B2 RNA Research Articles

Synthesis and processing of small B2 transcripts in mouse embryonal carcinoma cells.

B2 genes are short repeated sequences which are transcribed by RNA polymerase III. Abundant transcripts accumulate in embryonic and transformed cells, but transcripts are rare or absent from normal differentiated cell types. During retinoic acid-induced differentiation of P19 embryonal carcinoma cells, an early transient increase in B2 RNA levels is followed by a rapid drop in expression. The marked changes in B2 RNA levels are most likely due to transcriptional modulation since B2 RNA stabilities are unaffected by differentiation. At least four short-lived B2 RNAs with apparent lengths of 150, 180, 240, and 500 nucleotides were characterized. The two larger RNAs are polyadenylated and are more stable in cells. A cDNA of a B2 gene was isolated which was over 99% identical to the consensus sequence. This B2 cDNA can be transcribed in human cells and yields at least two distinct transcripts. We propose a model for B2 RNA metabolism which describes transcription, posttranscriptional modification and processing, and nucleocytoplasmic transport.

Synthesis and Processing of Small B2 Transcripts in Mouse Embryonal Carcinoma Cells

B2 genes are short repeated sequences which are transcribed by RNA polymerase III. Abundant transcripts accumulate in embryonic and transformed cells, but transcripts are rare or absent from normal differentiated cell types. During retinoic acid-induced differentiation of P19 embryonal carcinoma cells, an early transient increase in B2 RNA levels is followed by a rapid drop in expression. The marked changes in B2 RNA levels are most likely due to transcriptional modulation since B2 RNA stabilities are unaffected by differentiation. At least four short-lived B2 RNAs with apparent lengths of 150, 180, 240, and 500 nucleotides were characterized. The two larger RNAs are polyadenylated and are more stable in cells. A cDNA of a B2 gene was isolated which was over 99% identical to the consensus sequence. This B2 cDNA can be transcribed in human cells and yields at least two distinct transcripts. We propose a model for B2 RNA metabolism which describes transcription, posttranscriptional modification and processing, and nucleocytoplasmic transport.

Synthesis of a 180 kDa protein is a molecular marker of Xenopus oocyte meiotic maturation

Xenopus oocyte maturation can be induced in vitro by a number of agents including hormones, proto-oncogenes and cyclins. Xenopus oocytes matured by a variety of treatments, including progesterone, insulin, testosterone and injection of Xenopus c- mos RNA, Xenopus cyclin B2 RNA and egg extracts, synthesize a predominant 180 kDa protein (p180). p180 synthesis begins during maturation prior to germinal vesicle breakdown (GVBD) but is prominent only in oocytes that have undergone GVBD. p180 synthesis is thus a useful, reliable molecular marker of oocyte maturation. p180 continues to be synthesized in eggs and embryos up to the gastrula stage of embryogenesis, but not in the neurula or tailbud embryo.

B2 RNA and 7SK RNA, RNA polymerase III transcripts, have a cap-like structure at their 5′ end

We found that hydrolysates of poly(A)+ RNA from Ehrlich ascites carcinoma cells which were transcribed by RNA polymerase III contained an unusual component designated as X. It was part of B2 RNA representing a transcript of B2 retroposon, typical of rodents. The component X possesses a cap-like structure, xppp5'G, where x has a non-nucleotide structure. About half of all B2 RNAs contained this group at the 5' end. Previously, Epstein et al. (1) detected a similar structure at the 5' end of small nuclear U6 RNA. Later, Singh and Reddy (2) showed methyl to be the blocking group in the component x of U6 RNA. Besides B2 RNA, we found 5' ends containing methyl groups in 7SK RNA.

End-stage dilated cardiomyopathy. Persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response.

Tissue from the explanted hearts of 21 patients with idiopathic dilated cardiomyopathy or 19 patients with other specific heart muscle diseases were investigated for presence of enterovirus-specific RNA with an enterovirus group-specific cDNA probe. This was complementary to coxsackievirus B2 RNA sequences between nucleotide numbers 6,550 and 7,400, which are highly conserved between enteroviruses. Hearts from six patients with dilated cardiomyopathy and one patient with ischemic heart disease were found to contain virus RNA. Serology revealed that only one patient (dilated cardiomyopathy group) was positive for coxsackievirus B-specific IgM but negative for virus RNA in the myocardium. Quantitation of leukocytes and T-lymphocytes in the myocardium and expression of major histocompatibility locus antigens revealed no significant differences associated with persistence of virus RNA. These data demonstrate that enterovirus RNA persists in myocardium of a significant proportion of patients with end-stage dilated cardiomyopathy in the absence of a continuing cell-mediated or humoral immune response.

