Functional Mapping of the U3 Small Nucleolar RNA from the Yeast Saccharomyces cerevisiae

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C′ and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5′ portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.     

Nucleolar targeting of 5S RNA in Xenopus laevis oocytes: Somatic-type nucleotide substitutions enhance nucleolar localization

In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, ³²P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, ³²P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of ³⁵S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule. J. Cell. Biochem. 69:490–505, 1998. © 1998 Wiley-Liss, Inc.     

Reiterated transfer RNA genes of Xenopus laevis

The proportion of the Xenopus laevis genome complementary to “7 S” RNA, unfractionated transfer RNA and some selected aminoacyl-tRNAs, and the sequence complexity of these RNA species, have been determined by following the kinetics of RNA-DNA hybridization on filters under conditions of RNA excess at optimum rate temperature. For hybridization of aminoacyl-labelled tRNAs, conditions for optimum aminoacylation were first determined and, where necessary, aminoacyl-tRNAs were treated with nitrous acid to prevent discharge during annealing. Neither the extent nor rate of hybridization was affected by this treatment.“7 S” RNA, coded for by 580 genes per haploid complement of chromosomes, reacts like a single family of nucleotide sequences, whereas about 43 basic tRNA sequences are coded for by at least 7800 genes. If hybrids are not treated with RNase A, the apparent tDNA redundancy is some 23% greater but no more nucleotide sequences are detectable. Taken together, the results suggest that each tRNA sequence is, on average, 200-fold reiterated.The reiteration varies, however, for different aminoacyl-tRNAs. Thus, hybridization resolves only one valyl-tRNA which is coded for by 240 genes, but at least four leucyl-tRNA sequences can be distinguished by hybridization, each of which is on average 90-fold reiterated. Reiteration also varies for the two methionyl-tRNAs detectable both by hybridization and by reversed phase chromatography: tRNA₁^Met and tRNA₂^Met are 310- and 170-fold reiterated, respectively, and each is kinetically homogeneous. These saturation values are almost exactly additive and are not influenced by the presence of other tRNA species. Thus the results suggest that Xenopus tRNAs are no more heterogeneous than would be predicted by the genetic code, despite the high but variable multiplicity of tRNA cistrons.     

Polyadenylic acid-containing RNA in Xenopus laevis oocytes

The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.     

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis.

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.     

植物核仁小分子RNA及其研究进展

自从６０年代末Ｗｅｉｎｂｅｒｇ等在哺乳动物中发现了第一个核仁小分子ＲＮＡ（ｓｍａｌｎｕｃｌｅｏｌａｒＲＮＡ,ｓｎｏＲＮＡ）Ｕ３以来,这一领域的研究特别是９０年代以来的进展引起了人们的广泛关注。这些富集于核仁区的代谢稳定的ｓｎｏＲＮＡ暗示了它们在核糖体...     

Small nuclear U-ribonucleoproteins in Xenopus laevis development. Uncoupled accumulation of the protein and RNA components

The accumulation of protein and RNA components of small nuclear U-ribonucleoprotein particles is non-co-ordinate during oogenesis and early embryogenesis in Xenopus laevis. Northern blot hybridization of a cloned Xenopus U2-RNA gene to oocyte and embryo RNAs demonstrates that the amount of small nuclear U2-RNA per oocyte reaches a plateau early in oogenesis (at the start of yolk deposition); further accumulation is not observed in oogenesis, nor in embryogenesis until the late blastula stage. In contrast, we show by immunoblot analysis that the proteins that bind to small nuclear U-RNAs continue to be accumulated after vitellogenesis begins, reaching maximum amounts only at the end of oocyte development. No further accumulation of these proteins is seen during embryogenesis. The consequences of this non-co-ordinate synthesis of small nuclear RNA and small nuclear RNA-binding proteins are as follows: a 10- to 20-fold excess of the protein components of the small ribonucleoprotein particles over small nuclear RNA exists in large oocytes; the bulk of the protein is cytoplasmic, while the RNA is nuclear. Thus the excess protein in the cytoplasm is uncomplexed with RNA. The imbalance between protein and RNA is not corrected until the late blastula or early gastrula stages of embryogenesis, when a tenfold increase in the amount of small nuclear U2-RNA is detected. Thus the protein, but not the RNA, components of small nuclear U-ribonucleoprotein particles are stockpiled in oocytes for later use in embryonic development. During the course of these studies, we also found that there are tissue-specific differences in the Sm-antigenic proteins of X. laevis.     

Very long-lived messenger RNA in ovaries of Xenopus laevis

It is possible to label with radioactivity newly synthesized ovarian RNA after intraperitoneal injection of [³H]guanosine and [³H]uridine into immature Xenopus laevis, if ovaries in which only previtellogenic stage 1 oocytes are present. Following the amount of radioactivity in the ovarian pool of acid-soluble precursors indicates a complete clearance of acid-soluble radioactivity within 15–20 days after injection. Incorporation of radioactivity into total RNA (which is almost exclusively 4 and 5S RNAs at this stage) and poly(A)⁺ RNA ceases between 15 and 20 days after injection, but the total amount of radioactivity in these RNA fractions does not decline appreciably over the next 18 months. During this time, the ovary grows and develops since stage 6 oocytes eventually appear and there is a 10- to 20-fold increase in total RNA content, which changes in composition from almost exclusively (95%) 4 and 5S RNAs to mainly (75%) 18 and 28S RNAs. Thus, despite continued growth and development, radioactive RNA molecules synthesized during previtellogenesis survive for lengths of time commensurate with the length of oogenesis (1–2 years). Although very limited (<7%) reincorporation of radioactivity into RNA is detected, it cannot alone account for the stability of the label in poly(A)⁺ RNA. These results are interpreted as indicative of synthesis during previtellogenesis of tRNA, 5SrRNA, and messenger RNA molecules which are very long-lived.