Low molecular weight nuclear RNA in human lymphocytes : A comparison of PHA-treated and control cells

Seven species of low molecular weight nuclear (LMN) RNA were identified in human peripheral lymphocytes. These were designated as A, B, C, D, D′, E and F. The same 7 species of LMN RNA were obtained from resting lymphocytes and from lymphocytes exposed to PHA for 16 and 60 h, respectively. Thus, no detectable qualitative changes were seen in the spectrum of LMN RNAs during PHA-induced lymphocyte transformation. However, the amount of species A, D-D′, E and F per nucleus in fully transformed cells was greater than in untreated lymphocytes. This increase had not yet occurred after 16 h treatment with PHA. ³H-Uridine was incorporated into all species of LMN RNA of resting and PHA-treated lymphocytes. Furthermore, all species of LMN RNA except C (5S RNA) were methylated in both resting and transformed cells. The ³H and ¹⁴C specific activities of LMN RNAs following an 8 h exposure to ³H-uridine and ¹⁴C-methyl methionine were higher in PHA-treated cells than in untreated lymphocytes. For several species of LMN RNA (A, D-D′, E, F) the highest ³H and ¹⁴C spec. act. were observed after 16 h exposure to PHA. The possibility that quantitative alterations in the synthesis and methylation of LMN RNAs may occur during lymphocyte transformation is discussed.     

Manipulation of the amount of RNA in post-mitotic nuclei

Because all (or almost all) nuclear RNAs are liberated to the cytoplasm during mitosis and then return to the post-mitotic nuclei, we expected that if cytoplasm were amputated from mitotic cells the post-division nuclei would possess less than normal amounts of RNA. Experiments performed with amebae (A. proteus) show that this is in fact what happens. Furthermore, since the enucleate fragment cut from a mitotic cell possesses an “excess” of returnable nuclear RNAs, a normal interphase nucleus implanted into such mitotic cytoplasm might be expected to acquire above-normal amounts of RNA. Experiments reported here show that this expectation also is realized. Thus, the regulation of the normal nuclear concentration of these RNAs involves mechanisms other than a limited number of intranuclear “binding” sites and most likely is restricted by the rate of synthesis of these RNAs.The demonstration that nuclei can be depleted or enriched for RNAs, many of which are unique to nuclei, makes it possible to determine the consequences for cell metabolism of altered amounts of nuclear RNA. Hopefully, such studies will reveal the function(s) of these RNAs.     

Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.     

Nuclear ribonucleoprotein complexes of amphibian liver. I. Characterization of the complex and its small molecular weight RNA moiety.

Nuclear RNA-protein complexes containing small molecular weight RNAs were isolated from hepatic nuclei of Rana catesbeiana tadpoles and frogs according to a procedure normally used for the isolation of heterogeneous nuclear ribonucleoprotein complexes from other eukaryotic tissues. Preliminary characterization of the tadpole nuclear RNP indicated a particle size of 50--70 S in sucrose density gradients and a buoyant density of 1.40 gm/ml in CsCl gradients. When analyzed on SDS-polyacrylamide gels, this complex was observed to contain at least 40 polypeptides ranging in molecular weight from 15,000 to 200,000. Nuclear RNA-protein complexes were also isolated from adult frog hepatic nuclei by the same protocol and the RNA moiety which had been purified from the frog complex was compared with the nuclear RNA isolated from the tadpole particles. Electrophoretic analysis of the nuclear RNA-protein-associated RNA revealed minor qualitative and quantitive differences in the more than 25 discrete bands (4--9 S) associated with each particle. Base analysis of tadpole and frog nuclear RNA revealed a nucleotide composition of approximately 50% adenosine plus uridine nucleotides, with an unusually high content of cytosine residues (approximately 30%). Comparison of the two RNA samples demonstrated a large increase in the adenosine content of frog unclear RNA, and the presence of a minor base in frog nuclear RNA which was absent in the tadpole sample. These results indicated that changes in the RNA content of the amphibian nuclear RNP complex had occurred during bullfrog development.     

