Nob1p is required for cleavage of the 3' end of 18S rRNA

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.     

The ATPase and helicase activities of Prp43p are stimulated by the G‐patch protein Pfa1p during yeast ribosome biogenesis

Prp43p is a RNA helicase required for pre‐mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G‐patch protein Pfa1p, a component of pre‐40S pre‐ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G‐patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre‐40S pre‐ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre‐rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre‐rRNA processing defect of Δpfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre‐rRNA‐to‐18S rRNA conversion.     

Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly

The Saccharomyces cerevisiae splicing factors Ntr1 (also known as Spp382) and Ntr2 form a stable complex and can further associate with DExD/H-box RNA helicase Prp43 to form a functional complex, termed the NTR complex, which catalyzes spliceosome disassembly. We show that Prp43 interacts with Ntr1-Ntr2 in a dynamic manner. The Ntr1-Ntr2 complex can also bind to the spliceosome first, before recruiting Prp43 to catalyze disassembly. Binding of Ntr1-Ntr2 or Prp43 does not require ATP, but disassembly of the spliceosome requires hydrolysis of ATP. The NTR complex also dynamically interacts with U5 snRNP. Ntr2 interacts with U5 component Brr2 and is essential for both interactions of NTR with U5 and with the spliceosome. Ntr2 alone can also bind to U5 and to the spliceosome, suggesting a role of Ntr2 in mediating the binding of NTR to the spliceosome through its interaction with U5. Our results demonstrate that dynamic interactions of NTR with U5, through the interaction of Ntr2 with Brr2, and interactions of Ntr1 and Prp43 govern the recruitment of Prp43 to the spliceosome to mediate spliceosome disassembly.     

Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein

Numerous non-ribosomal trans-acting factors involved in pre-ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation. Here we demonstrate that Rio1p/Rrp10p is such a factor. Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man. We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation. We show that RIO1 encodes a cytoplasmic non-ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre-rRNA accumulation. In situ hybridization reveals that, in Rio1p depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre-rRNA at site D, producing mature 18S rRNA. Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10). Rio1p/Rrp10p is the first non-ribosomal factor characterized specifically required for 20S pre-rRNA processing.     

Ribosomal Protein S14 of Saccharomyces cerevisiae Regulates Its Expression by Binding to RPS14B Pre-mRNA and to 18S rRNA

Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-Em^RR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure.     

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA.

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

Cloning and sequence analysis of the 18 S ribosomal RNA gene of tomato and a secondary structure model for the 18 S rRNA of angiosperms

Summary The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (ycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 by long with a G+C content of 49.6%. Its sequence exhibits 94%–96% positional identity when it is colinearly aligned with the previously reported sequences of the 17–18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.     

Cross-linking of tobacco mosaic virus RNA and capped polyribonucleotides to 18S rRNA in wheat germ ribosome-mRNA complexes

Tobacco mosaic virus RNA, forming 40S or 80S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly(A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18S ribosomal RNA in 40S or 80S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40S complexes but not 80S complexes with ribosomes, was most efficiently cross-linked to 18S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40S complexes would be then converted to 80S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18S ribosomal RNA.     

Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

The loop of a stem structure close to the 5' end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A(1), the 5' end of the 18S rRNA, and at site A(2), located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3-pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5' external transcribed spacer (5' ETS). Cleavage at site A(0) in the yeast 5' ETS strictly requires base pairing between U3 and a sequence within the 5' ETS. In contrast, the U3-18S interaction is not required for A(0) cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A(1) cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A(1) cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.     

The 18S and 5S ribosomal RNA genes in Oenothera mitochondria: Sequence rearrangments in the 18S and 5S rRNA genes of higher plants

Summary The 18S and 5S ribosomal RNA genes are separated by a 582-nucleotide-long spacer region in the Oenothera mitochondrial genome. The 5S rRNA gene is 7 bp shorter than the maize and 3 bp shorter than the wheat sequences due to a 4 bp deletion in a side arm of the secondary structure model. The 18S rRNA molecule can be folded analogously to the maize and wheat mitochondrial and Escherichia coli models for this rRNA. Most of the sequence variations between the wheat and Oenothera molecules are located in the variable domains identified for the wheat 18S rRNA.The comparison of the 18S rRNA from the mitochondria of Oenothera as a representative of dicotyledonous plants with that of the monocotyledons wheat and maize provides an indication of the rate of diversity in higher plant mitochondrial genes and gives direct evidence for sequence rearrangements within the 18 S rRNA genes.     

