Expression of a mouse U1b gene in mouse L cells

Moussa, N.M.; Lobo, Susan; Marzluff, William F.

doi:10.1016/0378-1119(85)90186-6

Cited by 16 publications

(18 citation statements)

References 24 publications

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“…4A, lane 2). L cells that had been transformed with the U1.l and U1.2 genes for U l b RNA and then were radiolabeled for 16 h showed comparable levels of radioactive mature U l b and U l a RNA (Moussa et al, 1985). We obtained similar results when a labeling pulse was followed by 20 h of chase in cells transformed with the U1.2 gene (Fig.…”

Section: Effect Of Uv Irradiation Of L Cells On Thesupporting

confidence: 82%

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RNA synthesis and stability in UV‐irradiated and nonirradiated mouse L cells

Jones

Eliceiri

1989

Journal Cellular Physiology

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In mouse L cells, relatively low doses of UV light (e.g., about 35 J/m2) induced the rapid breakdown of the molecules of many RNA species transcribed shortly before irradiation. This included 28S, 18S, 5.8S, and 5S rRNA, U1, U2, U3, U4, and U5 small nuclear RNA, but not the main band of transfer RNAs or 7SL RNA. At higher UV doses, an RNA band that contains tRNAleu was also degraded rapidly after UV irradiation. RNA molecules synthesized long before irradiation (e.g., 22 h for small RNAs, 4 h for large rRNAs) were not affected. Our results suggest that the maturation and/or assembly into fully mature ribonucleoprotein particles of several small RNA species is not completed 4 h after transcription. The effect of UV radiation occurred in mouse L cells, but not in human HeLa or KB cells. In a previous report, L cells were transformed by DNA transfection with two mouse U1b RNA genes, named U1.1 and U1.2. We observed now that, in L cells transformed with the U1.2 gene, the ratio of radioactivity in the apparent U1b and U1a RNA precursors after 5 min of labeling was about 20 times higher than a) this ratio in briefly labeled L cells that had been transformed with the U1.1 gene, and b) the ratio of radioactive mature U1b and U1a RNA after 20 h of chase in L cells transformed with the U1.2 gene. These results suggest that very high levels of U1b RNA are transcribed from the exogenous U1.2 gene copies, followed by the rapid degradation of most of these transcripts.

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Section: Effect Of Uv Irradiation Of L Cells On Thesupporting

confidence: 82%

“…Mouse L cells produce over three times more U l a RNA than Ulb RNA (Moussa et al, 1985). Two genes for mouse Ulb RNA have been cloned, named U1.l and U1.2, which are located in opposite DNA strands about 5 kb apart (Marzluff et al, 1983).…”

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confidence: 99%

RNA synthesis and stability in UV‐irradiated and nonirradiated mouse L cells

Jones

Eliceiri

1989

Journal Cellular Physiology

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“…A more interesting possibility is that the concentration of U4X RNA, or changes in the U4X-to-U4B RNA ratio, plays a role in the selection of developmentally regulated alternative splice sites in mRNA precursors. The developmental or cell typespecific expression of snRNA sequence variants has also been reported for other animals besides the chicken (4,7,(10)(11)(12)15). It has been suggested that changes in the concentrations or ratios of splicing factors (e.g., variants of the snRNPs) may play a role in alternative splicing of mRNAs (1,4,5,9,11,12).…”

Section: U'mentioning

confidence: 81%

Developmental and tissue-specific expression of U4 small nuclear RNA genes.

Korf¹,

Botros²,

Stumph³

1988

Mol. Cell. Biol.

