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.     

The status of small nuclear RNA in the ribonucleoprotein fibrils containing heterogeneous nuclear RNA

hnRNP are made of two classes of unit, monoparticles and heterogeneous complexes. The monoparticles are much more easily dissociated by salt than the heterogeneous complexes. We made use of this differential salt sensitivity to determine the localization of snRNA in hnRNP.

• 1.1, About 50% of the snRNA were released by NaCl under the conditions of dissociation of monoparticles. U₁ RNA which was enriched in monoparticles was preferentially released.
• 2.2, When the proteins resistant to salt dissociation were digested with proteinase K, an additional small proportion of snRNA was released, in particular a species designated as 5 S_a RNA. Therefore, 5 S_a RNA seems to be preferentially associated with the proteins of heterogeneous complexes.
• 3.3, 40% of the snRNA remained associated with the hnRNA in the absence of any detectable protein. U₁ and U₂ RNA were the major RNAs in this fraction. The same RNA pattern was obtained for phenol-extracted RNA.

The results indicate that all snRNA species are associated with the proteins of monoparticles, with those of heterogeneous complexes and with hnRNA. The existence of these pools of snRNA may reflect different functional states.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence of Dictyostelium small nuclear RNA Dd8 not homologous to any other sequenced small nuclear RNA

The cellular slime mold Dictyostelium discoideum, a lower eukaryote, was shown to contain several species of small nuclear RNA (Takeishi, K., and Kaneda, S. (1981) J. Biochem. (Tokyo) 90, 299-308; Wise, J. A., and Weiner, A. M. (1981) J. Biol. Chem. 256, 956-963). One of these RNAs, Dd9 or D2, was sequenced and found to be homologous to mammalian nucleolar U3 RNA. In the present study, the nucleotide sequence of another Dictyostelium small nuclear RNA Dd8 was determined by direct analysis. The sequence is: (formula; see text) Dd8 RNA contains high proportions of A (34%) and U (30%). No modified nucleotide could be detected in the internal region. Computer-assisted analysis of sequence homology indicated that Dd8 RNA is not homologous to any other small nuclear RNA species sequenced so far.     

Precursors of U4 small nuclear RNA

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.     

A small RNA of Mycoplasma capricolum that resembles eukaryotic U6 small nuclear RNA.

Mycoplasma capricolum, a parasitic prokaryote, contains several small stable RNAs, besides rRNAs and tRNAs. One of them, designated MCS4 RNA (125 nucleotides in length), has been isolated and sequenced. This RNA is abundant in the cell, and is encoded by two genes. Unexpectedly, MCS4 RNA has been found to reveal extensive sequence similarity to eukaryotic U6 snRNAs. This finding suggests that MCS4 and U6 snRNAs are derived from a common ancestral RNA that has existed before the divergence of prokaryotes and eukaryotes.     

Ultraviolet light-induced inhibition of small nuclear RNA synthesis

Two apparently distinct types of inhibition of the synthesis of U1, U2, U3, U4, and U5 small nuclear RNA, induced by ultraviolet (UV) radiation, have been described before: immediate and delayed. Our present observation can be summarized as follows: a) neither the immediate nor the delayed inhibition appear to be mediated by the formation of cyclobutane pyrimidine dimers, since they were not prevented by photoreactivating light, in ICR 2A frog cells; b) the inhibition of U1 RNA synthesis, monitored in HeLA cells within the first few minutes after irradiation, extrapolated to a substantial suppression at time zero of postirradiation cell incubation, providing further support for the proposal that the immediate inhibition is a reaction separate from the delayed UV light-induced inhibition of U1 RNA synthesis; c) the transition from the pattern of the immediate inhibition to that of the delayed inhibition (disappearance of the UV-resistant fraction of U1 RNA synthesis and increased rate of inhibition) occurred gradually, without an apparent threshold, within the first 2 hr of incubation after irradiation; and d) the incident UV dose that resulted in a 37% level of residual U1 RNA synthesis (D37) during the delayed inhibition was about 7 J/m2, with an apparent UV dose threshold, and was about 60 J/m2 for the immediate inhibition.     

Architecture of the U5 small nuclear RNA.

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.     

Rn7SK small nuclear RNA is involved in cellular senescence

Rn7SK is a conserved small nuclear noncoding RNA which its function in aging has not been studied. Recently, we have demonstrated that Rn7SK overexpression reduces cell viability and is significantly downregulated in stem cells, human tumor tissues, and cell lines. In this study, we analyzed the role of Rn7SK on senescence in adipose tissue-derived mesenchymal stem cells (AD-MSCs). For this purpose, Rn7SK expression was downregulated and upregulated via transfection and transduction, respectively, in AD-MSCs and subsequently, various distinct characteristics of senescence including cell viability, proliferation, colony formation, senescence-associated β galactosidase activity, and differentiation potency was analyzed. Our results demonstrated the transient knockdown of Rn7SK in MSCs leads to delayed senescence, while its overexpressions shows opposite effects. When osteogenic differentiation was started, however, they exhibited a greater differentiation potential than the original MSCs, suggesting a potential tool for stem cell-based regenerative medicine.