Nucleic Acids Research

We have isolated and partially characterized a family of small nuclear UNAs (snRNAs) from three different species of the protozoan Tetrahymena. We find six distinct snRNAs ranging in size from 100 to 250 nucleotides. The two largest snRNAs, as well as an abundant, heterogenous group of smaller snRNAs are found in the nucleolar RNA fraction. None of the snRNAs are transcription products of the ribosomal RNA gene or its flanking regions, as shown by hybridization tests. The snRNAs are metabolically stable as determined by pulse/ chase experiments and several of them contain a number of modified nuclotides. The snRNAs from Tetrahymena all have slightly different sizes from mammalian snRNAs. The cap structure of the snRNAs from Tetrahymena differs from that of the snRNAs from mammalian cells, but has not yet been fully characterized. The relative amount of snRNAs to total RNA is less in Tetrahymena (>0.1%) than in mammalian cells (.-2%). INTRODUCTION Eucaryotic cells contain a set of discrete, metabolically stable small nuclear RNAs (snRNAs) ranging in size from 100 to 300 nucleotides (for reviews, see ref. 1,2). The most abundant of the snRNAs (U1-U6) have an unusual cap-structure, contain many modified nucleotides, are transcription products of RNA polymerase II (3) and are found as ribonucleoprotein particles (snRNP). The U-snRNP particles can be precipitated by specific human auto-antibodies (4). An increasing number of data indicates that the UlsnRNP (and possibly U2-snRNP) is involved in the splicing of premessenger RNA (5,6,7,8). Although the U-snRNA family has been studied extensively in mammalian cells, the function of the other U-snRNAs still remains elusive. The nucleolar snRNA U3 has been found hydrogenbonded to pre-28S ribosomal RNA (9,10) and recent models based on sequence data suggest a functional role of U3 RNA in the processing of pre-28S ribosomal RNA (11,12). © I RL Press Limited, Oxford, England. 4203 Nucleic Acids Research Gel electrophoretic characterization of nuclear RNA have demonstrated the presence of snRNAs in a variety of lower eucaryotic cells (13, 14, 15). Detailed analyses have been made on snRNAs from the slime mold Dictyostelium (16,17) the yeast Saccharomyces cerevisiae (18), the dinoflagellate Crypthecodinium cohnii (19) and pea plants (20). These studies show that lower eukaryotes and plants have U-snRNAs in the same size range as that of mammalian cells and that these snRNAs contain a specific cap-structure as well as a number of modified nucleotides. The U-like snRNAs in lower eukaryotes have slightly different sizes than their mammalian counterparts and appear in fewer copies per cell. The nucleotide sequence of the different U-snRNAs is extremely well conserved among vertebrates (1). The sequence data of snRNAs in lower eukaryotes are limited to the sequence of a U3-like snRNA in Dictyostelium (called D2) (21), a U4/U6-like snRNA in yeast (called snR3) (22) and U5-like snRNAs in pea plants (20), dinoflagellate (23) and Tetrahymena (24). The nucleotide sequence data suggest very similar secondary structures of the homologous snRNAs from lower and higher eukaryotes with extensive nucleotide homology in areas proposed to be single-stranded. It is therefore likely that the U-snRNAs have important and conserved functions. Contradictory to this view are the observations that Dictyostelium has a capped snRNA with a nucleotide sequence that is not homologous to any of the mammalian snRNAs (25) and that the U4-like snRNA in yeast is dispensable for this organism (22). Our aim was to obtain further information on small nuclear RNAs from a lower eukaryote. The protozoan Tetrahymena was chosen for several reasons: 1) it is well characterized metabolically; 2) nuclei and nucleoli can be isolated in pure and transcriptionally active forms; 3) the ribosomal RNA gene and the processing of ribosomal RNA have been well characterized (26) and a large part of the ribosomal RNA gene has been sequenced, making Tetrahymena a particularly suitable organism for studying nucleolar snRNAs; and 4) early publications have indicated the existence of at least three snRNAs in Tetrahymena (27,28). Furthermore, one snRNA from Tetrahymena has been characterized in detail (24). It was shown to have a cap, a high nucleotide sequence homology to mammalian US and could be assigned the same secondary structure as U5.