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...

The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[('I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions. L-3,5,3'-Triiodothyronine (T3), the active form of thyroid hormone, modulates the expression of a number of genes (1, 2), and this is ascribed to the association of T3 with receptors localized in chromatin (3). In rat pituitary tumor cells the concentration of growth hormone (GH) mRNA increases in response to physiological concentrations ofT3 (refs. 4 and 5).Although additional mechanisms have not been ruled out, this response reflects an increased rate of transcription of the GH gene (6, 7), presumably through a direct effect of the receptor (8). Moreover, T3 acts synergistically with glucocorticoid hormones in stimulating GH gene transcription (6, 9). Binding regions for the rat (10, 11) and human (12) glucocorticoid receptors have been identified in the human GH gene upstream and downstream from the transcription initiation site (cap site). Homologous binding regions have also been detected (12) in the human placental lactogen (chorionic somatomammotropin, CS) gene, which has a 92% overall nucleotide sequence homology with the human GH gene (13). In contrast, the question of specific recognition sites for thyroid hormone receptors on any DNA has remained open. We have now searched for such sites in the human GH and CS genes (12) using the human thyroid hormone receptor in a cell-free system. This system contained [1251]T3-labeled nuclear extracts from cultured human lymphoblastoid cells of the IM-9 line and cloned fragments from the genes of interest. MATERIALS AND M...

The hypothesis that the glucocorticoid hormone receptor interacts with RNA has been tested in cultured rat hepatoma cells. The receptor was covalentIy labeled with radioactive dexamethasone mesylate, and putative RNA-receptor complexes were stabilized by either cell-free crosslinking using formaldehyde or irradiation of intact cells. After chemical cross-linking in vitro, the receptor displayed the buoyant density of a ribonucleoprotein in CsCl gradients. After photochemical crosslinking in cells labeled with radioactive uridine, the receptor analysed by polyacrylamide gel electrophoresis was carrying labeled ribonucleotides.

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (R,) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate. In contrast, the highly purified glucocorticoid receptor yielded a sharp symmetrical peak of R, = 7.1 nm. We demonstrate that the 7.1-nm component could not result from a proteolytic degradation of the 8.3-nm receptor form. The same receptor heterogeneity was observed in thymus cytosol which contains less proteases than liver. After labeling with [3H]dexamethasone 21 -mesylate and SDSjPAGE the same 94-kDa receptor band was revealed in both the 8.3-nm and 7.1-nm forms. Immunoblotting experiments showed that both the 94-kDa hormone-binding subunit and the 90-kDa heat-shock protein were present in the two different receptor forms. The 8.3-nm receptor form was converted to the 7.1-nm receptor form after treatment by ribonuclease A in the presence of molybdate and this effect was dose-dependent, being completely prevented by placental ribonuclease inhibitor (RNasin). In contrast, in the presence of molybdate, the 7.1-nm receptor form was ribonuclease-insensitive. Treatment of cytosol with RNase A in the absence of molybdate, partially shifted the untransformed receptor towards the 5.2-nm transformed receptor form. This effect was abolished by placental ribonuclease inhibitor. RNase S protein, an enzymatically inactive proteolytic fragment of RNase A, or S1 nuclease, which is specific for single-stranded nucleic acids, were ineffective when used instead of RNase A. In contrast, cobra venom endonuclease, which preferentially attacks double-stranded regions of small RNAs, caused a complete conversion of the 7 -8-nm untransformed receptor to the 5.2-nm transformed receptor form. These results were not observed in the presence of molybdate. Addition of RNasin prior to heating cytosol in the absence of molybdate did not prevent the receptor from dissociating to the 5.2-nm form, suggesting that an endogenous RNase is not involved in the transformation process. The 7.1-nm receptor form was shifted to a 9.2-nm complex when incubated with an excess of GR 49 antireceptor antibody, whereas the 8.3-nm receptor form did not bind to the antibody. Furthermore, both these receptor forms were shifted when incubated with the monoclonal AC 88 anti-(heat-shock protein) antibody. These results domonstrate that a ribonuclease-sensitive cytosolic factor is closely associated to the receptor protein inside the heteromeric untransformed glucocorticoid receptor. To identify the receptor-associated RNA, the untransformed receptor was purified by protamine sulfate precipitation, affinity chromatography and size-exclusion HPLC. Samples were extracted with phenol and RNA was end-labeled with 32P. Receptor purification led to a specific enrichment in a 120-nucleotide RNA band which could not be detected when a mock purification was performed. Taken together these results suggest tha...

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