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Filippini D Renzi F Bozzoni I Caffarelli E 《Biochemical and biophysical research communications》2001,288(1):16-21
The Xenopus laevis Nop56 gene (XNOP56), coding for a snoRNP-specific factor, belongs to the 5'-TOP gene family. XNOP56, as many 5'-TOP genes, contains an intron-encoded snoRNA. This previously unidentified RNA, named U86, was found as a highly conserved species in yeast and human. While in human it is also encoded in an intron of the hNop56 gene, in yeast it has an unprecedented gene organization: it is encoded inside an open-reading frame. Both in X. laevis and yeast, the synthesis of U86 snoRNA appears to be alternative to that of the cotranscribed mRNA. Despite the overall homology, the three U86 snoRNAs do not show strong conservation of the sequence upstream from the box D and none of them displays significant sequence complementarity to rRNA or snRNA sequences, suggesting a role different from that of methylation. 相似文献
3.
Intronic U14 snoRNAs of Xenopus laevis are located in two different parent genes and can be processed from their introns during early oogenesis. 总被引:3,自引:0,他引:3
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L Xia J Liu C Sage E B Trexler M T Andrews E S Maxwell 《Nucleic acids research》1995,23(23):4844-4849
U14 is a member of the rapidly growing family of intronic small nucleolar RNAs (snoRNAs) that are involved in pre-rRNA processing and ribosome biogenesis. These snoRNA species are encoded within introns of eukaryotic protein coding genes and are synthesized via an intron processing pathway. Characterization of Xenopus laevis U14 snoRNA genes has revealed that in addition to the anticipated location of U14 within introns of the amphibian hsc70 gene (introns 4, 5 and 7), additional intronic U14 snoRNAs are also found in the ribosomal protein S13 gene (introns 3 and 4). U14 is thus far a unique intronic snoRNA in that it is encoded within two different parent genes of a single organism. Northern blot analysis revealed that U14 snoRNAs accumulate during early oocyte development and are rapidly expressed after the mid-blastula transition of developing embryos. Microinjection of hsc70 pre-mRNAs into developing oocytes demonstrated that oocytes as early as stages II and III are capable of processing U14 snoRNA from the pre-mRNA precursor. The ability of immature oocytes to process intronic snoRNAs is consistent with the observed accumulation of U14 during oocyte maturation and the developmentally regulated synthesis of rRNA during oogenesis. 相似文献
4.
E Caffarelli A Fatica S Prislei E De Gregorio P Fragapane I Bozzoni 《The EMBO journal》1996,15(5):1121-1131
A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r-protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre-mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre-mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA. 相似文献
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Eukaryotic nucleoli contain a large family of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs) that are involved in processing and site-specific methylation of pre-rRNA. Several proteins have been reported to be common factors of box C/D snoRNPs in lower and higher eukaryotes; nevertheless none of them has been clearly shown to directly interact with RNA. We previously identified in Xenopus laevis, by means of UV crosslinking in vivo, two proteins associated with box C/D snoRNAs, fibrillarin and p68. Here we show that fibrillarin interacts directly and specifically with the U16 box C/D snoRNA in a X. laevis oocyte nuclear extract and that it does not require p68 for binding. Specific binding is also obtained with a recombinant fibrillarin demonstrating that the protein is able to bind directly and specifically to U16 snoRNA by itself. 相似文献
7.
In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.
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E Caffarelli M Arese B Santoro P Fragapane I Bozzoni 《Molecular and cellular biology》1994,14(5):2966-2974
It was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions. 相似文献
8.
The sequences and structural features of Xenopus laevis U3 small nucleolar RNA (snoRNA) necessary for pre-rRNA cleavage at sites 1 and 2 to form 18 S rRNA were assayed by depletion/rescue experiments in Xenopus oocytes. Mutagenesis results demonstrated that the putative stem of U3 domain I is unnecessary for 18 S rRNA processing. A model consistent with earlier experimental data is proposed for the structure of domain I when U3 is not yet bound to pre-rRNA. For its function in rRNA processing, a newly discovered element (5' hinge) was revealed to be important but not as critical as the 3' hinge region in Xenopus U3 snoRNA for 18 S rRNA formation. Base-pairing is proposed to occur between the U3 5' hinge and 3' hinge and complementary regions in the external transcribed spacer (ETS); these interactions are phylogenetically conserved, and are homologous to those previously described in yeast (5' hinge-ETS) and trypanosomes (3' hinge-ETS). A model is presented where the base-pairing of the 5' hinge and 3' hinge of U3 snoRNA with the ETS of pre-rRNA helps to correctly position U3 boxes A'+A for their function in rRNA processing. Like an earlier proposal for yeast, boxes A' and A of Xenopus may base-pair with 18 S sequences in pre-rRNA. We present the first direct experimental evidence in any system that box A' is essential for U3 snoRNA function in 18 S rRNA formation. The analysis of insertions and deletions indicated that the spacing between the U3 elements is important, suggesting that they base-pair with the ETS and 18 S regions of pre-rRNA at the same time. 相似文献
9.
