Globin mRNA in Ffsriend cells: its structure, function and synthesis

347

Biochimica et Biophysica A cta, 605 (1980) 347-364 © Elsevier/North-Holland Biomedical Press

BBA 87084

GLOBIN m R N A IN FRIEND CELLS: ITS S T R U C T U R E , F U N C T I O N A N D SYNTHESIS PETER J. CURTIS * The Wistar Institute o f Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104 (U.S.A.J (Received September 3rd, 1979)

Contents I.

Introduction

II.

Structure of the adult globin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

............................................

347 348

III.

Synthesis of globin mRNA precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

IV.

Processing of the nuclear precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

353

V.

Structure and function of globin mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. 5' Noncoding region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. 3' Noncoding region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Coding region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Secondary structure of globin mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355 355 356 356 357

VI.

Stability of globin mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

358

VII.

Kinetics ofglobin mRNA induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

359

VIII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

359

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

360

References

360

.................................................

I. Introduction T r e a t m e n t o f m o u s e e r y t h r o l e u k e m i c (MEL) cells w i t h a variety o f c o m p o u n d s , such as d i m e t h y l s u l p h o x i d e , butyric acid, h a e m i n , results in a series o f changes characteristic for the differentiation steps leading to m a t u r e o r t h o c h r o m a t i c erythroblasts. The m o s t striking feature is the synthesis o f globin m R N A f o l l o w e d by the a c c u m u l a t i o n o f n o r m a l m o u s e adult h e m o g l o b i n up to 20% o f the cell protein. In this review we shall limit ourselves to a description o f the structure o f m o u s e adult globin genes, their transcription, the processing o f the transcript, and the structure, f u n c t i o n and stability o f globin m R N A * Formerly of the Institute for Molecular Biology, University of Zurich, Zurich, Switzelland. Abbreviations: MEL cells, mouse erythroleukemic ceUs; SV40, simian virus 40; HMBA, hexamethylene bisacetamide.

348 in induced MEL cells, but we shall also include data obtained from other systems such as mouse foetal liver cells and mouse anaemic spleen cells to extend and to compare with the data derived from induced MEL cells. We shall also refer to data on globin mRNA from rabbit and human so that we can make inferences concerning the role of specific sequences. II. Structure of the adult globin genes Several mouse strains have at least one (possibly two) adult a-globin genes and two fl-globin genes, fl major and fl minor [ I - 4 ] , which are expressed in the induced MEL cells [5,6]. The structure of the globin genes has been determined following the cloning of appropriate chromosomal DNA. For cloned chromosomal DNA containing fl-globin sequences, two separate clones were characterised on the basis of restriction analysis followed by sequence analysis which identified the clone XgtWESMflG2 as the gene for fl major and by implication XgtWESMflG3 as the gene for/3 minor [7-10]. The two adult /3-globin genes are each interrupted by two intervening sequences of DNA, a small sequence of 116 base pairs at the codons for amino acids 30-31 and a larger sequence of 650 base pairs at the codons for amino acids 104-105, as shown by restriction analysis, R loop formation with globin mRNA and sequence analysis. The rabbit/3-globin gene also contains two intervening sequences at identical positions [11,12], an arrangement which is found in the DNA from several different rabbit tissues both erythroid and nonerythroid. Intervening sequences have also been found in adenovirus [13-15], SV40 [16], mouse immunoglobulin [17], yeast tRNA [18,19] and chick ovalbumin [20,21 ], and this appears to be a common feature of eukaryotic gene organisation (see Ref. 22 for review). With respect to the surrounding sequences of the closely linked/3-globin genes, heteroduplex mapping revealed that/3 major and/3 minor had homologous sequences only in the globin mRNA sequence with perhaps approx. 100 additional nucleotides at the 5' and 3' ends as well as the 5' half of the large intervening sequence [10]. It was suggested that divergence of sequences surrounding the /3-globin genes would reduce the possibility of recombination between the two genes a.fter their appearance by gene duplication. There is another important result with regard to the fl-globin gene structure by Miller et al. [23]. They showed that the large intervening sequence was found only in the fl-globin gene and no other place in chromosomal DNA. We will discuss possible function or relevance of the intervening sequences later. Genetic studies have established that the a-globin gene is located on chromosome 11, while/3 minor and/3 major are closely linked on chromosome 7. A lepore type/3 chain has been characterised in a species of mouse (Mus musculus caroli) that contains at its 5' end /3 minor sequence and/3 major at the 3' end [24] so that it is assumed that/3 minor gene precedes fl major in the direction of transcription. Such an arrangement corresponds to that found for the human/3-like genes,/3- and 6-globin, both by genetic data [25,26] and more recently, by the physical linkage, inter-gene distance and relative orientation of the 6- and/3-globin genes, as has been determined by cloning [27,28]. More recently the a-globin gene has been cloned [2,4]. a-Globin sequences were detected in three different locations in chromosomal DNA. By a combination of R loop formation with globin mRNA and sequence analysis, two intervening sequences have been located in positions that are homologous with respect to their amino acids. The intervening sequence corresponding to the large sequence of/3-globin is located between amino acids 9 9 - 1 0 0 and is approx. 150 base pairs, and the other somewhat smaller intervening