Human 1A6/DRIM, the homolog of yeast Utp20, functions in the 18S rRNA processing

1A6/DRIM is a nucleolar protein with a nucleolar targeting sequence in its 3'-terminus. Bioinformatic analysis indicated that human 1A6/DRIM shares 23% identity and 43% similarity with yeast Utp20, which has been reported as a component of U3 snoRNA protein complex and has been implicated in 18S rRNA processing. In the present study, we found, by utilizing RT-PCR with RNA extracted from anti-1A6/DRIM immunoprecipitates and Northern blotting, that 1A6/DRIM is associated with U3 snoRNA. Pulse-chase labeling assays showed that silencing of 1A6/DRIM expression in HeLa cells resulted in a delayed 18S rRNA processing. Furthermore, immunoprecipitations revealed that 1A6/DRIM was also associated with fibrillarin, another U3 RNP component in HeLa cells. These results indicate that 1A6/DRIM is involved in 18S rRNA processing and is the bona fide mammalian Utp20.     

Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis.

The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation.     

Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Interaction with dyes specific for the secondary structure of native RNA and RNA subjected to hydrolysis by nuclease S1

The interaction of ethidium bromide (EtBr) with double-stranded (ds), and acridine orange (AO) with single-stranded (ss) fragments of 16S rRNA Escherichia coli in a wide range of ionic strength, at various pH, Zn2+ ion concentrations and partial hydrolysis by nuclease S1 was investigated. It was shown that about 90% of the RNA molecule is accessible to both dyes, when the ionic strength is near of 0.01 (pH 7). Approximately half of the RNA becomes inaccessible to dyes, when the ionic strength was increased up to 0.08-0.24 (pH 4.7-7), independent on the presence of Zn2+ ions (10(-3) M). About a half of the ds-, and a quarter of the ss-segments of the RNA, deduced from the secondary structure model were protected from the interaction with EtBr and AO. The hydrolysis of about a half of ss-segments upon addition of the Zn2+ (10(-3) M) ions did not affect the RNA tertiary structure. The experimental data obtained confirm the idea of the existence of some "nucleus" (or "nuclei") within the 16S rRNA molecule. The "nucleus" seems to be inaccessible to the dyes and is very stable to heat denaturation. It was supposed that this structure is organized by means of interaction of some of the parallelly oriented ds-segments, as it was suggested earlier for the phage MS2 RNA structure.     

The longest 18S ribosomal RNA ever known. Nucleotide sequence and presumed secondary structure of the 18S rRNA of the pea aphid, Acyrthosiphon pisum.

An EMBL4 recombinant phage which encodes one of the full length of the aphid ribosomal DNA has been isolated from the aphid genomic library. Determination of the complete nucleotide sequence of the aphid 18S rRNA gene revealed that it is 2469 bp with a G + C content of 59%. The aphid 18S rRNA gene studied here is the longest and has the highest G + C content among the 18S rRNA genes examined so far. Evidence provided by the S1 nuclease assay suggests that the aphid 18S rRNA gene examined in this study is not a pseudogene containing an insertion sequence. Based on the nucleotide sequence of the 18S rRNA gene, we constructed a presumed secondary-structure model of the aphid 18S rRNA. In the aphid 18S rRNA, the eucaryote-specific E21 and 41 region are supposed to be longer and more complex than the counterparts of other 18S rRNA.     

Rcl1p, the yeast protein similar to the RNA 3'-phosphate cyclase, associates with U3 snoRNP and is required for 18S rRNA biogenesis

RNA 3'-terminal phosphate cyclases are evolutionarily conserved enzymes catalysing conversion of the 3'-terminal phosphate in RNA to the 2',3'-cyclic phosphodiester. Their biological role remains unknown. The yeast Saccharomyces cerevisiae contains a gene encoding a protein with strong sequence similarity to the characterized cyclases from humans and Escherichia coli. The gene, named RCL1 (for RNA terminal phosphate cyclase like), is essential for growth, and its product, Rcl1p, is localized in the nucleolus. Depletion or inactivation of Rcl1p impairs pre-rRNA processing at sites A(0), A(1) and A(2), and leads to a strong decrease in 18S rRNA and 40S ribosomal subunit levels. Immunoprecipitations indicate that Rcl1p is specifically associated with the U3 snoRNP, although, based on gradient analyses, it is not its structural component. Most of Rcl1p sediments in association with the 70-80S pre-ribosomal particle and a 10S complex of unknown identity. Proteins similar to Rcl1p are encoded in genomes of all eukaryotes investigated and the mouse orthologue complements yeast strains depleted of Rcl1p. Possible functions of Rcl1p in pre-rRNA processing and its relationship to the RNA 3'-phosphate cyclase are discussed.     

Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.