Sequence homologies in mammalian 5.8S ribosomal RNA.

Oligonucleotide products of complete pancreatic or T1 RNase digestion or partial T1 RNase digestion of HeLa cell (human) and MPC-11 cell (mouse) 5.8S rRNA are identical with those obtained from Novikoff hepatoma (rat) 5.8S rRNA except for minor differences at the termini. pCp is the only major 5' terminus of both human and mouse RNAs; both pGp and pCp 5' termini were found in rat 5.8S RNA. Furthermore, HeLa cells contain C-U-U at the 3' end rather than the C-U terminus of mouse and rat. The results indicate that the nucleotide sequence has been highly conserved during the evolution of mammals and suggest that, as reported for 5S rRNA, this sequence is essentially constant throughout the Mammalia.     

Subcellular localization of low-molecular RNA in the cells of chick embryos

The subcellular distribution of chick embryo low molecular weight RNAs has been studied by the thermal phenol fractionation procedure. The major part of 8SII RNA, earlier discovered in some oncornaviruses and normal cells, was extracted by phenol at 4%. Our results thus give evidence of the cytoplasmic localization of this RNA. Another part of 8SII RNA, which has been extracted at 65 degrees, is thought to consist of newly synthesized molecules located in the nuclei. A similarity in subcellular distribution of low molecular weight RNAs from chick embryo and rat liver has been obtained, i. e. such a distribution may be of universal nature.     

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.     

Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome

Anti-Ro autoantibodies precipitate several small cytoplasmic ribonucleoproteins from mammalian cells. The RNA components of these particles, designated hY1-hY5 in human cells and mY1 and mY2 in mouse cells, are about 100 nucleotides long. We have analyzed a genomic clone that appears to contain true RNA-coding regions for two of the human Ro RNAs, hY1 and hY3. These RNAs exhibit many sequence and secondary structure homologies, both with each other and with the recently sequenced hY5 RNA. The hY2 RNA is a slightly truncated form of hY1; several shorter versions of hY3 are also detected in cell extracts and immunoprecipitates. The human hY1 and hY3 genes cross-hybridize with the mouse Ro RNAs, mY1 and mY2, respectively; we show that the mouse Ro RNAs are exclusively contained in Ro particles. The genes for hY1 and hY3 are transcribed in vitro by RNA polymerase III. In contrast with all other mammalian class III genes described, they appear to be present as single copies in the human genome.     

Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

Gene expression in chemically transformed mouse embryo cells: selective enhancement of the expression of C type RNA tumor virus genes.

Treatment of a nontumorigenic clone of AKR mouse embryo cells in culture with a variety of polycyclic aromatic hydrocarbons has resulted in the development of derivative clones which are highly tumorigenic and exhibit other characteristics of the transformed phenotype. A 3-methylcholanthrene-transformed derivative clone (clone MCA) has been compared to the parent clone (clone 2B) with respect to the abundance and diversity of polysomal poly(A)-containing mRNA sequences. Hybridization kinetic experiments show that the poly(A)-containing sequences of both clones are organized into indistinguishable abundance classes, and that the vast majority of the sequences are common to both the parent and derivative clones. The levels of two specific messenger RNAs (α- and β-globin mRNA) which characterize highly differentiated mouse erythroid cells were much less than 1 molecule per cell in either cell type. Titration of a balanced complementary DNA probe to AKR murine leukemia virus (AKR-MuLV) 70S RNA with purified polysomal poly(A)-containing RNA from both parent and derivative clones shows that approximately 5000 and 1200 viral 35S RNA equivalents are present in the cytoplasm of growing and resting clone MCA cells, respectively. Rapidly growing clone 2B cells contain less than about 30 viral 35S RNA equivalents per cell. Viral specific sequences therefore correspond to members of the high abundance class of poly(A)-containing RNA sequences in clone MCA cells and to the low abundance class of sequences in clone 2B cells. Within the limits of detection, this large increase in abundance is characteristic only of viral specific RNA sequences.     

Cytoplasmic RNA extraction from fresh and frozen mammalian tissues

The quality of collections of expressed sequence tags andfull-length cDNAs is adversely affected by the presence of "junk" clones derivedfrom unspliced or partially spliced RNAs present in conventional total RNA preparations. One can overcome this problem by using intact cytoplasmic RNA to create cDNA libraries, but the methods in the literature that describe the preparation of RNA only work well for extracting cultured cells. Cell lines are not as diverse as one would like, and to clone comprehensive sets of human and model organism full-length cDNAs, libraries have to be prepared from tissue samples. Thus, we have developed a robust and inexpensive method that allows intact cytoplasmic RNA to be extracted from both fresh and frozen mammalian tissues. A mouse full-length, cap-trapped cDNA library prepared with RNA using this new procedure had excellent characteristics.     

Mouse ubiquitous B2 repeat in polysomal and cytoplasmic poly(A)+RNAs: uniderectional orientation and 3''-end localization.