Induction of heat shock protein transcripts and B2 transcripts by various stresses in Chinese hamster cells

We have investigated the induction of known hsp (heat shock protein) RNA and other heat shock (HS) inducible transcripts in Chinese hamster cells by various stresses including DNA damaging agents. cDNA clones coding for at least 14 different HS-inducible transcripts were isolated. By DNA sequence analysis and homology with cDNA clones of other species, some of these cDNA clones were identified as coding for hsp27, hsp89 alpha, hsp89 beta, two different hsp70s, ubiquitin, and the HS-inducible RNA polymerase III transcript B2. In addition, hsp-related cDNA clones, hsp60 and four with hsp70 homology, were isolated which coded for transcripts which were not induced by HS or other stresses in two different Chinese hamster cell lines. After HS or treatment with the HS-mimetic agent ethanol, there was coordinate induction of all 14 transcripts. With severe HS treatments which produced substantial cytotoxicity, the increase in all transcripts except B2 RNA was delayed and, in some cases, suppressed. The only DNA damaging agent, which induced many HS-inducible transcripts, was high-dose methylmethane sulfonate (MMS). However, induction by MMS was not coordinate for all transcripts as it was for HS, and B2 RNA was not induced. hsp27 RNA induction differed from the others in several respects including induction by irradiation and other agents which produce high levels of DNA damage repaired by nucleotide excision repair. The implications of these findings in cellular events such as cytotoxicity, thermotolerance, and regulation of stress responses will be discussed.

Small B2 RNAs in mouse oocytes, embryos, and somatic tissues

RNAs of full-grown mouse oocytes, ovulated eggs, embryos, and somatic tissues have been analyzed on Northern blots for the presence of small transcripts homologous to the B2 element, a repetitive 180-nucleotide (N) sequence in the genome, using a single stranded RNA probe. In addition to the heterogeneous 200- to 600-N polyadenylated group reported by others, cytoplasmic RNA contains discrete nonadenylated species of B2-related RNA, approximately 100, 120, 155, and 180 N in length. During meiotic maturation of oocytes, the 200- to 600-N group declines and the 155-N species becomes more prominent. Upon hybridization to oligo(dT) and cleavage with RNase H to remove poly(A) regions, the 200- to 600-N group is removed, the 180-N species increased greatly, and the 155-N species increased slightly. Essentially all of the 200- to 600-N species bind to poly(U) sepharose. We conclude that polyadenylation rather than run-on transcription of B2 elements accounts for most of the heterogeneity of the 200- to 600-N group and that some deadenylation and cleavage take place during maturation. Small B2 RNAs make up 0.04% of total RNA in oocytes and eggs, 6- to 9-fold more than in brain. For comparison, a known small RNA, 4.5 S RNA, is relatively sparse in oocytes and almost absent in eggs. The 100-N B2-related species has been tentatively identified as 4.5 S I RNA; relative to total RNA, it remains approximately constant in oocytes and somatic tissues. During development to the blastocyst stage, small B2 RNAs per embryo increase severalfold, but decline as a fraction of total RNA. In postimplantation development, they continue to decline toward the level found in brain. Expression of B2 transcripts in hnRNA rises around 10 days of development to the level found in brain. The time course of expression of small B2 RNAs suggests an important role in development.

Enhanced B2 transcription in simian virus 40-transformed cells is mediated through the formation of RNA polymerase III transcription complexes on previously inactive genes.

RNA polymerase III (pol III) transcribes the highly repeated murine B2 elements. We showed previously that the B2 RNAs are induced 4-fold in normal growing cells and 20-fold in simian virus 40-transformed cells relative to the levels in normal confluent cells. By employing chromatin as a template in a partially purified pol III transcription system, we now demonstrate that the augmented expression results from the formation of pol III transcription complexes on previously inactive B2 genes. Extracts prepared from normal growing cells and transformed cells transcribed cloned pol III templates 5-fold more efficiently than extracts from normal confluent cells. This increase was attributed to 5-fold greater levels of factor IIIC; the levels of pol III and factor IIIB were the same in all extracts. We discuss how the levels of IIIC and differing accessibility of this factor to repressed B2 genes mediate the formation of pol III transcription complexes in normal growing and transformed cells.

Proteoglycan-19, laminin and collagen type IV production is correlated with the levels of mRNA in F9 cell aggregates differentiating in the presence or absence of cyclic AMP

