Ribonucleoprotein organization of eukaryotic RNA

Pederson, Thoru; Munroe, Stephen H.

doi:10.1016/0022-2836(81)90377-6

Cited by 22 publications

(8 citation statements)

References 32 publications

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“…The arrow at 30S marks the position of a peak of 30S particles, prepared from nuclei of 165-28 cells pulse-labeled with [3H]uridine as described previously (12,13,21) and analyzed in a parallel gradient. tamination of nuclear fractions containing globin RNA has been considered (19).…”

Section: Discussionmentioning

confidence: 99%

“…High-molecular-weight nuclear RNPs are broken down with EDTA and RNase to 30S particles (30). In contrast, the same treatment of cytoplasmic RNP results in complete degradation of the RNA (19). When CAD RNP was extracted from nuclei in the presence of EDTA, digested with pancreatic RNase, and analyzed in a sucrose gradient, most CAD sequences were found in a peak with a sedimentation coefficient of approximately 30S (Fig.…”

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confidence: 99%

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Abundant nuclear ribonucleoprotein form of CAD RNA.

Sperling¹,

Sperling²,

Levine³

et al. 1985

Mol. Cell. Biol.

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Previous studies of large nuclear ribonucleoproteins (RNPs) containing mRNA and precursors of mRNA have been hampered by the heterogeneity of the material. Any one molecular species of RNA is generally present at a level of less than 1% of the total. Biochemical information about large nuclear RNPs is therefore limited to bulk properties, for the most part derived from analysis of a breakdown product of the RNP that sediments at about 30S in a sucrose gradient. This 30S complex, made up of a fragment of RNA and a set of basic proteins, is regarded as a subunit of the native structure (12,13,22).In the studies reported here, we have investigated cells containing amplified genes as a source of an abundant large nuclear RNP, with a view to the possible isolation and analysis of homogeneous material. We have used a mutant, simian virus 40 (SV40)-transformed Syrian hamster cell line in which the gene for a multifunctional enzyme abbreviated CAD (for carbamoyl-phosphate synthetase, aspartate transcarbamylase, dihydro-orotase) is amplified about 200-fold (7,11,16,27). The levels of CAD mRNA and protein show a corresponding increase, CAD mRNA making up about 1% of the cytoplasmic polyadenylated RNA (16,27). One of our first objectives was to determine whether CAD sequences are similarly enriched in nuclear RNA.A further feature of the CAD gene pertinent to studies of nuclear RNP is its size and complexity. The structure of the gene indicates that the primary transcript is 25 kilobases (kb) in length and contains 37 or more intervening sequences (17). Hybridization analysis reveals a mature mRNA of about 7.9 kb (16, 27). The great length of these molecules heightens interest in the mechanism of their packaging, processing, and transport and at the same time poses difficulties for biochemical studies, for example, the problem of instability due to a large target size for RNase. were grown in culture as described previously (24).Nuclear and cytoplasmic fractions. (i) Procedure 1. Approximately 108 cells scraped from subconfluent plates were washed three times with ice-cold 137 mM NaCI-8 mM Na2HPO4-1.5 mM KH2PO4-2.5 mM KCI, allowed to swell for 10 min in 2 ml of 10 mM Tris-hydrochloride -3 mM MgCl2 (pH 7.5) (TM), and lysed with 10 strokes of a Dounce homogenizer (B pestle) at 4°C. Nuclei were pelleted and washed, once with 1 ml of TM containing 0.5% Triton X-100 and once with 1 ml of TM. The postnuclear supernatant and detergent wash were combined and designated the cytoplasmic fraction.(ii) Procedure 2. Subconfluent plates were washed with 125 mM KCI-30 mM Tris-hydrochloride (pH 7.5;) mM magnesium acetate-1 mM 2-mercaptoethanol-2 mM ribonucleoside vanadyl complex (2)-0.15 mM spermine-0.05 mM spermidine at 4°C, and cells scraped from the plates were washed twice with the same buffer. Approximately 108 cells were allowed to swell for 10 min in 2.5 ml of swelling buffer (same as wash buffer except the KCl concentration was 10 mM), lysed with 20 strokes of a Dounce homogenizer (B pestle), overlaid on an equal volume of swellin...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Abundant nuclear ribonucleoprotein form of CAD RNA.

Sperling¹,

Sperling²,

Levine³

et al. 1985

Mol. Cell. Biol.

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“…The procedures used for pulse labeling hnRNA with [3Hjuridine, RNA-protein crosslinking in vivo, and hnRNA-ribonucleoprotein particle (hnRNP) isolation have been published in detail (4,(8)(9)(10). All of the crosslinking experiments reported here involved irradiation of cell suspensions (107/ml) in a stainless steel Petri dish for 15 min (40C) at 3.6 X 105 ergs/ mm2 (1 erg = 0.1 .J).…”

Section: Methodsmentioning

confidence: 99%

Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.