A cDNA library in pBR322 was prepared with cytoplasmic poly(A)+RNA from mouse liver cells. From 1 to 1.5% of clones hybridized to either B1 or B2 ubiquitous repetitive sequences. Several clones hybridizing to a B2 repeat were partially sequenced. The full-length B2 sequence was found at the 3'-end of abundant 20S poly(A)+RNA (designated as B2+mRNAx) within the non-coding part of it. B2+mRNAx is concentrated in mouse liver polysomes and absent from cytoplasm of Ehrlich carcinoma cells. The B2 sequence seems to be located at the 3'-end of some other mRNAs as well. To determine the orientation of the B2 sequence in different RNAs, its two strands were labeled, electrophoretically separated, and used for hybridization with Northern blotts containing nuclear, cytoplasmic and polysomal RNAs. In nuclear RNA, the B2 sequence is present in both orientations; in polysomal and cytoplasmic poly(A)+RNAs, only one ("canonical") strand of it can be detected. Low molecular weight poly(A)+B2+RNA [1] also contains the same strand of the B2 element. The conclusion has been drawn that only one its strand can survive the processing. This strand contains promoter-like sequences and AATAAA blocks. The latter can be used in some cases by the cell as mRNA polyadenylation signals.     

Isolation and characterization of two linked mouse U1b small nuclear RNA genes.

A 6.9 kilobase Eco R1 fragment containing genes for two U1 RNAs has been isolated from a library of mouse DNA. The two genes code for an RNA which is very similar, if not identical, to mouse U1b RNA as judged by S1 nuclease mapping. This RNA is one base longer than the mouse U1a RNA, human U1 RNA, and rat U1 RNA and differs in six nucleotide substitutions from rat U1 RNA. The two genes are five kilobases apart and the U1 RNAs are coded for on opposite strands of the DNA with the 5' ends juxtaposed. The sequences flanking the genes are identical for 700 bases 5' to the gene and at least 80 bases 3' to the gene.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

4.5S RNA is encoded by hundreds of tandemly linked genes, has a short half-life, and is hydrogen bonded in vivo to poly(A)-terminated RNAs in the cytoplasm of cultured mouse cells.

4.5S RNA is a group of RNAs 90 to 94 nucleotides long (length polymorphism due to a varying number of UMP residues at the 3' end) that form hydrogen bonds with poly(A)-terminated RNAs isolated from mouse, hamster, or rat cells (W. R. Jelinek and L. Leinwand, Cell 15:205-214, 1978; F. Harada, N. Kato, and H.-O. Hoshino, Nucleic Acids Res. 7:909-917, 1979). We have cloned a gene that encodes the 4.5S RNA. It is repeated 850 (sigma = 54) times per haploid mouse genome and 690 (sigma = 59) times per haploid rat genome. Most, if not all, of the repeats in both species are arrayed in tandem. The repeat unit is 4,245 base pairs long in mouse DNA (the complete base sequence of one repeat unit is presented) and approximately 5,300 base pairs in rat DNA. This accounts for approximately 3 X 10(6) base pairs of genomic DNA in each species, or 0.1% of the genome. Cultured murine erythroleukemia cells contain 13,000 molecules per cell of the 4.5S RNA, which can be labeled to equilibrium in 90 min by [3H]uridine added to the culture medium. The 4.5S RNA, therefore, has a short half-life. The 4.5S RNA can be cross-linked in vivo by 4'-aminomethyl-4,5',8-trimethylpsoralen to murine erythroleukemia cell poly(A)-terminated cytoplasmic RNA contained in ribonucleoprotein particles.     

Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF.

Previous experiments with purified mouse and bovine nerve growth factor (NGF) have shown that the biological activities of these two NGFs are identical, whereas the immunological cross-reactivity of antibodies produced against the two NGF molecules is very limited. This observation, together with the fact that antibodies to mouse NGF do not affect the development of sympathetic and sensory neurons in chick embryos, suggests that the domain of the NGF molecules responsible for the biological action has been highly conserved during evolution, whereas other domains determining the immunological properties were under less rigorous evolutionary constraint. The nucleotide sequences of bovine and chick NGF were determined from a cDNA clone prepared from mRNA of bovine seminal vesicles and from cloned chick genomic DNA, and the amino acid sequences deduced therefrom were compared with the available sequences of mouse and human NGF. All six cysteine residues were conserved in agreement with the previous finding that the biological activity of NGF is conformation-dependent requiring intact disulfide bridges. Amino acid changes are mainly confined to hydrophilic regions expected to be potential antigenic determinants, thus providing an explanation for the poor immunological cross-reactivities between the different NGFs. One single hydrophilic region is conserved in all NGFs and this region could be involved in the biological activity. The carboxy termini of bovine and chick NGF differ from that of mouse NGF, the changes in the amino acid sequences suggest that chick and bovine NGF are probably not processed by the gamma-subunit and that no 7S complex can be formed as in the mouse submandibular gland.     

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.