Androgen-induced gene activation in the rat prostate

The effect of androgens on gene activation in the rat prostate has been investigated by examining precursor incorporation into RNA, by DNA-RNA hybridization of RNA transcribed $\text{in}\text{vitro}$ from prostate chromatin, and by thermal denaturation of prostatic chromatin. The results show a selective synthesis of nuclear RNA and a changed thermal melting profile of prostatic chromatin as a result of testosterone administration. Further, the $\text{in}\text{vitro}$ synthesized RNA transcribed from prostatic chromatin of androgen-treated rats contained new RNA species that were not transcribed from chromatin of untreated castrated controls. The data provide direct evidence for an activated state of the prostatic chromatin stimulated by androgens.     

Subnuclear particles containing a small nuclear RNA and heterogeneous nuclear RNA

Five of the stable low molecular weight RNA species in the HeLa cell nucleus have been localized in RNP complexes in the cell nucleus. The two abundant species C and D and the three minor species F, G′ and H are found in RNP particles following two different methods of preparation. Sonication of nuclei releases the five small RNAs and also the hnRNA in RNPs that sediment in a range from 10 to 150 S. Alternatively, incubation of intact nuclei at elevated temperature and pH releases four of the small RNAs and degraded hnRNA in more slowly sedimenting structures.When nuclear RNPs obtained by sonication are digested with RNAase in the presence of EDTA, the hnRNA is degraded and the hnRNPs sediment at 30 S. The structures containing the small RNA species D are similarly shifted to 30 S particles by RNAase and EDTA but not by either agent alone. In contrast, the sedimentation of complexes containing species G′ and H are not altered by exposure to RNAase/EDTA and small RNA species C and F are unstable under these conditions.In isopycnic metrizamide/²H₂O gradients species D and hnRNA accumulate at a density characteristic of RNP particles. They have a similar but not identical distribution.Species D is released from large RNPs by salt concentrations of 0.1 m-NaCl or greater, while the hnRNA remains in large RNP particles. In contrast, the structures containing species G′ and H are stable in 0.3 m-NaCl. All five of the small nuclear RNA species and the hnRNAs are released from rapidly sedimenting complexes by the ionic detergent sodium deoxycholate.It is suggested that the low molecular weight RNA species play a structural role in RNP particles in the cell nucleus and that a subpopulation of species D may be associated with the particles that package the hnRNA.     

In vitro association of unique species of cellular 4S RNA with 35S RNA of RNA tumor viruses

Unique 4S RNA species from AKR mouse embryo cells hybridize with AKR murine leukemia virus and avian myeloblastosis virus 35S RNAs $\text{in vitro}$. Analyses by reversed-phase column chromatography indicate that the major 4S species that hybridize with the two viral RNAs are probably the same. A 4S RNA species with similar chromatographic properties is a major component of the AKR viral 4S RNA which associates with the viral 70S RNA $\text{in vivo}$.     

Evidence for distinct mRNAs for ferritin subunits

Poly A enriched RNA from iron loaded HeLa cells and rat liver were translated separately and together in wheat germ lysates to investigate the origins of the H and L subunits of ferritin. Most of the ferritin translated from the HeLa RNA was of the H type, while that from the liver RNA was mostly L type. Mixtures of these RNAs gave $\text{H}\text{L}$ ratios which correlated with the relative amounts of added HeLa and rat RNAs. These results indicate that the H and L subunits of ferritin are not derived by post-translational modification but from distinct mRNA species.     

Small RNA species of the HeLa cell: Metabolism and subcellular localization

The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the “nuclear skeleton.” SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.     

Crosslinking of proteins to ribosomal RNA in HeLa cell polysomes by sodium periodate.

HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in $\text{Escherichia}$,$\text{coli}$ 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs.     

Ribonucleoprotein particles containing non-coding Y RNAs, Ro60, La and nucleolin are not required for Y RNA function in DNA replication

Background

Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.

Methodology/Principal Findings

We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.

Conclusions/Significance

We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins.     

