Non-coding snoRNA host genes in Drosophila: expression strategies for modification guide snoRNAs.

Modification guide snoRNAs either are encoded within introns and co-transcribed with the host gene pre-mRNA or are independently transcribed as mono- or polycistronic units. Different eukaryotic kingdoms utilize these coding strategies to various degrees. Intron-encoded and polycistronic snoRNAs are released from primary transcripts as pre-snoRNAs by the spliceosome or by an RNase III-like activity, respectively. In the spliceosomal pathway, the resulting intron lariat is then linearized by a debranching activity. The leader and trailer sequences of pre-snoRNAs are removed by exonucleolytic activities. The majority of snoRNA host genes encode proteins involved in the synthesis, structure or function of the translational apparatus. Several vertebrate snoRNA host genes do not appear to code for functional proteins. We have identified two unusually compact box C/D multi-snoRNA host genes in D. melanogaster, dUHG1 and dUHG2, similar in their organization to the corresponding vertebrate non-protein-coding host genes. In dUHG1 and dUHG2, the snoRNA sequences are located within introns at a conserved distance of about 75 nucleotides upstream of the 3' splice sites. Both genes initiate transcription with TOP-like sequences that share unique features with previously reported Drosophila snoRNA host genes. Although the spliced dUHG RNAs are relatively stable, they exhibit little potential for protein coding.

Modification of U6 spliceosomal RNA is guided by other small RNAs.

The vertebrate spliceosomal snRNAs are highly modified by pseudouridylation and 2'-O-methylation. We have identified novel conserved small RNAs that can direct addition of two methyl groups in U6 snRNA, at A47 and C77. These guide RNAs, mgU6-47 (methylation guide for U6 snRNA residue 47) and mgU6-77 contain boxes C, C', D, and D' and associate with fibrillarin. Each RNA can form a duplex with U6 snRNA positioning A47 and C77 for 2'-O-methylation. The antisense element of mgU6-77 can also position C2970 of 28S rRNA for 2'-O-methylation. Depletion of mgU6-77 from Xenopus oocytes prevents 2'-O-methylation of both C77 in U6 and C2970 in 28S; methylation can be restored by injecting in vitro transcribed mgU6-77. Thus, mgU6-77 appears to function in the 2'-O-methylation of two distinct classes of cellular RNA, snRNA, and rRNA.

A small nucleolar RNA requirement for site-specific ribose methylation of rRNA in Xenopus.

Vertebrate cells contain a large number of small nucleolar RNA (snoRNA) species, the vast majority of which bind fibrillarin. Most of the fibrillarin-associated snoRNAs can form 10- to 21-nt duplexes with rRNA and are thought to guide 2'-O-methylation of selected nucleotides in rRNA. These include mammalian UHG (U22 host gene)-encoded U25-U31 snoRNAs. We have characterized two novel human snoRNA species, U62 and U63, which similarly exhibit 15- (with one interruption) and 12-nt complementarities and are therefore predicted to direct 2'-O-methylation of A590 in 18S and A4531 in 28S rRNA, respectively. To establish the function of antisense snoRNAs in vertebrates, we exploited the Xenopus oocyte system. Cloning of the Xenopus U25-U31 snoRNA genes indicated that they are encoded within multiple homologs of mammalian UHG. Depletion of U25 from the Xenopus oocyte abolished 2'-O-methylation of G1448 in 18S rRNA; methylation could be restored by injecting either the Xenopus or human U25 transcript into U25-depleted oocytes. Comparison of Xenopus and human U25 sequences revealed that only boxes C, D, and D', as well as the 18S rRNA complement, were invariant, suggesting that they may be the only elements required for U25 snoRNA stability and function.

A mammalian gene with introns instead of exons generating stable RNA products.

The nucleoli of eukaryotic cells are the sites of ribosomal RNA transcription and processing and of ribosomal subunit assembly. They contain multiple small nucleolar RNAs (snoRNAs), several of which are essential for rRNA maturation. The U3, U8 and U13 snoRNA genes are transcribed independently, whereas U14-U24, as well as E3, are located within introns of protein-coding genes, most of whose functions are linked to translation. These snoRNAs are co-transcribed with their host pre-mRNAs and released by processing from excised introns. Here we show that, in addition to U22, seven novel fibrillarin-associated snoRNAs, named U25-U31, are encoded within different introns of the unusually compact mammalian U22 host gene (UHG). All seven RNAs exhibit extensive (12-15 nucleotides) complementarity to different segments of the mature rRNAs, followed by a C/AUGA ('U-turn') sequence. The spliced UHG RNA, although it is associated with polysomes, has little potential for protein coding, is short-lived, and is poorly conserved between human and mouse. Thus, the introns rather than the exons specify the functional products of UHG.

