Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.     

Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing

Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethyl-guanosine (m3G), which is present in the 5'-terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing

The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5'-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit.     

Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease.

Autoantibodies against U3 small nuclear ribonucleoprotein are associated with scleroderma autoimmune disease. They were shown to react with fibrillarin, a 34- to 36-kilodalton protein that has been detected in all eukaryotes tested from humans to yeasts. We isolated a 1.6-kilobase cDNA encoding fibrillarin from a Xenopus laevis cDNA library. The protein contains a 79-residue-long Gly-Arg-rich domain in its N-terminal region and a putative RNA-binding domain with ribonucleoprotein consensus sequence in its central portion. This is the first report of cloning of fibrillarin, and the deduced protein sequence is in agreement with the involvement of the protein in a ribonucleoprotein particle.     

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.     

Comparative sequence analysis of a single-gene conserved segment in mouse and human

Comparative mapping and sequencing of the mouse and human genomes have defined large, conserved chromosomal segments in which gene content and order are highly conserved. These regions span megabase-sized intervals and together comprise the vast majority of both genomes. However, the evolutionary relationships among the small remaining portions of these genomes are not as well characterized. Here we describe the sequencing and annotation of a 341-kb region of mouse Chr 2 containing nine genes, including biliverdin reductase A (Blvra), and its comparison with the orthologous regions of the human and rat genomes. These analyses reveal that the known conserved synteny between mouse Chromosome (Chr) 2 and human Chr 7 reflects an interval containing one gene (Blvra/BLVRA) that is, at most, just 34 kb in the mouse genome. In the mouse, this segment is flanked proximally by genes orthologous to human chromosome 15q21 and distally by genes orthologous to human Chr 2q11. The observed differences between the human and mouse genomes likely resulted from one or more rearrangements in the rodent lineage. In addition to the resulting changes in gene order and location, these rearrangements also appear to have included genomic deletions that led to the loss of at least one gene in the rodent lineage. Finally, we also have identified a recent mouse-specific segmental duplication. These finding illustrate that small genomic regions outside the large mouse–human conserved segments can contain a single gene as well as sequences that are apparently unique to one genome. The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned the accession numbers AC074224 and AC074041.     

Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya. The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein. Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition. To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants. This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the protein.     

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing.

To probe functions of the U1 small nuclear ribonucleoprotein particle (snRNP) during in vitro splicing, we have used unusual splicing substrates which replace the 5' splice site region of an adenovirus substrate with spliced leader (SL) RNA sequences from Leptomonas collosoma or Caenorhabditis elegans. In agreement with previous results (J.P. Bruzik and J.A. Steitz, Cell 62:889-899, 1990), we find that oligonucleotide-targeted RNase H destruction of the 5' end of U1 snRNA inhibits the splicing of a standard adenovirus splicing substrate but not of the SL RNA-containing substrates. However, use of an antisense 2'-O-methyl oligoribonucleotide that disrupts the first stem of U1 snRNA as well as stably sequestering positions of U1 snRNA involved in 5' and 3' splice site recognition inhibits the splicing of both the SL constructs and the standard adenovirus substrate. The 2'-O-methyl oligoribonucleotide is no more effective than RNase H pretreatment in preventing pairing of U1 with the 5' splice site, as assessed by inhibition of psoralen cross-link formation between the SL RNA-containing substrate and U1. The 2'-O-methyl oligoribonucleotide does not alter the protein composition of the U1 monoparticle or deplete the system of essential splicing factors. Native gel analysis indicates that the 2'-O-methyl oligoribonucleotide inhibits splicing by diminishing the formation of splicing complexes. One interpretation of these results is that removal of the 5' end of U1 inhibits base pairing in a different way than sequestering the same sequence with a complementary oligoribonucleotide. Alternatively, our data may indicate that two elements near the 5' end of U1 RNA normally act during spliceosome assembly; the extreme 5' end base pairs with the 5' splice site, while the sequence or structural integrity of stem I is essential for some additional function. It follows that different introns may differ in their use of the repertoire of U1 snRNP functions.     

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA.

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.     

Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.

The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.     

Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during normal cellular metabolism, and incorporation into DNA causes transversion mutation. Organisms possess an enzyme, 8-oxo-dGTPase, which catalyzes the hydrolysis of 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing the occurrence of mutation. There are highly conserved amino acid sequences in prokaryotic and eukaryotic proteins containing this and related enzyme activities. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site- directed mutagenesis of the cloned cDNA for human 8-oxo-dGTPase, and the activity and stability of mutant forms of the enzyme were examined. When lysine-38 was replaced by other amino acids, all of the mutants isolated carried the 8-oxo-dGTPase-negative phenotype. 8-Oxo-dGTPase-positive revertants, isolated from one of the negative mutants, carried the codon for lysine. Using the same procedure, the analysis was extended to other residues within the conserved sequence. At the glutamic acid-43, arginine-51 and glutamic acid-52 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild type protein. We propose that Lys-38, Glu-43, Arg-51 and Glu-52 residues in the conserved region are essential to exert 8-oxo-dGTPase activity.