U21, a novel small nucleolar RNA with a 13 nt. complementarity to 28S rRNA, is encoded in an intron of ribosomal protein L5 gene in chicken and mammals. 总被引:2,自引:2,他引:0
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L H Qu M Nicoloso B Michot M C Azum M Caizergues-Ferrer M H Renalier J P Bachellerie 《Nucleic acids research》1994,22(20):4073-4081
Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit. 相似文献
10.
Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease. 总被引:5,自引:0,他引:5
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An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNAs. Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing. To identify the factors necessary for processing of intron-encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA- coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing-deficient phenotype. Specific depletion of Nop1p completely prevents U18 snoRNA synthesis, but does not affect processing of a dicistronic snoRNA-coding unit that has a canonical Rnt1p site. Correct cleavage of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p. Pull-down experiments showed that the two proteins interact physically. These data indicate that cleavage of U18, snR38 and possibly other intron-encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred. 相似文献
11.
The U18 small nuclear RNA (snRNA) is one of several newly discovered intron-encoded nucleolar RNAs whose function is unknown. We have studied the accumulation and function of the U18 snRNA in oocytes of the vertebrate, Xenopus laevis. The U18 snRNA contains 13 nt complementary to a highly conserved sequence in 28S ribosomal RNA (rRNA). Three oligonucleotides, selected to contain all or some of the complementary sequence, deplete the U18 snRNA upon injection into Xenopus oocytes. Injection of two of the oligonucleotides has no effect on pre-rRNA processing or ribosome transport. Injection of the third oligonucleotide does interrupt pre-18S rRNA processing, but this is due to coincidental simultaneous depletion of the U22 snRNA. The U18 snRNA is the first nucleolar snRNA that is not essential for ribosome biogenesis in vertebrates. 相似文献
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Nucleolar localization elements in U8 snoRNA differ from sequences required for rRNA processing. 总被引:1,自引:0,他引:1
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U8 small nucleolar RNA (snoRNA) is essential for metazoan ribosomal RNA (rRNA) processing in nucleoli. The sequences and structural features in Xenopus U8 snoRNA that are required for its nucleolar localization were analyzed. Fluorescein-labeled U8 snoRNA was injected into Xenopus oocyte nuclei, and fluorescence microscopy of nucleolar preparations revealed that wild-type Xenopus U8 snoRNA localized to nucleoli, regardless of the presence or nature of the 5' cap on the injected U8 snoRNA. Nucleolar localization was observed when loops or stems in the 5' portion of U8 that are critical for U8 snoRNA function in rRNA processing were mutated. Therefore, sites of interaction in U8 snoRNA that potentially tether it to pre-rRNA are not essential for nucleolar localization of U8. Boxes C and D are known to be nucleolar localization elements (NoLEs) for U8 snoRNA and other snoRNAs of the Box C/D family. However, the spatial relationship of Box C to Box D was not crucial for U8 nucleolar localization, as demonstrated here by deletion of sequences in the two stems that separate them. These U8 mutants can localize to nucleoli and function in rRNA processing as well. The single-stranded Cup region in U8, adjacent to evolutionarily conserved Box C, functions as a NoLE in addition to Boxes C and D. Cup is unique to U8 snoRNA and may help bind putative protein(s) needed for nucleolar localization. Alternatively, Cup may help to retain U8 snoRNA within the nucleolus. 相似文献
14.