Economidis¹,

Pederson²

1983

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

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Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) CeU 15,1477-1493, lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly -+ poly(A) addition --splicing.Over 36 years ago the crystallographer William T. Astbury stated "Biosynthesis is supremely a question of fitting molecules or parts of molecules one against another, and one of the great biological developments of our time is the realization that probably the most fundamental interaction of all is that between the proteins and the nucleic acids. " (p. 70 of ref. 1). The prescience of this view was amply borne out during the next 20 years by the demonstration that all nucleic acids are complexed with proteins in the cell, either stably (e.g., ribosomes) or transiently (e.g., transfer RNA with aminoacyl tRNA synthetases). "Heterogeneous nuclear RNA (hnRNA)" is the term given to eukaryotic RNA polymerase II transcripts and their processed intermediates. Like most other species of cellular RNA, hnRNA is thought to be complexed with proteins (2). hnRNA can be isolated in association with nuclear particles (3, 4), and ultrastructural analysis of spread chromatin also demonstrates that nascent hnRNA is complexed with protein (5-7).The cell fractionation and ultrastructural approaches have their respective advantages and disadvantages. The biochemical isolation of hnRNA-ribonucleoprotein complexes permits identification of the associated proteins but, like all cell fractionation studies, does not address the in vivo authenticity of these particles. Conversely, the ultrastructural data provide compelling evidence that hnRNA has a ribonucleoprotein structure in vivo but do not identify the proteins or their mode of interaction with the RNA. A recent attempt (8) to resolve these questions exploited UV light to catalyze RNA-protein crosslink formation in nuclear ribonucleoprotein in vivo.In the present study we applied this method to identify the particular proteins with which hnRNA is in contact in living cells and to probe the immediacy of ribonucleoprotein formation after hnRNA transcription.EXPERIMENTAL PROCEDURES Mouse erythroleukemia cells were cultured as described by Pede...

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“…19). The assembly of hnRNA transcripts into ribonucleoprotein (RNP) particles (hnRNP) appears to be a very early posttranscriptional event, as indicated by the RNP-like ultrastructure ofnascent hnRNA (20) and by the presence of very briefly pulse-labeled hnRNA transcripts in RNP structures (17). This raises the question ofwhether hnRNA transcripts synthesized in vitro in isolated nuclei become assembled into hnRNP particles resembling those found in vivo.…”

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confidence: 99%

Assembly of nuclear ribonucleoprotein particles during in vitro transcription.

Economidis¹,

Pederson²

1982

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

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The assembly of heterogeneous nuclear RNA (hnRNA) into ribonucleoprotein (RNP) particles has been investigated during in vitro transcription in isolated nuclei. Approximately 80% of the in vitro transcription observed in mouse Friend erythroleukemia cell nuclei is attributable to the activity of RNA polymerase 11. In vitro hnRNA transcripts are assembled into particles having the same properties as the nuclear ribonucleoprotein (hnRNP) particles in which hnRNA is found in vivo. Direct contact of hnRNP proteins with newly transcribed hnRNA was demonstrated by nuclease protection experiments and by the covalent transfer of 32P-labeled nucleotides from [a-32P]UTP-labeled hnRNA transcripts to specific proteins by RNA-protein crosslinking followed by nuclease digestion and electrophoresis of the nucleotide-bearing proteins. The availability of an in vitro system for hnRNP assembly opens a new route for investigating the functional relationship between nuclear structure and mRNA processing.Isolated nuclei are capable of transcription when incubated under the proper conditions (1-4). For example, nuclei isolated from HeLa cells infected with adenovirus serotype 2 (Ad2) synthesize RNAs of the size expected for primary transcripts from the major late promoter at 16.5 map units on the Ad2 genome (5-7). Some of these in vitro Ad2 transcripts are correctly capped, polyadenylated, and spliced (6, 8, 9), indicating that the mRNA processing machinery is at least partially active in the isolated nucleus.The RNA polymerase II transcripts of eukaryotic genes, known collectively as heterogeneous nuclear RNA (hnRNA), are complexed with a specific set of nuclear proteins in the cell (10-18, reviewed in ref. 19). The assembly of hnRNA transcripts into ribonucleoprotein (RNP) particles (hnRNP) appears to be a very early posttranscriptional event, as indicated by the RNP-like ultrastructure ofnascent hnRNA (20) and by the presence of very briefly pulse-labeled hnRNA transcripts in RNP structures (17). This raises the question ofwhether hnRNA transcripts synthesized in vitro in isolated nuclei become assembled into hnRNP particles resembling those found in vivo.To examine this issue, we have investigated the RNP status of hnRNA transcripts synthesized in vitro by endogenous RNA polymerase II in isolated nuclei, using the system of Manley et al. (8). Our results show that hnRNP assembly takes place in vitro, that the particle proteins make direct contact with the newly synthesized hnRNA, and that the in vitro-assembled particles are identical to native hnRNP by several criteria. This opens up an experimental system for exploring the relationship between nuclear RNP assembly and mRNA processing. METHODSCell Fractionation, in Vitro Transcription, and hnRNP Isolation. Stocks ofmouse Friend erythroleukemiacells (clone 745) were maintained in monolayer cultures and grown in suspension culture for each experiment (13). Erythroid differentiation was induced for 84 hr with 2% dimethyl sulfoxide. In some cases, cells were pulse labeled ...

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Ribonucleoprotein organization of eukaryotic RNA

Cited by 22 publications

References 32 publications

Abundant nuclear ribonucleoprotein form of CAD RNA.

Abundant nuclear ribonucleoprotein form of CAD RNA.

Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.

Assembly of nuclear ribonucleoprotein particles during in vitro transcription.

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