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)     

A common maturation pathway for small nucleolar RNAs.

We have shown that precursors of U3, U8 and U14 small nucleolar RNAs (snoRNAs) are not exported to the cytoplasm after injection into Xenopus oocyte nuclei but are selectively retained and matured in the nucleus, where they function in pre-rRNA processing. Our results demonstrate that Box D, a conserved sequence element found in these and most other snoRNAs, plays a key role in their nuclear retention, 5' cap hypermethylation and stability. Retention of U3 and U8 RNAs in the nucleus is saturable and relies on one or more common factors. Hypermethylation of the 5' caps of U3 RNA occurs efficiently in oocyte nuclear extracts lacking nucleoli, suggesting that precursor snoRNAs are matured in the nucleoplasm before they are localized to the nucleolus. Surprisingly, m7G-capped precursors of spliceosomal small nuclear RNAs (snRNAs) such as pre-U1 and U2, can be hypermethylated in nuclei if the RNAs are complexed with Sm proteins. This raises the possibility that a single nuclear hypermethylase activity may act on both nucleolar and spliceosomal snRNPs.     

NLS peptide conjugated molecular beacons for visualizing nuclear RNA in living cells

Imaging the expression and localization of RNAs in live-cell nucleus can provide important information on RNA synthesis, processing, and transport. Here, we report the development of a bifunctional molecular beacon (NLS-MB) composed of a single nuclear localization sequence (NLS) peptide conjugated to a molecular beacon for efficient delivery and imaging of endogenous RNAs in the nuclei of living cells. We characterized the NLS-MBs by comparing their signal-to-noise ratios with unmodified molecular beacons and determined their efficiency of nuclear import. We demonstrated the specificity and sensitivity of the method by observing in living cells the localization and colocalization of small nuclear RNAs (snRNA) U1 and U2 at discrete foci in the nucleoplasm, and the localization of small nucleolar RNA U3 in the nucleolus. These snRNAs were chosen because of their essential roles in RNA biogenesis. The results were validated using in situ hybridization as positive control and random beacons as negative control. This novel approach may be applied to imaging other nuclear RNAs and pre-mRNAs in living cells.     

The small nuclear RNAs of the cellular slime mold Dictyostelium discoideum. Isolation and characterization

Three species of small nuclear RNA from the lower eucaryote Dictyostelium discoideum have been isolated and characterized with regard to size, cellular abundance, modified nucleotide content, and 5'-end structures. Previous studies had shown that the nuclei of mammalian cells contain a number of discrete low molecular weight, nonribosomal, nontransfer RNA molecules known as small nuclear RNAs. The mammalian small nuclear RNAs range in size from approximately 100 to 250 nucleotides and are quite abundant, in some cases approaching ribosomal RNA in number of copies/cell. Some of these molecules have an unusual cap structure at their 5'-ends similar to that found on eucaryotic messenger RNAs, and a number contain a characteristic set of internal modifications as well. Our results indicate that the small nuclear RNAs of Dictyostelium resemble their counterparts in higher eucaryotic cells structurally, but are present in significantly fewer copies/cell. The implications of these findings for small nuclear RNA function are discussed.     

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.     

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in HeLa cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs.The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w     

U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle.

U4 and U6 RNAs of mammalian cells possess extensive intermolecular sequence complementarity and hence have the potential to base pair. A U4/U6 RNA complex, detectable in nondenaturing polyacrylamide gels, is released when human small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 RNAs are dissociated with proteinase K in the presence of sodium dodecyl sulfate. The released RNA/RNA complex dissociates with increasing temperature, consistent with the existence of specific base-pairing between the two RNAs. Since U6 RNA is selectively released from intact snRNPs under the same conditions required to dissociate the U4/U6 RNA complex, the RNA-RNA interaction may be sufficient to maintain U4 and U6 RNAs in the same snRNP particle. The biological implications of these findings are discussed.     

A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro

RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease. We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs.