Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.

Kish, Valerie M.; Pederson, Thoru

doi:10.1073/pnas.74.4.1426

Cited by 30 publications

(7 citation statements)

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“…In addition, it has been possible to demonstrate that these latter hnRNP preparations contain specific proteins bound to defined nucleotide sequences, such as poly(A) (15,20,22), and that they FIGURE 8 Northern blot hybridization of 8-globin gene transcripts in hnRNP . RNA was recovered from nuclei, cytoplasm, or purified hnRNP particles by Proteinase K-phenol deproteinization and electrophoresed in agarose gels after denaturation in glyoxal (see Materials and Methods) .…”

Section: Identification Of Covalently Intact (3-globin Mrna Precursomentioning

confidence: 99%

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Pederson¹,

Davis²

1980

The Journal of Cell Biology

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To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-2005 and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of 38,000 mol wt . The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. The authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the particles to dissociation during isopycnic banding in C5 2SO 4 gradients without prior aldehyde fixation . Hybridization analysis with cloned mouse 8-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the a-globin gene . Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32 P-labeled cloned mouse fl-globin DNA reveals the presence in hnRNP of two size classes of /3-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S 8-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S a-globin mRNA . These results establish, for the first time, that the nuclear transcripts of a specific, welldefined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing .The transcription of structural genes in the eukaryotic cell nucleus takes place at a nucleoprotein level of organization . This is true of both the templates for transcription, chromatin (reviewed in reference 36), and the products of transcription, heterogeneous nuclear RNA, which is rapidly assembled into ribonucleoprotein particles known as hnRNP (33,34). However, the functional significance of these nucleoprotein environments for transcription and hnRNA metabolism is not understood . As a step toward defining the relationships between messenger RNA (mRNA) processing and nuclear structure, hnRNP particles have been purified from globin-producing mouse Friend erythroleukemia cells and characterized with particular reference to transcripts of the 8-globin gene. The THE JOURNAL OF CELL BIOLOGY -VOLUME 87 OCTOBER 1980 47-54 © The Rockefeller University Press -0021-9525/80/10/0047/08 $1 .00 major conclusion is that both pre-spliced and spliced mRNA sequences have a ribonucleoprotein structure in the nucleus. MATERIALS AND METHODSCell Culture, Radioisotopic Labeling, and Cell Fractionation Clone 745 mouse Friend erythroleukemia cells were maintained in monolayer culture usingJoklik-mod...

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Section: Identification Of Covalently Intact (3-globin Mrna Precursomentioning

confidence: 99%

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Pederson¹,

Davis²

1980

The Journal of Cell Biology

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“…hnRNP particles were first seen as nascent RNP fibrils on the lateral loops of amphibian oocyte lampbrush chromosomes (2,3) and later by electron microscopy in a variety ofeukaryotic cells (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) . hnRNP particles have been isolated (15,16), and their constituent RNA and proteins have now been characterized in some detail (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32).…”

mentioning

confidence: 99%

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.

Mayrand¹,

Setyono²,

Greenberg³

et al. 1981

The Journal of Cell Biology

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The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A) + hnRNA in intact HeLa cells, as revealed by a novel in situ RNA-protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the >42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A) + hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in CS2S04 density gradients . These results establish that poly(A) + hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo . This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modu ;ating hnRNA structure and processing in the cell .Eukaryotic messenger RNA is derived from precursors collectively known as heterogeneous nuclear RNA (hnRNA) . The conversion of hnRNA to mRNA involves a number of welldefined steps, including the addition of poly(A) and 5'-caps, and the removal of intervening DNA sequence transcripts through a series of coupled cleavage-ligation reactions known as splicing . These mRNA processing steps take place while hnRNA is tightly complexed with nuclear proteins in ribonucleoprotein particles known as hnRNP (reviewed in reference 1). hnRNP particles were first seen as nascent RNP fibrils on the lateral loops of amphibian oocyte lampbrush chromosomes (2, 3) and later by electron microscopy in a variety ofeukaryotic cells (4-14) . hnRNP particles have been isolated (15, 16), and their constituent RNA and proteins have now been characterized in some detail (15-32).It is likely that the structure of hnRNP is functionally related to mRNA processing (1) . We have been studying the nucleoprotein structure of defined RNA sequences in hnRNP using nucleases and other probes. For example, the use of nucleases specific for either single-or double-stranded RNA has revealed that intramolecular double-stranded regions of hnRNP are essentially free of associated protein, in contrast to singlestranded hnRNA which is extensively complexed with protein (27). Recently, we have found that messenger RNA-homologous sequences in hnRNP are extensively complexed with protein as revealed by nuclease protection experiments (31, 33) . The recent demonstration that hnRNP from mouse Friend erythroleukemia cells contains the prespliced 15S precursor of ß-globin mRNA (30) supports the idea that hnRNP particles are the sites of mRNA processing in the cell.In this investigation we used a novel methodology (32) to address another important aspect of hnRNP structure : the number of different protein species that are complexed with hnRNA . Beca...

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“…Cells that had taken up the control oligo(dA) showed no signal ( Figure 2D), indicating that the oligo(dA) does not hybridize appreciably to RNA in the cell. (HeLa cell nuclear RNA contains some oligo(U) tracts, but these are present at a much lower concentration than poly(A); Kish and Pederson, 1977). …”

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Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Politz

Tuft

Prasanth

et al. 2006

MBoC

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Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy. INTRODUCTIONNuclear speckles are morphologically distinct regions of the nucleoplasm that contain pre-mRNA splicing components as well as poly(A) RNA (Carter et al., 1993;Zhang et al., 1994;Lamond and Spector, 2003). They are operationally defined by their immunostaining with a variety of pre-mRNA splicing factor antibodies, and they also show in situ hybridization signal using probes for poly(A). When these sites are viewed in the electron microscope, most of them are found to represent interchromatin granule clusters (Fakan et al., 1984. However, RNA polymerase II transcription sites are distributed throughout the nucleus, indicating that although some of the RNA present in interchromatin granules/speckles is nascent, transcription is not restricted to this compartment, and much of the poly(A) RNA there has likely been transcribed previously and/or at distant sites (Fakan and Nobis, 1978;Wansink et al., 1993, Zeng et al., 1997, Neugebauer and Roth, 1997, Cmarko et al., 1999Guillot et al., 2004). Indeed, although transcripts that are being rapidly produced or that contain many introns are sometimes observed in speckles at the light microscopy level (Johnson et al., 2000;Shopland et al., 2003), the majority of studies over the years has indicated that most speckles are not primary sites of pre-mRNA splicing (for reviews, see Mattaj, 1994;Huang and Spector, 1996a;Neugebauer and Roth, 1997;Lamond and Spector, 2003). In two cases, a single species of pre-mRNA has been followed from its transcription site to the nuclear pore, and in neither case did the RNA seem to accumulate in nuclear subcompartments (Singh et al.,...

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Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.

Cited by 30 publications

References 19 publications

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

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