Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells

Abstract

In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. A database analysis of human box C͞D snoRNAs revealed conservation of their intronic location, with a preference for 70 -80 nt upstream of the 3 splice site. Transfection experiments showed that synthesis of gas5-encoded U75 and U76 snoRNAs dropped significantly for mutant constructs possessing longer or shorter spacers between the snoRNA and the 3 splice site. However, the position of the snoRNA did not affect splicing of the host intron. Substitution mutations within the spacer indicated that the length, but not the specific sequence, is important. A in vitro system that couples pre-mRNA splicing and processing of U75 has been developed. U75 synthesis in vitro depends on its box C and D sequences and requires an appropriate spacer length. Further mutational analyses both in vivo and in vitro, with subsequent mapping of the branch points, revealed that the critical distance is from the snoRNA coding region to the branch point, suggesting synergy between splicing and snoRNA release.T he nucleoli of eukaryotic cells contain large numbers of small nucleolar ribonucleoprotein (snoRNP) particles that function in the nucleolytic processing and nucleotide modification of precursor rRNAs. Currently, more than 150 small nucleolar RNA (snoRNA) species have been identified. SnoRNAs can be divided into two classes: those that possess boxes C (RUG-AUGA) and D (CUGA), required for association with an abundant nucleolar protein fibrillarin, and those that possess boxes H (ANANNA) and ACA, which mediate the binding of Gar1 protein (reviewed in refs. 1 and 2). Box C͞D snoRNAs and box H͞ACA snoRNAs target specific sites in pre-rRNA for 2Ј-O-methylation and pseudouridylation, respectively (3-8). The methylation reaction is guided by an extensive region (10-21 nt) of complementarity between the box C͞D snoRNA and rRNA sequences flanking the modification site (3, 5, 9-11).Vertebrate snoRNAs are encoded within the introns of snoRNA host genes, which can be either protein coding or noncoding (reviewed in refs. 12 and 13). A common feature of mammalian snoRNA host gene transcripts is a 5Ј terminal oligopyrimidine sequence, whose precise function with respect to snoRNA synthesis is unknown (14, 15). In most cases, it appears that snoRNAs are released from excised, debranched introns by exonucleolytic trimming (16-18); a minor pathway involves endonucleolytic cleavage of flanking intron sequences (19,20). Many yeast snoRNAs are transcribed as monocistronic or polycistronic precursors from independent transcription units, and proteins involved in their processing have been characterized (21-23). On the other hand, no factors involved in snoRNA release have been identified in mammalian cells.The processing of intronic snoRNAs is directed by elements residing within the snoRNA coding region. Exonucleolytic trimming and accumulation of vertebrate box C͞D snoRNAs depend on the C and D boxes and an adjacent 4-to 5-...

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

“…However, few chicken and human ncRNA genes are paired in regions of conserved synteny (Table 2), relative to the high level of shared gene order observed for protein-coding genes (see below). Those classes of ncRNAs that are most often syntenic are microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs), which are often found in the introns of protein-coding genes (or, rarely, of specialized 'host' genes 35 ). Most ncRNA genes thus seem to have been translocated to distant genomic sites during vertebrate evolution, without accumulating large numbers of pseudogenes, as would be expected were this process to occur via retrotransposition.…”

Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells

“…RPAs were performed as described (Hirose and Steitz 2001). A PCR fragment that covers each ncRNA region was cloned into pGEM-T Easy vector (Promega), followed by digestion with an appropriate restriction enzyme for in vitro transcription.…”

Genome-wide analyses of two families of snoRNA genes from Drosophila melanogaster, demonstrating the extensive utilization of introns for coding of snoRNAs

“…6). Taking account of sequences necessary for snoRNA processing (Hirose and Steitz 2001), the intron hosts are only sufficiently long enough to hold a snoRNA gene or gene cluster without any redundant sequence. Interestingly, this compact structure of the host introns appears strictly maintained to encode the two families of snoRNAs in different host genes.…”

“…The distance from the snoRNA gene/gene cluster to the 3 0 splice sites averaged between 60 and 80 bp, which was very similar to the intronic positioning of box C/D snoRNA genes in mammals. This distance has been proven to be important for the effective processing of the snoRNAs from their host mRNA precursor (Hirose and Steitz 2001).…”