Abstract F9 embryonal carcinoma cells differentiate to embryoid bodies containing an outer epithelial layer of visceral endoderm cells when cultured as aggregates in medium containing retinoic acid (RA). Another pathway of differentiation to parietal endoderm is followed when dibutyryl cyclic AMP (cAMP) is added to the medium. We have measured the accumulated levels of RNA transcripts from a chrondroitin sulfate proteoglycan gene (PG-19), the type IV collagen α, α2 subunit genes, and laminin B1, B2 subunit genes during these differentiation processes. Laminin B2 gene is uniquely regulated among the extracellular matrix component genes studied. The level of laminin B2 RNA remains almost invariant during RA induction of differentiation but is induced 11-fold by cAMP with RA. In contrast, laminin B1, collagen IV α, and α2 genes are induced in two stages with six- to sevenfold accumulation of RNA induced by RA and fourfold greater levels by cAMP (19-to 28-fold overall). All of these matrix-encoding genes except proteoglycan are expressed at low levels in unstimulated F9 cells, whereas PG-19 is completely undetectable and is observed only after 2 days of stimulation with RA. Its increased expression with RA and cAMP induction is at least 100-fold during F9 differentiation. Extracellular matrix transcripts are relatively stable and this accounts in part for high accumulated levels during differentiation. We conclude that several kinds of gene regulation occur among the matrix components and other differentiation markers, and this makes the F9 model system useful to study the differential effects of hormone treatments on cellular events leading to differentiation and loss of tumorigenicity.

The transcription of B2 repeated sequences is regulated during the transition from quiescent to proliferative state in cultured rodent cells

The RNA polymerase III-dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus-induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus-transformed cells. In both systems, nuclear run-off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2-specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation.

Induction of Specific Transcription by RNA Polymerase III in Transformed Cells

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.

Induction of specific transcription by RNA polymerase III in transformed cells.

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.

Induction of B2 RNA polymerase III transcription by heat shock: enrichment for heat shock induced sequences in rodent cells by hybridization subtraction

When hybridization subtraction was used to enrich for sequences induced by heat shock in Chinese hamster cells, B2 sequences were found to be one of the major sequences enriched. With cloned B2 probes, we found that the level of the short, 0.1 to 0.6 kb, polyadenylated RNA polymerase III transcripts of this repetitive genetic element increased approximately 10 to 20 fold after heat shock. Transcription of B2 RNA by RNA polymerase III was rapidly induced after heat shock based on time course studies and nuclear runoff experiments. The induction of B2 RNA was not a nonspecific response to lethality or cellular injury because maximum B2 RNA induction was observed with even nontoxic heating while no induction occurred with other agents such as UV or X-radiation. Since B2 RNA increased after heat shock in several different Chinese hamster and mouse cell lines, induction of B2 RNA by heat shock is probably common in rodent cells. B2 RNA may also be the most abundant transcript induced by heat shock because the level of B2 RNA was substantially higher than several other abundant transcripts induced by heat shock including a rodent HSP70. Our finding of the induction of high levels of RNA polymerase III B2 transcripts in different rodent cells raise the possibility of a role in the heat shock response.

Activation of putative transposition intermediate formation in tumor cells.

The transcription levels of two families of mouse repetitive elements namely intracisternal A particle (IAP) genes, and B2 sequences were analyzed in different tumor cells and normal tissues. These sequences belong to two major classes of mobile elements present in the mouse genome. The Northern blots containing poly(A) + RNAs from tumor cells and normal tissues were hybridized to the cloned IAP gene and B2 sequence. The content of IAP gene transcripts in tumor cells is much higher than in normal cells. A 10- to 100-fold difference was found. The predominant IAP-gene specific RNAs in all investigated tumor cells were 9.5, 6.8 and 5.3 kb long. Additional RNA species were found in some of the tumors. The active synthesis of small cytoplasmic B2 RNA transcribed by RNA polymerase III was also detected in most tumor cells tested. Usually it was higher than in normal cells. Free closed circular DNAs hybridizing to IAP gene probes were cloned from Ehrlich ascites carcinoma cells. We speculate that the data obtained indicate the enhanced transposition of mobile elements in tumor cells which may be an important factor of tumor progression.

RNAs containing B2 repeated sequences are transcribed in the early stages of mouse embryogenesis.

An in situ hybridization technique was used to detect RNAs containing B2 sequences in the early mouse embryo. Accumulation of B2 sequences occurs early from the one cell stage. The level of B2 RNA decreases in the late two cell embryo, and then increases at the moment of second cleavage. In the blastocyst, inner cell mass cells contain more B2 transcripts than trophectoderm cells. In 7.5-day embryos the expression of B2 sequences is restricted to ectoderm and mesoderm. At all stages, transcription of the B2+ strand is greater than B2- strand. We detected B2+ RNAs in the nucleus and cytoplasm, whereas B2- RNAs were present only in the nucleus.