Mapping of ribosomal protein S3 and internally nested snoRNA U15A gene to human chromosome 11q13.3-q13.5.

The mammalian ribosome is a massive structure composed of 4 RNA species and about 80 different proteins. One of these ribosomal proteins, S3, appears to function not only in translation but also as an endonuclease in repair of UV-induced DNA damage. Moreover, the first intron of human RPS3 transcripts is processed to generate U15A, a small nucleolar RNA. We localized the nested RPS3/U15A genes to the immediate vicinity of D11S356 and D11S533 on human chromosome 11q13.3-q13.5 using a combination of somatic cell hybrid analysis, fluorescence in situ hybridization, and YAC/STS content mapping. These findings add to the evidence that genes encoding ribosomal proteins are scattered about the human genome.

Requirement for intron-encoded U22 small nucleolar RNA in 18S ribosomal RNA maturation.

The nucleoli of vertebrate cells contain a number of small RNAs that are generated by the processing of intron fragments of protein-coding gene transcripts. The host gene (UHG) for intro-encoded human U22 is unusual in that it specifies a polyadenylated but apparently noncoding RNA. Depletion of U22 from Xenopus oocytes by oligonucleotide-directed ribonuclease H targeting prevented the processing of 18S ribosomal RNA (rRNA) at both ends. The appearance of 18S rRNA was restored by injection of in vitro-synthesized U22 RNA. These results identify a cellular function for an intron-encoded small RNA.

Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry.

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.

tRNA splicing in yeast and wheat germ. A cyclic phosphodiesterase implicated in the metabolism of ADP-ribose 1",2"-cyclic phosphate.

Adenosine diphosphate (ADP)-ribose 1",2"-cyclic phosphate (Appr > p) is produced as a result of transfer RNA (tRNA) splicing in the yeast Saccharomyces cerevisiae and probably in other eukaryotes. Endonucleolytic cleavage and ligation result in a mature length tRNA with a 2'-phosphate at the splice junction. This 2'-phosphate is transferred to NAD to produce Appr > p. Metabolism of Appr > p requires hydrolysis of the 1",2"-cyclic phosphate linkage. We show here that yeast has a unique cyclic phosphodiesterase that can hydrolyze Appr > p, ribose 1,2-cyclic phosphate, and ribose 1,3-cyclic phosphate to the corresponding ribose 1-phosphate derivatives. The cyclic phosphodiesterase is highly specific for Appr > p; there is 20-fold less activity on ribose 1,3-cyclic phosphate and no detectable activity on nucleoside 2',3'-cyclic phosphates. A similar cyclic phosphodiesterase is present in wheat germ. The wheat germ cyclic phosphodiesterase activity co-chromatographs with a 2',3'-cyclic nucleotide 3'-phosphodiesterase that was previously identified and purified. The purified wheat germ enzyme has a distinct preference for Appr > p and ribose cyclic phosphate compared to guanosine 2',3'-cyclic phosphate and shares other biochemical characteristics with the yeast enzyme.

A small nucleolar RNA is processed from an intron of the human gene encoding ribosomal protein S3.

A human small nucleolar RNA, identified previously in HeLa cells by anti-fibrillarin autoantibody precipitation and termed RNA X, has been characterized. It comprises two uridine-rich variants (148 and 146 nucleotides), which we refer to as snRNA U15A and U15B. Secondary structure models predict for both variants a U15-specific stem-loop structure, as well as a new structural motif that contains conserved sequences and can also be recognized in the other fibrillarin-associated nucleolar snRNAs, U3, U14, and RNA Y. The single-copy gene for human U15A has been found unexpectedly to reside in intron 1 of the ribosomal protein S3 gene; the U15A sequence appears on the same strand as the S3 mRNA and does not exhibit canonical transcription signals for nuclear RNA polymerases. U15A RNA is processed in vitro from S3 intron 1 transcripts to yield the correct 5' end with a 5'-monophosphate; the in vitro system requires ATP for 3' cleavage, which occurs a few nucleotides downstream of the mature end. The production of a single primary transcript specifying the mRNA for a ribosomal or nucleolar protein and a nucleolar snRNA may constitute a general mechanism for balancing the levels of nucleolar components in vertebrate cells.