Nuclear RNA - Protein Interactions Messenger RNA Processing

Eucaryotic messenger RNA precursors are processed in nuclear ribonucleoprotein particles (hnRNP). Here recent work on the structure of hnRNP is reviewed, with emphasis on function. Detailed analysis of a specific case, the altered assembly of hnRNP in heatshocked Drosophila and mammalian cells, leads to a general hypothesis linking hnRNP structure and messenger RNA processing. A unifying principle emerging from the modern era of biology is the realization that cellular processes can be understood in terms of chemical binding equilibria among macromolecules, such as those between nucleic acids and proteins. Here I review recent progress in the area of eucaryotic messenger RNA (mRNA) ~ biosynthesis, with particular emphasis on nuclear RNA-protein interactions involved in mRNA processing. Eucaryotic genes that code for mRNA are copied by RNA polymerase II into transcripts collectively termed heterogeneous nuclear RNA (hnRNA). The great majority of these nuclear transcripts undergo subsequent covalent modifications, through which some (but, importantly, not all) are converted into mRNA (1). The posttranscriptional modifications of hnRNA include addition of inverted guanosine nucleotides ("caps") at the 5' termini of most transcripts, specific base and ribose methylations, 3' processing followed by addition of poly(A) sequences at the 3' ends of some hnRNA molecules, and the excision of intervening DNA sequence transcripts followed by ligation of the mRNA sequences (splicing). In addition to these covalent modifications of the RNA transcript, another important step in the maturation of mRNA is the assembly of heterogeneous nuclear RNA into This invited article is based on a lecture presented in the Symposium on "Transcription, RNA Processing, and Nuclear Structure" at the 22nd Annual Meeting of the American Society for Cell Biology, Baltimore, Maryland, November 30-December 4, 1982, and is paper No. 29 in a series entitled "Ribonucleoprotein Organization of Eucaryotic RNA." The author dedicates this paper to Harry Eagle, a pioneer of cell biology, on the occasion of his 78th birthday. ~ Abbreviations used in this paper: hnRNA, heterogenous nuclear RNA; hnRNP, ribonucleoprotein complexes containing heterogeneous nuclear RNA; mRNA, messenger RNA; mRNP, mRNAprotein complexes; RNP, ribonucleoprotein. ribonucleoprotein complexes, termed hnRNP particles (2). This begins while the transcript is still a nascent RNA chain (3-5). As 3'-OH poly(A) addition obviously cannot occur on nascent hnRNA chains (elongation proceeding 5' ~ 3'), and because poly(A) addition normally takes place before splicing (6), it follows that hnRNP assembly precedes both of these mRNA processing steps (7). The central question is the functional significance of hnRNP particles. There are two extreme possibilities, which are not mutually exclusive. One is that hnRNP is simply a metabolically inert packaging device, involving a regular array of stable hnRNA-protein contacts analogous to the nucleoprotein organization ofchromatin or viral nucleocapsids. The other possibility is that hnRNP particles reflect dynamic interactions of proteins at specific hnRNA sites related to mRNA processing, for example splicing. The former view of hnRNP has prevailed for many years, but recent evidence now points to the latter possibility. The distinction comes down to determining the extent to which the structure of hnRNP is nucleotide sequence-specific (8). Historical Perspective The study of hnRNP complexes has proceeded concurrently since about 1960 in the theaters of both cytology and biochemistry. Several reviews of both aspects of hnRNP research have been published recently (2, 9-12). The focus of the present article is on current and future directions in the field, for which the following synopsis is intended to serve as a background. The idea that eucaryotic gene transcripts exist in the cell as nuclear RNP particles arose from cytological studies of meiotic prophase ("lampbrush") chromosomes in amphibian oocytes. Rapidly labeled nascent hnRNA (13, 14) on the lateral loops of DNA was observed to be particulate (15), and THE JOURNAL OF CELL BIOLOGY • VOLUME 97 NOVEMBER 1983 1321-1326 © The Rockefeller University Press • O021-9525183111/1321106 $1.00 "1321 on July 9, 2017 jcb.rress.org D ow nladed fom the morphology of these RNP particles was seen to vary substantially from one loop to another (16). Subsequently, the RNP form of hnRNA has been confirmed and further detailed through ultrastructural studies (17-19), including the analysis of nascent hnRNP particles on chromatin spread from lysed nuclei by the procedures developed by Miller (see references 20-30). The biochemical isolation of nuclear RNP particles containing hnRNA was pioneered by G. P. Georgiev and colleagues (31) in the Soviet Union. They showed that if rat liver nuclei are incubated in an isotonic buffer at pH 8.0, a major fraction of the rapidly labeled nuclear RNA is extracted in the form of 30S RNP complexes (31). The metabolic instability of this particle-associated RNA fraction and its DNAlike base composition led the Moscow group to conclude correctly that the particles contain pre-messenger RNA. The 30S RNP complexes were termed "informofers" ("information bearers"), to contrast them from the cytoplasmic messenger RNA-protein complexes (mRNP) that Spirin had described several years earlier, which had been named "informosomes" ("information bodies"). The important work of the Moscow group on nuclear hnRNP and cytoplasmic mRNP, published mainly between 1965 and 1970, has been recently reviewed in detail (10, 32).