Classification of gas5 as a multi-small-nucleolar-RNA (snoRNA) host gene and a member of the 5'-terminal oligopyrimidine gene family reveals common features of snoRNA host genes.

In the nucleolus of eukaryotic cells, ribosomal DNA is transcribed by RNA polymerase I into long precursor (pre-rRNA) transcripts, which are modified by methylation and pseudo-uridylation, cleaved to yield 18S, 5.8S, and 28S rRNAs, and then assembled into the mature large and small ribosomal subunits prior to export to the cytoplasm (for reviews see references 22, 26, and 77). A large number of small nucleolar ribonucleoprotein (snoRNP) particles have emerged as key players in this biosynthetic process. Currently more than 70 snoRNA species have been identified (for reviews see references 51, 75, and 82). All snoRNAs, with the exception of MRP RNA, can be divided into two classes: those that possess boxes C (RUGAUGA) and D (CUGA), which are required for association with the abundant nucleolar autoantigen fibrillarin (see reference 51), and those that possess boxes H (ANANNA) and ACA, which mediate the binding of Gar1 protein (4, 8, 24, 37). Only a few snoRNAs have been found to be required for growth in yeast (U3 [6, 29], U14 [43], MRP [14, 73], snR10 [80, 81], and snR30 [5, 54]) or for specific pre-rRNA cleavage events in Xenopus oocytes (U3 [34, 72], U8 [63], and U22 [85]). Recently, box C/D snoRNAs and box H/ACA snoRNAs were found to target specific sites in pre-rRNA for 2′-O-methylation and pseudouridylation, respectively (for reviews see references 46, 48, 62, 75, and 82). These modification reactions are mediated by extensive regions (10 to 21 nt) of complementarity between the so-called “antisense” snoRNAs and sequences flanking the rRNA sites to be modified. Specifically, U24, U20, and U25 were shown to direct site-specific ribose methylation of pre-rRNA in HeLa cells (39), yeast (13), and Xenopus oocytes (87), respectively, and snR8, snR3, snR33, and snR5 (among others) were demonstrated to target pre-rRNA for pseudouridylation in yeast (23, 57). The presence of ∼200 modified nucleotides in vertebrate rRNA (47) suggests that more than half of the antisense snoRNAs remain to be identified. A unique feature of snoRNAs is that most are encoded within the introns of protein-coding genes (reviewed in reference 51). This economic use of introns is commonplace among intron-rich organisms, such as vertebrates, where antisense snoRNAs have been found to be exclusively intron encoded. By contrast, in Saccharomyces cerevisiae many snoRNAs are produced from independent transcription units. A vertebrate host gene intron encodes only a single snoRNA, whereas in yeast and plants, some snoRNA genes are located in polycistronic arrays without exons separating the snoRNA sequences (41). In all species investigated, the intron-encoded snoRNAs are transcribed from their host genes by RNA polymerase II as portions of the pre-mRNA. The functional snoRNAs are then produced by exonucleolytic trimming that follows either splicing (12, 38, 86) or endonucleolytic cleavage of intron sequences (9, 10). The mode by which snoRNA sequences became inserted into the introns of their host genes is not known. Interestingly, the host gene for a particular snoRNA can differ even among closely related vertebrates, suggesting that intron-encoded snoRNAs may be highly mobile genetic elements (see reference 51). Likewise, the reason particular genes have been chosen as hosts for intron-encoded snoRNAs has been unclear. Initially, it appeared that all snoRNA host genes generate protein products that function in ribosome biogenesis or in translation; ribosomal proteins (rp) L1, L5, L7a, S8, nucleolin, and eIF4AI are a few examples (see references 66, 68, 67, 59, 58, and 24, respectively). Such genetic organization could provide coregulation of protein components of the translational machinery and snoRNAs, which contribute to rRNA maturation (76). However, the discovery of other snoRNA host genes lacking obvious ribosome-related functions (for example, ATP synthase β [39]) suggested that host genes may have been chosen merely to meet the need for transcription rates high enough to produce a sufficient level of snoRNAs (∼104 copies/cell) to base pair with the cell’s nascent pre-rRNA molecules. UHG (U22 host gene) is an unusual snoRNA host gene because it does not appear to specify a protein product. It generates, in addition to U22, seven different box C/D antisense snoRNAs (U25 to U31) (84). Comparison of mouse and human UHG sequences revealed that its introns are more conserved than its exons, suggesting that the snoRNAs may be the only functional portions of the transcript. Both human and mouse UHG messages are riddled with stop codons in all three reading frames; the longest open reading frames (ORFs) would produce peptides of 51 and 40 amino acids for human and mouse UHG, respectively. Nonetheless, the UHG transcript resembles a typical rp mRNA in that it begins with a C residue, is spliced and polyadenylated, and is associated with ribosomes. However, unlike rp mRNAs, the spliced UHG RNA is almost undetectable in HeLa cells. Inhibition of translation in HeLa cells with the initiation inhibitor pactamycin or elongation inhibitors cycloheximide or puromycin results in a 15-fold increase in the level of spliced UHG transcript (84). This link between the levels of UHG RNA and active translation, in conjunction with its numerous stop codons, suggests that it may be a candidate for the nonsense-mediated decay pathway (49, 84). Here we report the identification of a second member of the UHG class of snoRNA host genes. Growth arrest-specific transcript 5 (gas5) was initially discovered in a screen for potential tumor suppressor genes expressed at high levels during growth arrest (74). The murine gas5 gene produces a ubiquitous, polyadenylated, alternatively spliced message which is almost undetectable in actively growing cells yet is highly expressed in cells undergoing serum starvation or density arrest (15, 16). We demonstrate that gas5 is a multi-snoRNA host gene which encodes 9 (in mouse) or 10 (in human) antisense snoRNAs. By mapping the 5′ end of the gas5 transcript and comparing it with other known snoRNA host genes, we observe that all known snoRNA host genes exhibit characteristics which define the 5′TOP (terminal oligopyrimidine) class of genes. We provide evidence that membership in the 5′TOP family explains why the abundance of the gas5 spliced product is growth dependent. Furthermore, the discovery that all snoRNA host genes contain 5′TOP sequences may illuminate why certain genes have been selected to serve as snoRNA host genes.