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U4 RNA is one of several small nuclear RNAs involved in the splicing of mRNA precursors. The domestic chicken has two genes per haploid genome that are capable of encoding U4 RNA. The U4X RNA gene (which encodes a sequence variant of U4 RNA that was unknown prior to the cloning of the gene) and the U4B RNA gene were both expressed in vivo in each of seven adult and three embryonic chicken tissues examined. However, the ratio of U4B RNA to U4X RNA can vary more than sevenfold in both a tissue-and stage-specific manner.The small nuclear RNAs (snRNAs) of the U family (Ul to U10) are evolutionarily conserved and metabolically stable RNA molecules present in the nuclei of eucaryotic cells (16). The Ul, U2, U4, U5, and U6 snRNAs are involved in the splicing of mRNA precursors (14,18,19). In metazoans, there are multiple copies of the genes for each of the major snRNAs (usually 5 to 1,000 gene copies per family) (3). However, in the chicken there are only two genes per haploid genome that are capable of encoding U4 RNA (6). The organization of the U4X and U4B RNA genes in the chicken genome is diagrammed at the top of Fig. 1. The U4B and U4X genes are closely linked and are in the same transcriptional orientation (6). The sequences of the encoded RNA transcripts are shown at the bottom of Fig. 1. The U4B and U4X RNAs differ in sequence at seven nucleotide positions. U4B is a major U4 RNA sequence variant that has been identified in chickens and other vertebrates (8, 16), but the U4X RNA transcript has not previously been characterized.Differential accumulation of U4B and U4X RNA in adult tissues. To study the differential accumulation of these U4 snRNAs, we synthesized two DNA 19-mer oligonucleotides ( Fig. 1, U4B probe and U4X probe) that were specifically complementary to the 3' ends of U4B RNA and U4X RNA, respectively. To examine the relative accumulation of U4B and U4X transcripts in various tissue types, we isolated total cellular RNA from seven tissue samples from an adult hen by using the guanidinium-hot phenol method described by Maniatas et al. (13). Purified RNA was dissolved in 99% formamide, run on a 6% polyacrylamide-7 M urea gel, and electroblotted to GeneScreen Plus (Du Pont, NEN Research Products) at 60 V for 1 h and then at 30 V for 45 min, with a chamber buffer consisting of 12 mM Tris acetate, 6 mM sodium acetate, and 0.3 mM EDTA (pH 7.5). The filter was prehybridized at 45°C in 0.9 M NaCl-1% sodium dodecyl sulfate-10 mM Tris hydrochloride (pH 7.6)-0.5 mM EDTA-8 ,ug of Escherichia coli DNA-20 ,ug of ATP per ml. Fig. 2, lower panel). To ensure the hybridization specificity of the U4B and U4X probes for the respective U4 RNA variants, artificial U4X-like (SP6:U4X) and U4B-like (SP6:U4B) transcripts, approximately 178 and 233 nucleotides in length, respectively, were included in the gels as internal controls. These were generated by transcribing the cloned genes in vitro with SP6 RNA polymerase as specified by the supplier (Promega). Relevant portions of the templates (cloned into the pSP64 vector) a...

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“…The filters were then counted in a liquid scintillation counter to monitor hybridization efficiency. The hybridized RNA was eluted and analyzed by gel electrophoresis under conditions which separate Ula and Ulb RNA (27).…”

Section: Methodsmentioning

confidence: 99%

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

Lobo¹,

Marzluff²

1987

Mol. Cell. Biol.

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The transcription of Ul RNA genes was studied in isolated nuclei from mouse myeloma cells. Using a cloned Ulb gene as a probe, we showed that isolated nuclei synthesize both Ulb and Ula RNA. The Ul RNAs were initiated in vitro, as measured by incorporation of adenosine 5'-O-(2-thiotriphosphate) into Ul RNA. There was transcription of the 3'-flanking region but no transcription of regions directly 5' to the Ul genes. In addition to Ul RNAs of the correct length which were released from the nuclei, there were larger RNAs, presumably resulting from transcription into the 3'-flanking region, which were retained in the nuclei. Chase experiments showed that these longer transcripts were not precursors to mature Ul RNA, a finding consistent with the idea that 3'-end formation is coincident with transcription. During the chase, there was maturation of the 3' ends of Ula and Ulb RNAs from slightly longer precursors. In addition to accurate transcription of Ul RNA, there was also synthesis of U2 and U3 RNA. All three of these RNAs were transcribed by RNA polymerase II, as measured by their sensitivity to a-amanitin.The U series of small nuclear RNAs (snRNAs) is one of the most abundant transcripts in mammalian cells. These RNAs are encoded by multiple genes and contain an unusual 5' cap structure, 2,2,7 trimethyl guanosine (3). These RNAs are synthesized by RNA polymerase II, as measured by the sensitivity of their synthesis to low concentrations of aamanitin (8,25,33). An exception is U6 RNA, which lacks the cap structure found at the 5' ends of other U RNAs and is believed to be transcribed by RNA polymerase III (13,32). Some aspects of the synthesis of Ul, U2, and U3 RNAs differ from those of other RNAs synthesized by RNA polymerase II. While cell-free systems have been described which transcribe most other genes accurately, the cell-free systems currently available from mammalian cells do not accurately transcribe the Ul or U2 genes (28, 39). We have previously described the development of both nuclear (25) and DNA-dependent (26) systems from sea urchin embryos which transcribe the sea urchin Ul RNA gene accurately.The promoters of the vertebrate U snRNAs differ from those of other genes transcribed by polymerase II. There is a sequence about 200 nucleotides 5' to the gene which is essential for transcription in vivo (19) and on injection of the genes into Xenopus oocytes (29,33,39). When the genes are used as a template in a DNA-dependent system, there is transcription which initiates about 25 bases downstream from this upstream promoter element (as if it were functioning like a TATAA box in this system) but no transcription which initiates at the proper start site (28). The promoters of the sea urchin U RNA genes are not utilized in Xenopus oocytes (37), unlike the promoters of other sea urchin genes transcribed by RNA polymerase II.The formation of the 3' end of the U snRNAs is unusual in two respects. There is initial formation of a transcript a few nucleotides longer than the mature RNA (6,18,41 (18).Dissecting...

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Expression of a mouse U1b gene in mouse L cells

Cited by 16 publications

References 24 publications

RNA synthesis and stability in UV‐irradiated and nonirradiated mouse L cells

RNA synthesis and stability in UV‐irradiated and nonirradiated mouse L cells

Developmental and tissue-specific expression of U4 small nuclear RNA genes.

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

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