stable expression of a tagged U4 RNA we demonstrate that the tRN A-like gene, but not the upstream tRNA genes, is essential for U4 expressio n and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo, Mapping the 2'-O-methyl groups on U4 and U6 small nucl ear RNAs suggests the presence of modifications in canonical positions. How ever, the number of modified nucleotides is fewer than in mammalian homolog ues, The U4 genomic organization including both tRNA-Iike and tRNA genes ma y represent a relic whereby trypanosomatids "hired" tRNA genes to provide e xtragenic promoter elements, The close proximity of tRNA genes to the tRNA- like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.