Processing of intron-encoded box C/D small nucleolar RNAs lacking a 5',3'-terminal stem structure 总被引:1,自引:0,他引:1
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The C and D box-containing (box C/D) small nucleolar RNAs (snoRNAs) function in the nucleolytic processing and 2'-O-methylation of precursor rRNA. In vertebrates, most box C/D snoRNAs are processed from debranched pre-mRNA introns by exonucleolytic activities. Elements directing accurate snoRNA excision are located within the snoRNA itself; they comprise the conserved C and D boxes and an adjoining 5',3'-terminal stem. Although the terminal stem has been demonstrated to be essential for snoRNA accumulation, many snoRNAs lack a terminal helix. To identify the cis-acting elements supporting the accumulation of intron-encoded box C/D snoRNAs devoid of a terminal stem, we have investigated the in vivo processing of the human U46 snoRNA and an artificial snoRNA from the human beta-globin pre-mRNA. We demonstrate that internal and/or external stem structures located within the snoRNA or in the intronic flanking sequences support the accumulation of mammalian box C/D snoRNAs lacking a canonical terminal stem. In the intronic precursor RNA, transiently formed external and/or stable internal base-pairing interactions fold the C and D boxes together and therefore facilitate the binding of snoRNP proteins. Since the external intronic stems are degraded during snoRNA processing, we propose that the C and D boxes alone can provide metabolic stability for the mature snoRNA. 相似文献
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U16 belongs to the family of box C/D small nucleolar RNAs (snoRNAs) whose members participate in ribosome biogenesis, mainly acting as guides for site-specific methylation of the pre-rRNA. Like all the other members of the family, U16 is associated with a set of protein factors forming a ribonucleoprotein particle, localized in the nucleolus. So far, only a few box C/D-specific proteins are known: in Xenopus laevis, fibrillarin and p68 have been identified by UV crosslinking and shown to require the conserved boxes C and D for snoRNA interaction. In this study, we have identified an additional protein factor (p62), common to box C/D snoRNPs, that crosslinks to the internal stem region, distinct from the conserved box C/D "core motif," of U16 snoRNA. We show here that, although the absence of the core motif and, as a consequence, of fibrillarin and p68 binding prevents processing and accumulation of the snoRNA, the lack of the internal stem does not interfere with the efficient release of U16 from its host intron and only slightly affects snoRNA stability. Because this region is likely to be the binding site for p62, we propose that this protein plays an accessory role in the formation of a mature and stable U16 snoRNP particle. 相似文献
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A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA. 总被引:18,自引:1,他引:17
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We report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components. 相似文献
18.
Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D. 总被引:19,自引:4,他引:15
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Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing. 相似文献
19.
The U14 snoRNA is required for 2'-O-methylation of the pre-18S rRNA in Xenopus oocytes. 总被引:2,自引:0,他引:2
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We have studied the role of the U14 small nucleolar RNA (snoRNA) in pre-rRNA methylation and processing in Xenopus oocytes. Depletion of U14 in Xenopus oocytes was achieved by co-injecting two nonoverlapping antisense oligonucleotides. Focusing on the earliest precursor, depletion experiments revealed that the U14 snoRNA is essential for 2'-O-ribose methylation at nt 427 of the 18S rRNA. Injection of U14-depleted oocytes with specific U14 mutant snoRNAs indicated that conserved domain B, but not domain A, of U14 is required for the methylation reaction. When the effect of U14 on pre-rRNA processing is assayed, we find only modest effects on 18S rRNA levels, and no effect on the type or accumulation of 18S precursors, suggesting a role for U14 in a step in ribosome biogenesis other than cleavage of the pre-rRNA. Xenopus U14 is, therefore, a Box C/D fibrillarin-associated snoRNA that is required for site-specific 2'-O-ribose methylation of pre-rRNA. 相似文献
20.
The sequence of the 5' end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation. 总被引:3,自引:0,他引:3
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B A Peculis 《Molecular and cellular biology》1997,17(7):3702-3713
Ribosome biogenesis in eucaryotes involves many small nucleolar ribonucleoprotein particles (snoRNP), a few of which are essential for processing pre-rRNA. Previously, U8 snoRNA was shown to play a critical role in pre-rRNA processing, being essential for accumulation of mature 28S and 5.8S rRNAs. Here, evidence which identifies a functional site of interaction on the U8 RNA is presented. RNAs with mutations, insertions, or deletions within the 5'-most 15 nucleotides of U8 do not function in pre-rRNA processing. In vivo competitions in Xenopus oocytes with 2'O-methyl oligoribonucleotides have confirmed this region as a functional site of a base-pairing interaction. Cross-species hybrid molecules of U8 RNA show that this region of the U8 snoRNP is necessary for processing of pre-rRNA but not sufficient to direct efficient cleavage of the pre-rRNA substrate; the structure or proteins comprising, or recruited by, the U8 snoRNP modulate the efficiency of cleavage. Intriguingly, these 15 nucleotides have the potential to base pair with the 5' end of 28S rRNA in a region where, in the mature ribosome, the 5' end of 28S interacts with the 3' end of 5.8S. The 28S-5.8S interaction is evolutionarily conserved and critical for pre-rRNA processing in Xenopus laevis. Taken together these data strongly suggest that the 5' end of U8 RNA has the potential to bind pre-rRNA and in so doing, may regulate or alter the pre-rRNA folding pathway. The rest of the U8 particle may then facilitate cleavage or recruitment of other factors which are essential for pre-rRNA processing. 相似文献