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

Abstract

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...

Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells.

“…5}. When the cross-linked material is digested extensively with ribonuclease A, the protein-oligoribonucleotide adducts usually comigrate with, or migrate slightly behind, the untreated polypeptides on SDS-PAGE {Greenberg 1980; Economides and Pederson 1983;Choi and Dreyfuss 1984;Garcia-Blanco et al 1989). In the case of SF2, the mobility of the labeled adducts appears slightly slower than that of the untreated 33-kD doublet.…”

Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

“…The first described spliceosome proteins were called hnRNP proteins, based on their association with large heterogeneous nuclear RNA transcripts now known to include unspliced pre-mRNAs (6)(7)(8)(9)(10)(11)(12). The C-group hnRNP proteins are abundant nuclear proteins of Mr 42,000-44,000 that have been implicated in splicing (13,14) and are known to be phosphorylated in vivo (8,(15)(16)(17) by a casein kinase II-type activity (18,19).…”

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

“…This is due to the fact that nonspecific interactions between RNA and proteins are likely to occur in vitro (2, 14, 38). UV cross-linking of RNA to protein in intact cells overcomes these difficulties and allows the identification of proteins which are in direct contact with hnRNA and mRNA in vivo (10,34,35,50,51,53).The in vivo UV cross-linking approach relies on the fact that UV light of sufficient intensity generates photoreactive species of RNA which react virtually indiscriminately with molecules, including proteins, which are in direct contact with it. Cross-linked complexes of polyadenylated [poly(A)+] mRNA were isolated under protein-denaturing conditions to ensure that only proteins covalently linked to mRNA were purified with it, eliminating nonspecific associations of proteins with mRNA (51, 53).…”