Studies on dissociation and reconstitution of nuclear 30-S ribonucleoprotein particles containing pre-mRNA

Kulguskin, V.V.; Krichevskaya, A. A.; Lukanidin, E. M.; Georgiev, G.P.

doi:10.1016/0005-2787(80)90115-x

Cited by 9 publications

(11 citation statements)

References 16 publications

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“…This does not necessarily mean that hnRNP particles are not associated with other nuclear elements in vivo, but it does indicate that the first level of hnRNP structure can assemble in a soluble in vitro system. This conclusion is compatible with the previous demonstration that hnRNP, dissociated into RNA and protein by 2 M NaCl, can reassociate after dialysis to low ionic strength (38)(39)(40) A major unsolved problem in the area of hnRNP function is the extent to which the structure of these particles is based on sequence-specific RNA-protein interactions. Recently, a detailed study has appeared of RNP structure on an adenovirus E2 transcript, the DNA-binding protein mRNA precursor (52).…”

Section: Discussionsupporting

confidence: 78%

In vitro assembly of a pre-messenger ribonucleoprotein.

Economidis¹,

Pederson²

1983

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

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Transcription of the Bal I E restriction fragment of adenovirus DNA by RNA polymerase H in a HeLa cell extract produces a RNA transcript 1,712 nucleotides in length. This transcript contains the first two elements of the tripartite leader that, in vivo, is spliced onto the late mRNAs. We have found that this adenovirus 2 transcript forms a specific ribonucleoprotein complex (RNP) in this in vitro system. The RNP particle sediments in sucrose gradients as a monodisperse peak at 50 S and has a buoyant density of 1.34 g/cm3 in Cs2SO4, indicating the same 4:1 protein/RNA composition as native nuclear RNPs that contain premRNA sequences (hnRNP). Moreover, the in vitro-assembled RNP is resistant to concentrations of NaCl that are known to dissociate nonspecific RNA-protein complexes. The adenovirus 2 transcript is precipitated by a monoclonal antibody for hnRNP core proteins.In addition, RNA-protein crosslinking of [a-32P]UTP-labeled transcript/RNP complexes reveals that the major proteins in contact with the RNA are the Mr 32,500-41,500 species known to be associated with hnRNA in vivo. These results demonstrate the in vitro assembly of a specific RNA polymerase II transcript into RNP. Moreover, because the 1,712-nucleotide adenovirus 2 transcript lacks poly(A) addition sites and because the leader sequences are not spliced appreciably in this in vitro system, it follows that RNP formation requires neither polyadenylylation nor splicing, nor is it sufficient to cause the latter. mRNA processing takes place in nuclear ribonucleoprotein particles (RNPs) known as heterogeneous nuclear RNP, or hnRNP (reviewed in ref. 1). These particles are variable in morphology (2, 3), RNA sequence (4, 5), and protein composition (6-10). For this reason, most previous studies of hnRNP organization have succeeded only insofar as revealing major structural features common to hnRNP particles as a whole.To define hnRNA-protein interactions in greater detail, it would be useful to study a RNP particle containing a specific pre-mRNA transcript. One possibility is to isolate an especially prevalent pre-mRNA, as RNP, from the nuclei of cells producing abundant mRNAs. However, in most cases, the nuclear precursors of abundant cytoplasmic mRNAs turn out not to be particularly prevalent in the nuclear RNA (for P-globin mRNA, RNA Analysis. The desired portion of the transcription reaction, usually 5-50 ,ul, was mixed with 2.4 ml of a solution containing 50 mM sodium acetate (pH 5.2), 1% NaDodSO4, 10 mM EDTA, and Escherichia coli tRNA at 10 ,ug/ml; 2.4 g of CsCl was added and dissolved by heating to 60°C for 2 min. The solution was layered over 1.2 ml of 5.7 M CsCl/100 mM EDTA and centrifuged in a Beckman SW 50.1 rotor at 38,000 rpm for 24 hr at 200C (14). The RNA pellet was dissolved in 0.3 M sodium acetate (pH 5.2) and precipitated with 2 vol of 95% ethanol at -20°C. The RNA was collected by centrifugation and dissolved in 3 ,ul of water, 4 ,ul of freshly deionized glyoxal, 8 ,ul of dimethyl sulfoxide, and 1 ,ul of 0.16 M sodium phosp...

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Section: Discussionsupporting

confidence: 78%

In vitro assembly of a pre-messenger ribonucleoprotein.

Economidis¹,

Pederson²

1983

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

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“…Furthermore, addition of deoxycholate or high salt to reconstituted complexes causes their redissociation (results not shown), indicating that protein-protein and protein-RNA interactions are reversible as well as cooperative. Kulguskin et al (13) reported a high degree of cooperativity in the reversible transition between 30S protein cores and dissociated proteins at high ionic strength in the absence of RNA. However, upon adding RNA and lowering the ionic strength they found that only those RNP reconstituted from 30S cores and not RNP reconstituted from the protein subunits could be redissociated.…”

Section: Discussionmentioning

confidence: 99%

“…One difficulty with this model is that, when stripped of nucleic acid in vitro, core protein complexes dissociate unless previously cross-linked (18) or maintained at an extremely high concentration (13). Another problem is the variability in the ratios of the individual core proteins in different preparations (3), as well as the variability of 30S RNP particle size and morphology within a single preparation (18).…”

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confidence: 99%

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.

Pullman¹,

Martin²

1983

The Journal of Cell Biology

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Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous -~20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.Ultrastructural and biochemical lines of investigation have independently shown that heterogeneous nuclear RNA (hnRNA) is complexed with proteins throughout its existence in the cell nucleus (reviewed in reference 20). In electron micrographs of transcriptional complexes spread from lysed nuclei, hnRNA-protein complexes (hnRNP) frequently appear as fibrils consisting of 20-nm beads connected by RNA strands (1, 2, 21, 23). The biochemical entity isolated from purified nuclei by either isotonic extraction or mechanical disruption, referred to by us as "30S RNP" since it usually sediments more slowly than the 40S ribosomal subunit, is 18-25 nm in diameter and contains 700-1,000 nucleotides of rapidly labeled RNA and 40-60 copies of a group of four to six proteins of 34,000-40,000 mol wt with similar amino acid compositions and isoelectric points (pI = 8.0-9.0) (3,5,8, 11,17). These proteins are highly conserved throughout the vertebrates and turn over slowly in contrast with many other proteins (17-19). Since they only partially protect hnRNA from nuclease digestion (18), it has been suggested that these proteins form a core around which the nucleic acid is wrapped (3"0), and hence their designation as "core proteins." This hypothesis is attractive in that it allows for new transcripts to be mai...

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“…These six proteins also interact with one another through protein-protein contacts (17,46) and can be released from hnRNA by high ionic strength conditions as heterotypic protein-protein oligomers (46,47). Thus, these proteins apparently interact with hnRNA as a preassembled unit, not as individual proteins free in solution.…”

Section: Discussionmentioning

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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Studies on dissociation and reconstitution of nuclear 30-S ribonucleoprotein particles containing pre-mRNA

Cited by 9 publications

References 16 publications

In vitro assembly of a pre-messenger ribonucleoprotein.

In vitro assembly of a pre-messenger ribonucleoprotein.

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.

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

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