Units of transcription and translation: the relationship between heterogeneous nuclear RNA and messenger RNA.

Benjamin LewinCellThe MIT Press28 Carleton StreetCambridge, Massachusetts 02142Considerable uncertainty still exists about the deri-vation of messenger RNA from its presumed precur-sor, heterogeneous nuclear RNA. Because of thelimitations of experiments with eucaryotic systems,several models for messenger production are con-sistent with the available data. As expressed byDarnell, Jelinek, and Molloy (1973), a commonlyaccepted view is that "it appears that mRNA inmammalian cells . . . is derived . . . by posttran-scriptional modification of larger RNA precursormolecules . . . which . . . are part of a class of highmolecular weight nuclear RNA molecules (between5000 and 50,000 nucleotides long)." An alternativeis the iconoclastic view of Davidson and Britten(1973) that "at present we lack reliable knowledgeas to what fraction of hnRNA sequences are presentin mRNA, if any . . . Contrary to some recent claims,there is as yet no hard evidence that any high mo-lecular weight RNA is the precursor of a smallmRNA, or contains within it an mRNA sequence."My intention is to discuss recent evidence on thenature of the units of transcription and translationand in particular to consider whether part of thehnRNA population can be equated with primarytranscripts or intermediate precursors to mRNA.Here I discuss the evidence that relates hnRNA andmRNA, from kinetic data on mRNA production andanalysis of the addition of polyadenylic acid to nu-clear RNA, and consider the limitations that can beplaced on the construction of models for a precur-sor-product relationship between hnRNA andmRNA. Next month I shall consider the analysis ofsequence components present in hnRNA andmRNA and their relationship to the organization ofthe eucaryotic genome (Lewin, 1975).Kinetic Relationship between hnRNA and mRNAA small proportion (less than 5%) of the RNA in thecytoplasm of HeLa cells was first identified asmRNA because it is associated with ribosomes, sed-iments at a size (about 18S) appropriate to codefor proteins, and has a base composition generallyresembling that of DNA (Girard et al., 1965; Lathamand Darnell, 1965). Because preparations of poly-somes may be contaminated with cosedimenting ri-bonucleoproteins derived from some other (per-haps nuclear) source, mRNA cannot be directlyextracted from them but must be purified from thenonmessenger contaminants. Messenger RNA hastherefore usually been identified by the criteria de-veloped by Perry and Kelley (1968) and Penman,Vesco, and Penman (1968): it is released from poly-somes by EDTA in the form of slowly sedimentingribonucleoprotein particles, which can be separat-ed from the contaminating RNP particles that con-tinue to sediment rapidly (for review see Lewin,1974). Puromycin also specifically releasesribosomes from mRNA and may therefore be usedin isolation procedures. Another technique for iso-lating messenger RNA has been developed morerecently from the observation that most of the cellu-lar mRNA population possesses a length of poly(A)at its 31 terminus and may therefore be identifiedby its reaction with oligo(U) or oligo(dT)Almost all mammalian messengers fall within thesize range of 400-4000 nucleotides. The size distri-bution has a median of 1200 bases; Davidson andBritten (1973) showed that it is closely paralleledby the distribution of coding lengths predicted fromthe size distribution of HeLa proteins. The unit oftranslation thus is probably usually monocistronic,a conclusion supported by the properties of the fewspecific messengers that have been isolated (David-son and Britten, 1973; Lewin, 1975).The size distribution of heterogeneous nuclearRNA is very much larger. Over long periods, mostof a radioactive label enters the 45S nucleolar pre-cursor to ribosomal RNA and its subsequent matu-ration products. Pulse labels, or labeled precursorsincorporated in the presence of low doses of actino-mycin which preferentially inhibit nucleolar RNAsynthesis, enter RNA molecules with a heteroge-neous distribution, but apparently very large in size,varying from a maximum of about 50,000 nucleo-tides to a minimum in the range of mRNA sizes.Although the size distributions of hnRNA and mRNAoverlap, only a small proportion of the hnRNA mole-cules is of the same order of size as mRNA whilemost are from 10-100 times longer. Unless only thesmallest hnRNA molecules are implicated, anymodel which supposes that hnRNA provides the nu-clear precursors to cytoplasmic messengers mustimply that the unit of transcription is much largerthan the unit of translation .The hnRNA population appears to turn over veryrapidly within the nucleus and only a small part ofa label in it ever reaches the cytoplasm (see Scher-rer et al., 1970). Although estimates for its half lifevary, they are always low; a half life of 23 min hasrecently been estimated for L cell hnRNA by Brand-horst and McConkey (1974). Because of the rapiddegradation suffered by the hnRNA molecules, anddue to difficulties with chase experiments, it is notpossible to establish any kinetic relationship be-tween the hnRNA population and the cytoplasmicmRNA population. From these experiments it isclear only that at most a small proportion of the