Metabolism of U6 RNA species in nonirradiated and UV‐irradiated mammalian cells

Choudhury, Kanakendu; Choudhury, Indrani; Jones, Robert W.; Thirunavukkarasu, Chellaiah; Eliceiri, George L.

doi:10.1002/jcp.1041370319

Cited by 20 publications

(16 citation statements)

References 23 publications

(28 reference statements)

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“…The following procedures were done as described: preparation of nuclear and cytoplasmic fractions from HeLa cells (8); isolation ofnucleoli (9); isolation ofRNA (10); hybrid selection of RNA (8); 10%o polyacrylamide gel electrophoresis (8); electrophoresis of RNA in 1% agarose and 2.2 M formaldehyde (11); RNA sequencing by the dideoxynucleotide method, using HeLa cell nucleolar RNA, avian myeloblastosis virus reverse transcriptase, and sequence-specific, antisense radioactive primers (12); and immunoprecipitation assays (13). The recommendations of the corresponding manufacturers were followed for (i) hybridization with ZetaProbe membranes (Bio-Rad) and (ii) sequencing of supercoiled DNA by dideoxy chain termination using Sequenase (United States Biochemical) or Taq I DNA polymerase (Promega).…”

Section: Methodsmentioning

confidence: 99%

Three small nucleolar RNAs of unique nucleotide sequences.

Ruff

Rimoldi

Raghu

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Three small RNA species were detected in human cells, and their cDNAs were synthesized and cloned. These RNAs are nucleolar, are 207, 154, and 135 nucleotides long, and are named El, E2, and E3, respectively, and their unique nucleotide sequences suggest that they may belong to an additional family of small nucleolar RNAs. The 5' ends of these three RNAs do not appear to have a trimethylguanosine cap or another type of cap. Apparent homologs of these three RNAs were detected in mouse, rabbit, and frog cells, suggesting their universal importance. They are housekeeping RNA species, since they are present in all rabbit tissues analyzed.The formation of cytoplasmic ribosomes in eukaryotic cells involves a complex series of synthesis, processing, assembly, and transport steps, many of which are poorly understood (reviewed recently in refs. 1 and 2). Some small nucleolar RNA (snoRNA) species, such as U3 RNA and yeast snR10 and U14 RNA, are required for rRNA precursor (pre-rRNA) processing (3-7). It has been difficult to study some of the low-abundance small nuclear RNAs in the absence of specific DNA or antibody probes. We have made cDNA probes for three snoRNAs and report some of the properties of these snoRNAs.t MATERIALS AND METHODS General Methods. The following procedures were done as described: preparation of nuclear and cytoplasmic fractions from HeLa cells (8); isolation ofnucleoli (9); isolation ofRNA (10); hybrid selection of RNA (8); 10%o polyacrylamide gel electrophoresis (8); electrophoresis of RNA in 1% agarose and 2.2 M formaldehyde (11); RNA sequencing by the dideoxynucleotide method, using HeLa cell nucleolar RNA, avian myeloblastosis virus reverse transcriptase, and sequence-specific, antisense radioactive primers (12); and immunoprecipitation assays (13). The recommendations of the corresponding manufacturers were followed for (i) hybridization with ZetaProbe membranes (Bio-Rad) and (ii) Radioactive Hybridization Probes. The El, E2, and E3 sequences in the corresponding cDNA clones, minus the tails that had been added for cDNA synthesis and cloning, were amplified by the polymerase chain reaction (14). The amplified products were then used as templates for extension of El-, E2-, and E3-specific, antisense 3' end primers with the Klenow fragment of DNA polymerase I in the presence of [a-32PldATP to generate the respective labeled, sequencespecific DNA probes. The radioactive DNA probe for Ul RNA was made by extension of a Ul-specific primer using as template the human Ul gene plasmid, linearized near the 5' end ofthe Ul gene. The radiolabeled DNA probe for U3 RNA was made by random primer labeling (15) using the rat U3 RNA sequence plasmid as template. RESULTSThere were some minor small RNA bands in HeLa cell nuclear RNA (bands labeled 1, 2, and 3 in lane 1 of Fig. 1) that could not be hybrid selected with any of the available DNA clones for small RNAs. RNA from these three sections of the gel was used to make three cDNA libraries. Clones from these libraries were used to probe Northern bl...

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Section: Methodsmentioning

confidence: 99%

Three small nucleolar RNAs of unique nucleotide sequences.

Ruff

Rimoldi

Raghu

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…One initial hint of such an activity arose from studies demonstrating that the transcription of numerous snRNA genes by both RNA polymerase II and III is sharply down regulated in response to UV light treatment for an extended period beyond the time required for repair of lesions that might impair transcription [123][124][125][126][127][128]. This initial finding suggested that a factor activated in response to DNA damage was capable of restricting snRNA gene transcription or alternatively that an essential factor was lost after DNA damage.…”

Section: Human Snrna Gene Transcription In Response To Genomic Stressmentioning

confidence: 99%

Transcriptional regulation of human small nuclear RNA genes

Jawdekar

Henry

2008

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

108

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The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated.

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“…For electrophoretic analysis, RNAs were dissolved in 80% deionised formamide, heated to 85 ~ C for 3 min, chilled immediately in ice and electrophoresed on 12% polyacrylamide -7 M Urea gel as described in [28]. The gel was ethidium bromide stained and photographed.…”

Section: Subcellular Fractionation Rna Extraction and Usnrna Fractiomentioning

confidence: 99%

“…Quantitation by densitometry after silver staining was done under the condition that has given a linear response with the RNA concentration [29]. Relative abundance of each UsnRNA was calculated by normalizing the areas of the peaks with respect to that of corresponding 5S rRNA [28,[32][33][34].…”

Section: Subcellular Fractionation Rna Extraction and Usnrna Fractiomentioning

confidence: 99%

“…Bands in the X-ray film and in the negative of ethidium bromide stained gel were densitometrically scanned as before. Quantitation by densitometry was done following the procedure of Choudhury et al [28] to give a linear response both with X-ray film and the negative of ethidium bromide stained gel. To study the kinetics of labelling of each of the six UsnRNAs during liver regeneration, area of each labelled UsnRNA band was normalised to that of the total unlabelled area of the corresponding 5S rRNA [28,[32][33][34] and plotted against different period of incubation.…”

Section: In Vitro Labelling Of Usnrnas In Isolated Hepatocytesmentioning

confidence: 99%

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Changes in UsnRNA biosynthesis during rat liver regeneration

et al. 1994

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Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50 % upto 48 hrs of RH. followed by gradual increase to reach the normal level within one month of R H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of RH. but in 24 and 72 hrs of RH. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of RH. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30-50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of RH. Thus, it may be concluded that the changes in UsnRNAs' level during th e proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver. (Mol Cell Biochem 141: 71-77, 1994).

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Metabolism of U6 RNA species in nonirradiated and UV‐irradiated mammalian cells

Cited by 20 publications

References 23 publications

Three small nucleolar RNAs of unique nucleotide sequences.

Three small nucleolar RNAs of unique nucleotide sequences.

Transcriptional regulation of human small nuclear RNA genes

Changes in UsnRNA biosynthesis during rat liver regeneration

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