Biosynthesis and cytoplasmic distribution of small poly(A)-containing B2 RNA

Previously, we described a small polyadenylated RNA predominantly located in cytoplasm and hybridizing with the ubiquitous B2 sequence of the mouse genome (Kramerov, D.A., Lekakh, I.V., Samarina, O.P. and Ryskov, A.P. (1982) Nucleic Acids Res. 10, 7477–7491). This 180–300 nucleotide long RNA was designated B2 RNA. Here, we demonstrate that B2 RNA is complementary to just one of the strands of cloned B2 sequence. The synthesis of B2 is rather resistant to ultraviolet irradiation of Ehrlich ascites carcinoma cells. The treatment of the cells with α-amanitin at a concentration completely blocking the formation of small nuclear RNAs U1, U2 and U3 does not interfere with the B2 RNA synthesis. These results suggest that B2 RNA formation is directly transcribed with the aid of RNA polymerase III, rather than being formed in the course of the processing of large RNA molecules which are known to contain a lot of B2 sequences. We also surprisingly found that the synthesis of up to 50% of long poly(A) +RNA in Ehrlich carcinoma cells is rather resistant to α-amanitin. The possible role of genetic elements including B2 sequences able to promote large RNA-polymerase III transcripts is discussed. B2 RNA in the cytoplasm is incorporated into the ribonucleoprotein particles, both small (12–18 S) and heavy. The latter probably correspond to informosomes. After deproteinization of heavy particles, a major part of B2 RNA still cosediments with mRNA and is split from it only after denaturation. We suggest that the B2 RNA of heavy ribonucleoproteins is associated with mRNA by short complementary stretches. About half of the B2 RNA is recovered in the cytoskeletal fraction. The possible role of B2 RNA in mRNA transport or in translation regulation is discussed.

Nucleotide sequence of small polyadenylated B2 RNA

Small poly(A)-containing RNA molecules which hybridize to the ubiquitous short repetitive sequence B2 and which are transcribed by RNA polymerase III have been identified in the cytoplasm of mouse cells. Here, we describe the structure of this small B2 RNA. A cDNA library was prepared from low-molecular-weight cytoplasmic poly(A)+RNA isolated from Ehrlich carcinoma cells and the clones which hybridized to B2 sequence were selected. The clones were sequenced and shown to contain B2 sequences followed by a poly(A) tract. The sequences of the cloned B2 RNAs different from each other by 3-10%, being similar in this respect to genomic B2 copies. Thus, B2 RNA is transcribed from many different B2 sequences in the genome. The 5'-ends of B2 RNA at least in most molecules coincide with the beginning of B2 genomic sequence. The poly(A) segments located at the 3'-end of small B2 RNA are the same size as in mRNA molecules, suggesting posttranscriptional formation. In some clones, additional sequences were detected between the 3'-end of B2 sequence and the poly(A) stretch. They seem to result from a lesion in the RNA polymerase III terminator in the corresponding B2 sequences. The possible significance of B2 sequences and small B2 RNA is discussed.

The sequences homologous to major interspersed repeats B1 and B2 of mouse genome are present in mRNA and small cytoplasmic poly(A) + RNA.

Heavy nuclear RNA contains high amounts of transcripts from repetitive sequences B1 and B2. Cytoplasmic poly(A)+RNA and, particularly, polysomal poly(A)+RNA (mRNA) also include these sequences but in smaller amounts. The abundant 2 kb mRNA of mouse liver are found to have a B2 sequence. These sequences are also found in a new class of low-molecular-weight heterogeneous (200-400 nucleotide long) poly(A)+RNAs. These RNAs are located mostly in cytoplasm rather than in polysomes. The amount of small B2+ RNAs is noticeable larger than that of small B1+ RNAs. Tumour cells seem to contain more small B2 RNA than normal cells. The hybridization tests show that extended homology exists between the B1 sequence and 4.5S small nuclear RNA, which is predicted from comparison of their base sequences. Also, we have found homology between B1 and small cytoplasmic 7S RNA. Hybridization of B2 to sn or sc RNAs has not been observed, although the sequencing reveals partial homology between B2 and 4.5S sn RNAI /1/.

A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro.

The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro. RNA polymerase B purified from the mutant is altered both structurally and functionally. The enzyme is defective in the RNA chain initiation and elongation reactions. Enzyme-DNA binding is comparatively much less affected. These enzymological defects in the mutant enzyme are enhanced at elevated ionic strengths. Purified rpo B1 RNA polymerase B is lacking B32 and B16.5 subunits. However, the low activity of the mutant enzyme cannot be accounted for only by the loss of these two polypeptides. Wild type enzyme devoid of B32 and B16.5 subunits can be obtained after a mild urea treatment. This enzyme variant, called RNA polymerase B, does not share the enzymological properties of the mutant RNA polymerase. Immunoprecipitation of the enzyme from crude extracts shows that, in the rpo B1 mutant, a normal amount of RNA polymerase B is synthesized which contains the full complement of subunits. The polypeptide chain altered by the rpo B1 mutation was identified by partial proteolysis with proteinase K in the presence of sodium dodecyl sulfate. The 35S-labeled peptide pattern generated from the B220 subunit of the mutant enzyme differs markedly from the peptide pattern of the wild type subunit. The rpo B1 mutation therefore alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerase molecule.