Specific binding of Alu sequences by HeLa nuclear extracts

Plasmid Blur 8 which contains the 300bp human Alu consensus sequence and plasmid pBR322 were digested with restriction enzymes and the fragments obtained end labelled with 32P-gamma-ATP. The end labelled fragments were incubated with HeLa nuclear extracts and the incubation mixtures passed through a nitrocellulose filter. The 300bp alu consensus sequence was preferentially retained on the filter. The HeLa nuclear extract did not preferentially bind any fragments generated from pBR322 and histones which bind nonspecifically all DNA fragments did not preferentially bind the alu sequence. We conclude that the HeLa nuclear extract contains components which specifically bind the human alu sequence.     

Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA.

Ribonucleoprotein particles containing heterogeneous nuclear RNA (Pederson, 1974) were isolated from HeLa cells and digested with ribonucleases A and T₁ at high ionic strength. The nuclease-resistant material, comprising 9.4% of the initial acid-insoluble [³H]adenosine radioactivity, was further fractionated by poly(U)-Sepharose chromatography. The bound fraction eluted from the column with 50% formamide and banded in cesium sulfate gradients (without aldehyde fixation) at a buoyant density characteristic of ribonucleoprotein (1.45 g/cm³). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this material revealed two Coomassie blue-stained bands. The major polypeptide had a molecular weight of 74,000 a less prominent band had a molecular weight of 86,000. The RNA components contained 74.4 mol % AMP and 17.7 mol % UMP. Polyacrylamide gel electrophoresis of the RNA, labeled with [³H]adenosine, demonstrated the presence of molecules 150 to 200 nucleotides in length (poly(A)), as well as molecules 20 to 30 nucleotides long (oligo(A)). Both poly(A) and oligo(A) sequences have previously been identified in HeLa heterogeneous nuclear RNA. These data demonstrate that both the poly (A) and oligo(A) sequences in HeLa heterogeneous nuclear RNA exist in vivo tightly complexed with specific proteins.     

7SL RNA from Schizosaccharomyces pombe is encoded by a single copy essential gene

We have identified an abundant ribonucleoprotein particle from Schizosaccharomyces pombe with properties related to those of the vertebrate signal recognition particle (SRP), including cytoplasmic localization, association with microsomes and ribosomes at low, but not high, salt concentrations and high resistance to micrococcal nuclease. The 256-nucleotide RNA component carries a 5'-triphosphate group and shows close secondary structure, and limited primary sequence homology to vertebrate 7SL RNA. 7SL-like RNAs were also detected in a number of other fungi. The single copy gene (SRP7) encoding S.pombe 7SL was disrupted by insertion of a transposon carrying the selective marker LEU2, and the disrupted gene was used to replace one chromosomal SRP7 gene in a diploid strain. Haploid srp7[unk] strains fail to germinate.     

cis-Acting determinants of 7SL RNA packaging by HIV-1

The host noncoding RNA 7SL is highly enriched in the virions of retroviruses. We examined the regions of 7SL that mediate packaging by HIV-1. Both the Alu domain and the S domain were sufficient to mediate specific packaging when expressed separately as truncations of 7SL. However, while the Alu domain competed with endogenous 7SL for packaging in proportion to Gag, the S domain was packaged additively, implying that the Alu and S domains are packaged via separate mechanisms and that the Alu domain is packaged by the same mechanism as endogenous 7SL. Further truncations of the Alu domain or mutation of the Alu domain helix 5c region significantly reduced packaging efficiency, implicating helix 5c as critical for packaging, reinforcing the finding that 7SL packaging is highly selective, and confirming that 7SL is not passively acquired. Surprisingly, when the Alu domain was mutated so that it no longer contained a binding site for the SRP protein heterodimer SRP9/14, it was no longer packaged in a competitive manner but instead was packaged additively with endogenous 7SL. These data support a model in which 7SL RNA is packaged via interactions between Gag and a 7SL RNA structure that exists transiently at a discrete stage of SRP biogenesis. Our data further indicate that a secondary "additive" pathway exists that can result in the packaging of certain 7SL derivatives in molar excess to endogenously packaged 7SL.     

Determination of nucleotide sequences from double-stranded regions of HeLa cell nuclear RNA

Double-stranded regions which comprise about 4% of isolated HeLa cell heterogeneous nuclear RNA have been characterized by RNA fingerprinting and sequencing analysis. The simplicity of the pattern in two-dimensional RNA fingerprints suggests a sequence complexity of about 1000 nucleotides. The nucleotide sequences of six prominent RNase T₁-resistant oligonucleotides (ranging in size from 7 to 9 bases) have been determined using isolated double-stranded nuclear RNA labeled in vivo with ³²P-labeled inorganic phosphate. We conclude that (here exists a substantial subpopulation of simple, potentially complementary sequences common to much of the heterogeneous nuclear RNA population and interspersed with other kinds of sequences.     

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.     

Nucleotide sequence of 7 S RNA. Homology to Alu DNA and La 4.5 S RNA

7 S RNA, a component of normal higher eukaryotic cells and several oncornaviruses, was shown to be conserved in evolution (Erikson, E., Erikson, R. L., Henry, B., and Pace, N. R. (1973) Virology 53, 40-46). Recently, 7 S RNA was shown to be partially complementary to Alu family DNA sequences (Weiner, A. (1980) Cell 22, 209-218). In the present study the nucleotide sequence of Novikoff hepatoma 7 S RNA was determined to be: (formula, see text) Comparison of 7 S RNA, Alu and B1 family DNA, and La 4.5 S RNA sequences for homologies showed that 1) one-third of 7 S RNA, mainly the 5'-end, was homologous to Alu and B1 family sequences; 2) one 300-nucleotide long Alu family sequence contained two binding sites for 7 S RNA; and 3) the 5'-ends of 7 S RNA and La 4.5 S RNA also had extensive (60%) homologies. A model for the secondary structure of 7 S RNA based on maximal base pairing and preferential nuclease cleavage sites is also presented.     

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

A human-specific subfamily of Alu sequences.

Of a total of 500,000 Alu family members, approximately 500 are present as a human-specific (HS) subfamily. Each of the HS subfamily members shares a high degree of nucleotide identity and is not present at orthologous positions in other primate genomes, suggesting that HS subfamily members have recently inserted within the human genome. This confirms the hypothesis that the majority of Alu family members are amplified copies of a "master" gene(s). This master gene appears to be amplifying at a rate much slower than that seen earlier in primate evolution. Some of the HS Alu subfamily members have amplified so recently that they are dimorphic in the human population, making them a potentially powerful tool for studies of human populations.