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1.
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   

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Vertebrate U14 snoRNAs are encoded within hsc70 pre-mRNA introns and U14 biosynthesis occurs via an intron-processing pathway. We have shown previously that essential processing signals are located in the termini of the mature U14 molecule and replacement of included boxes C or D with oligo C disrupts snoRNA synthesis. The experiments detailed here now define the specific nucleotide sequences and structures of the U14 termini that are essential for intronic snoRNA processing. Mutagenesis studies demonstrated that a 5', 3'-terminal stem of at least three contiguous base pairs is required. A specific helix sequence is not necessary and this stem may be extended to as many as 15 base pairs without affecting U14 processing. The spatial positioning of boxes C and D with respect to the terminal stem is also important. Detailed analysis of boxes C and D revealed that both consensus sequences possess essential nucleotides. Some, but not all, of these critical nucleotides correspond to those required for the stable accumulation of nonintronic yeast U14 snoRNA. The presence of box C and D consensus sequences flanking a terminal stem in many snoRNA species indicates the importance of this "terminal core motif" for snoRNA processing.  相似文献   

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We have cloned and sequenced the ribosomal protein S13 gene from the Chinese hamster fibroblast HA-1 cells. The predicted protein encoded by this gene is identical to the human ribosomal protein S13, except for one amino acid substitution at residue 29, which is an alanine in the hamster protein and a threonine in that of humans. The physical organization of the six exons and five introns in the hamster S13 gene is also identical to that found in the human and Xenopus genes with respect to the amino acid codes, even though there are small differences in the lengths of the introns. The striking feature is that unlike its human and Xenopus counterparts, which encode two U14 snoRNAs in two separate introns, the hamster S13 gene encodes no U14 snoRNA. Instead, the hamster gene has a pseudo-U14 coding sequence in its third intron. Our data support the idea that the single copy of the hsc70/U14 gene, which we had previously characterized, is the only source for the production of both U14 snoRNA and hsc70 mRNA species in hamster HA-1 cells.  相似文献   

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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   

8.
Lim Y  Lee SM  Kim M  Lee JY  Moon EP  Lee BJ  Kim J 《Gene》2002,286(2):291-297
Analysis of the complete genomic structure of the human ribosomal protein S3 (rpS3) gene revealed the presence of a functional U15b snoRNA gene in its intron. Human ribosomal protein S3 (rpS3) gene of 6115 bp long has been identified to contain six introns and seven exons in this study. The first and fifth introns of human S3 gene contain functional U15 snoRNA genes. Although Xenopus and Fugu counterparts also have six introns and seven exons, S3 gene of Fugu contains two functional U15 snoRNAs in the fourth and sixth introns and two pseudo genes for U15 snoRNAs in the first and fifth introns. In Xenopus S1 gene encoding ribosomal protein S3, however, three of its six introns contain U15 snoRNA gene sequence. Sequence comparison of the U15 genes from Xenopus, Fugu and human revealed that the regions involved in binding to 28S rRNA and the consensus sequence (C, D and D' boxes) for snoRNAs are highly conserved among those genes from these three species. Human U15a and U15b RNAs which are derived from the first and the fifth introns, respectively, have been identified to be functional by microinjection of human U15a and U15b snoRNAs into Xenopus oocyte. Northern blot and primer extension analyses confirm that human U15b snoRNA is expressed in vivo.  相似文献   

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Multiple snoRNA gene clusters from Arabidopsis   总被引:9,自引:0,他引:9       下载免费PDF全文
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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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Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modifications (2'-O-ribose methylation and pseudouridylation). Their genomic organization show great flexibility: some are individually or polycistronically transcribed, while others are encoded within introns of other genes. Here, we present an evolutionary analysis of the U49 gene in seven species. In all species analyzed, U49 contains the typical hallmarks of C and D box motifs, and a conserved 12-15 nt sequence complementary to rRNA that define them as homologs. In mouse, human, and Drosophila U49 is found encoded within introns of different genes, and in plants it is transcribed polycistronically from four different locations. In addition, U49 has two copies in two different introns of the RpL14 gene in Drosophila. The results indicate a substantial degree of duplication and translocation of the U49 gene in evolution. In light of its variable organization we discuss which of the two proposed mechanisms of rearrangement has acted upon the U49 snoRNA gene: chromosomal duplication or transposition through an RNA intermediate.  相似文献   

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Among thousands of non-protein-coding RNAs which have been found in humans, a significant group represents snoRNA molecules that guide other types of RNAs to specific chemical modifications, cleavages, or proper folding. Yet, hundreds of mammalian snoRNAs have unknown function and are referred to as "orphan" molecules. In 2006, for the first time, it was shown that a particular orphan snoRNA (HBII-52) plays an important role in the regulation of alternative splicing of the serotonin receptor gene in humans and other mammals. In order to facilitate the investigation of possible involvement of snoRNAs in the regulation of pre-mRNA processing, we developed a new computational web resource, snoTARGET, which searches for possible guiding sites for snoRNAs among the entire set of human and rodent exonic and intronic sequences. Application of snoTARGET for finding possible guiding sites for a number of human and rodent orphan C/D-box snoRNAs showed that another subgroup of these molecules (HBII-85) have statistically elevated guiding preferences toward exons compared to introns. Moreover, these energetically favorable putative targets of HBII-85 snoRNAs are non-randomly associated with genes producing alternatively spliced mRNA isoforms. The snoTARGET resource is freely available at: (http://hsc.utoledo.edu/depts/bioinfo/snotarget.html).  相似文献   

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A novel gene organization: intronic snoRNA gene clusters from Oryza sativa   总被引:4,自引:0,他引:4  
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