Mapping of the major early endonuclease cleavage site of the rat precursor to rRNA within the internal transcribed spacer sequence of rDNA

The endonuclease cleavage of 41 S pre-rRNA to yield 32 S and 21 S pre-rRNA constitutes a major early step in the processing of pre-rRNA in rat liver. The 5'-terminus of 32 S pre-rRNA and the 3'-terminus of 21 S pre-rRNA were precisely located within the rDNA sequence by S1 nuclease protection mapping and use of appropriate rDNA restriction fragments. The 5'-terminus of 12 S pre-rRNA, an initial product of 32 S pre-rRNA processing, was also mapped within the rDNA sequence. The 5'-termini of 32 S and 12 S pre-rRNA coincide and map within a 14-residue T-tract (non-coding strand) at 161-163 bp upstream from the 5'-end of the 5.8 S rRNA gene. The 3'-terminus of 21 S pre-rRNA maps within the same T-tract. These results show that the endonuclease cleavage occurs within a U-tract in the internal transcribed spacer 1 sequence of 41 S pre-rRNA. The homogeneity of the 5'- or 3'-termini of 32 S, 12 S and 21 S pre-rRNA indicates also that the terminal processing of these molecules, if any, is markedly slower. The coincidence in the location of 32 S and 12 S pre-rRNA 5'-termini shows further that the endonuclease cleavage of 32 S pre-rRNA precedes the removal of its 5'-terminal segment to yield 5.8 S rRNA. The absence in the whole pre-rRNA internal transcribed spacer of sequences complementary to the target U-tract suggests that the endonuclease cleavage, generating 32 S and 21 S pre-rRNA, occurs in a single-stranded loop of U-residues.     

Localization and structure of endonuclease cleavage sites involved in the processing of the rat 32S precursor to ribosomal RNA.

The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5'- and 3'-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5'-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5'- and 3'-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5'-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3'-terminus of 5.8 S rRNA and the 5'-terminus of 28 S rRNA and (ii) the 5'-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.     

The effect of serum on the calcium content of BALB/c 3T3 mouse cells

The 5'-termini of purified rat liver nucleolar and cytoplasmic 28S ribosomal RNA (rRNA) are precisely located within the homologous rDNA sequence by S1 nuclease protection mapping using an appropriate rDNA restriction fragment. The 5'-termini of nucleolar 28S rRNA are heterogeneous in length. The bulk of the nucleolar 28S rRNA map within two CTC motifs in rDNA located in the internal transcribed spacer 2 at the 50-60 and 5-15 bp upstream from the site of the homogeneous 5'-terminus of the cytoplasmic 28S rRNA. These results provide direct proof that nucleolar 28S rRNA molecules contain excess sequences at their 5'-termini and require further processing to generate the mature cytoplasmic 28S rRNA.     

3'-terminal processing of ribosomal RNA precursors in mammalian cells.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.     

Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells

5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.     

Nucleotide sequences and mapping of novel heterogenous 5''-termini of adenovirus 2 early region 4 mRNA.

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.     

Maturation pathway for Novikoff ascites hepatoma 5.8 S ribosomal ribonucleic acid. Evidence for its presence in 32 S nuclear ribonucleic acid.

Evidence that 32 S nRNA contains 5.8 S rRNA was provided by studies on specific oligonucleotide sequences of these RNA species. Purified 32P-labeled 5.8 and 28 S rRNA and 32 S RNA were digested with T-1 ribonuclease, and the products were fractionated according to chain length by chromatography on DEAE-Sephadex A-25 at neutral pH. The oligonucleotides in Peak 8 were treated with alkaline phosphatase and the products were separated by two-dimensional electrophoresis on cellulose acetate at pH 3.5 and DEAE-paper in 7% formic acid. Seven unique oligonucleotide markers for 5.8 S rRNA including the methylated octanucleotide A-A-U-U-Gm-G-A-Gp were present in 32 S RNA but were not found in 28 S rRNA, indicating that 5.8 S rRNA is directly derived from the 32 S nucleolar precursor. These studies confirm a maturation pathway for rRNA species in which 32 S nucleolar RNA is a precursor of 5.8 S rRNA as well as 28 S rRNA.     

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

The late stages of 30S and 50S ribosomal subunits biogenesis have been studied in a wild-type (wt) strain of Escherichia coli (MC4100) subjected to a severe heat stress (45-46°C). The 32S and 45S ribosomal particles (precursors to 50S subunits) and 21S ribosomal particles (precursors to 30S subunits) accumulate under these conditions. They are authentic precursors, not degraded or dead-end particles. The 21S particles are shown, by way of a modified 3'5' RACE procedure, to contain 16S rRNA unprocessed, or processed at its 5' end, and not at the 3' end. This implies that maturation of 16S rRNA is ordered and starts at its 5'-terminus, and that the 3'-terminus is trimmed at a later step. This observation is not limited to heat stress conditions, but it also can be verified in bacteria growing at a normal temperature (30°C), supporting the idea that this is the general pathway. Assembly defects at very high temperature are partially compensated by plasmid-driven overexpression of the DnaK/DnaJ chaperones. The ribosome assembly pattern in wt bacteria under a severe heat stress is therefore reminiscent of that observed at lower temperatures in E. coli mutants lacking the chaperones DnaK or DnaJ.     

Functional analogues of bleomycin: DNA cleavage by bleomycin and hemin-intercalators

New hemin-intercalators (Hem-G's) that cleave DNA were synthesized, on the basis of 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole (Glu-P-1) as an intercalator moiety. Hem-G's, which possess an intramolecular ligand of the ferrous ion (a histidine or imidazole moiety), cleave DNA very efficiently and act at guanine-pyrimidine sequences preferentially. Bleomycin (BLM) also cleaved DNA with the same base-sequence selectivity shown by Hem-G's. The 5'-terminus of the DNA fragments cleaved by Hem-G's or by BLM is a phosphoryl group, while the 3'-terminus of the cleaved DNA fragments does not possess a 3'-phosphoryl group. There are more than three kinds of 5'-end 32P-labeled DNA fragments, which can be substrates of terminal deoxynucleotidyl transferase (TdT). One of the 3'-termini of the cleaved DNA fragments is a 3'-hydroxy group. The mobility of the 3'-end 32P-labeled DNA fragment cleaved by Hem-G's or by BLM corresponds to the removal of pyrimidine bases having guanine at the 5'-side. The mobility of one kind of the cleaved 5'-end 32P-labeled DNA fragments corresponds to the removal of guanine having pyrimidine at the 3'-side, followed by 3'-dephosphorylation. We propose that there exist plural mechanisms for DNA cleavage by Hem-G's or by BLM. The deduced structures of the cleaved DNA fragments suggest that one of the mechanisms involves deletion of two nucleotide units from DNA.     

Transcription and in vitro processing of yeast 5 S rRNA

A method is described for the isolation of a yeast chromatin fraction highly enriched in ribosomal DNA sequences. In the presence of exogenous yeast RNA polymerase III, this purified chromatin actively synthesizes a set of 5 S ribosomal RNAs all of which have 5'-sequences identical with mature 5 S RNA but which end with a variable number (up to 10) of additional residues at the 3'-terminus. These extra nucleotides are precisely removed by a processing nuclease found in the chromatin supernatant fraction.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.