Studies on Isolated Nuclei

Maggio, Rachele; Siekevitz, Philip; Palade, George E.

doi:10.1083/jcb.18.2.293

Cited by 86 publications

(11 citation statements)

References 34 publications

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“…2 as ones in contact specifically with hnRNA, as opposed to other classes of nuclear RNA, is supported by the following points. (i) Most of the ribosomal RNA precursors are removed with the nucleolar fraction prior to hnRNP particle isolation (2,4,19). (ii) As shown in Fig.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…These were useful findings on each protein's individual merit but obviously did not reveal the molecular breadth of the nucleolar protein landscape. In 2002, two groups refined the classical nucleolar isolation methods (notably that developed by Maggio et al 1963) and prepared high purity nucleoli from HeLa cells followed by proteomics (Andersen et al 2002;Scherl et al 2002;reviewed by Pederson 2002 andLeung et al 2003).…”

Section: The Parts List and Segueing Into The Cell Cyclementioning

confidence: 99%

The Nucleolus

Pederson

2010

Cold Spring Harbor Perspectives in Biology

360

275

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“…The nucleoplasmic fraction banding at the 0:30% sucrose interface was removed for further use. As previously described for mammalian cells (31,38,39), this Drosophila nuclear fraction contains chromatin and hnRNP particles (S. Mayrand, S. George, and T. Pederson, unpublished results).…”

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confidence: 99%

Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

Mayrand¹,

Pederson²

1983

Mol. Cell. Biol.

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Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S. A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25°C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25°C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations. Exposure of Drosophila embryos or cultured cells to elevated temperature elicits major changes in gene transcription and protein synthesis (2). Transcription of a small set of genes is activated by heat shock (4, 11), and the resulting heat shock mRNAs are rapidly exported to the cytoplasm where they are preferentially translated (19, 29, 47). Meanwhile, the appearance of new non-heat shock mRNAs in the cytoplasm ceases (46). It is not known whether this latter effect of heat shock reflects decreased rates of transcription, a reduced efficiency of mRNA processing, or both. We have previously reported that the rate of actin mRNA transcription rapidly decreases to a low level during heat shock in Drosophila cultured cells, suggesting that this particular mRNA may be regulated chiefly at the transcriptional level (11). However, there are other observations which indicate that transcription of heterogeneous nuclear RNA (hnRNA) continues during heat shock. First, although the rates of heat shock gene transcription are very high, these mRNAs nevertheless are found to comprise a surprisingly small fraction (2%) of the nonribosomal nuclear t Paper no. 22 in a series entitled "Ribonucleoprotein Organization of Eukaryotic RNA."RNA that is synthesized during heat shock (11). Second, the size distribution of nonribosomal nuclear RNA made during heat shock is heterogeneous and is similar to that of nuclear RNA from non-heat-shocked Drosophila cells (23)(24)(25)(26). In the present investigation we examined hnRNA transcr...

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Studies on Isolated Nuclei

Cited by 86 publications

References 34 publications

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

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

The Nucleolus

Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

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