Determination of the sequence homology between the four RNA species of cucumber mosaic virus by hybridization analysis with complementary DNA

The method of Taylor etal., (11) has been used to transcribe complementary DNA probes from the four major RNA species of cucumber mosaic virus (RNAs 1 - 4 in order of decreasing molecular weight). Analysis of the kinetics of hybridization of these probes in homologous and heterologous complementary DNA-RNA hybridization reactions has shown that the sequence of the smallest RNA (RNA 4), which contains the coat protein gene, is present within RNA 3. RNAs 1 and 2 are unique RNA molecules while each has a region of approximately 300 nucleotides in common with RNA 4.     

Microbial identification by immunohybridization assay of artificial RNA labels

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.     

Accumulation of unusual sterile transcripts of TCRβ in mouse hybridoma, murine tumour and non-human primate marmoset tumour

Accumulation of immunoglobulin Ig RNA (from several loci viz., C_H, C_α, J_k-C_k and S_μ during Igμ isotype switching) in B cells and T cell receptor (TCR) RNAs (α, β andγ) in T cells of unusual sizes emanating from germline and rearranged genes were reported to accumulate in human and mouse (and murine too). The precise mechanism and function of these sterile RNA species are yet to be delineated. Similar accumulation of RNA species of unusual sizes were identified with DNA-RNA hybridization and isolation of cDNA employing with DNA and antibody probes in mouse hybridoma, murine tumour, non-human primate marmoset tumour and human leukemic cells.     

Multiple forms of human dihydrofolate reductase messenger RNA: Cloning and Expression in Escherichia coli of their DNA coding sequence

The programming capacity for the synthesis of human dihydrofolic acid reductase in a rabbit reticulocyte lysate has been found to be greatly enhanced in the polysomal poly(A)-containing RNA from a methotrexate-resistant human cell variant (6A3), as compared to the RNA from its parental line (VA₂-B). A major fraction of this promoting activity is associated with a 3.8 × 10³ base RNA species detectable as a band in the ethidium bromide-stained electrophoretic pattern of the RNA from 6A3 cells, but not in the RNA from VA₂-B cells. Furthermore, sucrose gradient fractionation experiments have indicated that another substantial portion of the messenger activity is associated with RNA components around 10³ bases in size. Double-stranded complementary DNA synthesized from total poly(A)-containing RNA of 6A3 cells has been size fractionated, and both large (1400 to 3800 base-pairs) and small size complementary DNA (600 to 1400 base-pairs) species have been used separately to transform Escherichia coli χ2282 with pBR322 as a vector. Of 76 transformants obtained with the large size complementary DNA, identified by a differential colony hybridization assay, none has expressed the dihydrofolic acid reductase coding sequence in E. coli, as judged by resistance to trimethoprim. By contrast, eight trimethoprim-resistant transformants have been obtained using the small size complementary DNA, and their plasmids have been shown to contain the dihydrofolic acid reductase coding sequence by restriction mapping and DNA sequencing; moreover, immunoautoradiographic experiments have revealed the presence in the extracts of two of these transformants of a protein with the electrophoretic mobility and immunoreactivity of human dihydrofolic acid reductase. Restriction mapping and DNA transfer hybridization experiments have further indicated that the inserts of the chimaeric plasmids conferring trimethoprim resistance upon the host and of those lacking this capacity cover together a complementary DNA region of about 3.35 × 10³ base-pairs, in which the 564 base-pair dihydrofolic acid reductase coding stretch is located near the 5′ end of the sense strand. RNA transfer hybridization experiments using different cloned complementary DNA fragments as probes have shown the presence of three species of dihydrofolic acid reductase-specific messenger RNAs, with sizes of 3.8 × 10³, 1.0 × 10³ and 0.8 × 10³ bases, differing in the length of the 3′ untranslated region, in the poly(A)-containing RNA from two methotrexate-resistant variants, 6A3 and 10B3, and, in greatly reduced amounts, in the RNA from their respective parents, VA₂B and HeLa BU25.