349 sequence is located between amino acids 3 1 - 3 2 . The occurrence of intervening sequences at the same sites in the a-globin and/3-globin genes suggests that these sequences arose before the globin gene is thought to have undergone its first duplication which occurred at approx. 500 million years at a point early in vertebrate evolution. The globin genes are coordinately expressed in normal erythropoiesis and in induced MEL cells. The presence of sequences common to all globin genes might provide a possible mechanism to explain their coordinate expression, and such homologous regions might be visualised by heteroduplex mapping. Comparison of a-globin with/3 major by heteroduplex mapping showed a 165 base pairs segment of homology located approx. 1.5-2 kilobases from the 3' end of both coding sequences. No extensive homology was found between a-globin and/3 minor genes. However, the significance of this homology remains to be determined. Genes which are actively transcribed show an increased sensitivity to pancreatic DNAase I digestion of isolated nuclei, for example, globin genes in nuclei from erythroid cells are more sensitive than those in nuclei from nonerythroid cells [29]. Since no difference between active and inactive genes could be detected with micrococcal nuclease, it was assumed that DNAase I sensitivity reflected a change at a higher level of chromatin organisation than the nucleosome level. The globin genes in uninduced, induced and noninducible MEL cells showed the same increased sensitivity to DNAase I as active genes [30]. Such results imply that the rearrangement of the chromatin structure is necessary for globin gene transcription, but is not the only factor. III. Synthesis of globin mRNA precursors The accumulation of globin mRNA during induction of MEL cells or differentiation of normal erythroid cells could occur in a number of different ways. Control of the rate of transcription is considered to be a primary mechanism for regulating mRNA and evidence supporting this model has come from studies of globin RNA in induced MEL cells [ 3 0 34]. Gene amplification is involved in the accumulation of ribosomal RNA in Xenopus laevis [35], but it has been ruled out in the case of globin genes in MEL cells and normal erythroid cells [36]. Regulation of mRNA concentration could also occur by changes in the rate of post-transcriptional processes, e.g., rate and efficiency of processing of the primary transcript and transport of mRNA to the cytoplasm or by changes in the stability of the mRNA populations. We will now describe the evidence to indicate the contributions of various mechanisms for globin gene expression. A number of early reports dealt with the detection of globin-specific sequences in RNA sedimenting above 30 S in duck erythroblast nuclear RNA [37] and mouse foetal liver RNA [38]. In addition, Macnaughton et al. [39] identified globin-specific sequences in duck erythroblast nuclear RNA sedimenting at 14 S. These early studies were performed on unlabeled RNA using more or less pure labeled globin cDNA probes or by assaying for the capacity to direct globin synthesis in a protein-synthesising system. Such approaches, however, cannot establish a precursor-product relation between nuclear RNA and mRNA as they examine only the steady-state level. Examination of RNA from cells labeled for periods that are short compared with the half-life of the precursor to a large extent avoids the problem of aggregation as there is less contamination of high molecular weight RNA with labeled mature mRNA. Furthermore, it is possible to determine the rate of synthesis and degradation of the precursor, to perform pulse-chase experiments to establish a precursor-product relation and to charac-

350 terize the precursor with respect to its sequence arrangement. Recent studies using pulse-labeled RNA from MEL cells [40,41], as well as mouse foetal liver cells [42] and mouse anaemic spleen cells [43] have identified a 15-16 S RNA precursor specific for/3-globin mRNA and an 11 S RNA for ct-globin mRNA [44, 45]. In addition a 27 S RNA has been isolated from MEL cells using globin cDNA-cellulose [41 ] (see also Ref. 46). Competition hybridization and pulse-chase studies suggested that the 27 S RNA was indeed a precursor to globin mRNA. These results have been challenged by Haynes et al. [47] who also detected in induced MEL coils 27 S, 16 S and 10 S RNA that hybridised to globin cDNA-cellulose. Hybridisation of these RNA species to mouse a- and/3-globin cDNA containing plasmids demonstrated that 10 S RNA annealed to both 0~- and /3-globin cDNA-plasmid, 16 S RNA annealed to ~-globin cDNA-plasmid, while 27 S RNA annealed to neither the a- nor the/3-globin cDNA plasmid. It is thought that the 27 S RNA hybridizes to globin cDNA-cellulose because the globin mRNA used to synthesise globin cDNA-cellulose was contaminated by other RNA species. Convincing evidence for a precursor-product relationship between two RNA species is difficult to obtain in animal cells. One approach is to label cells for a short period with a radioactive nucleoside, then prevent further incorporation of radioactivity as completely as possible either by addition of a large excess of unlabeled nucleoside or by use of an inhibitor of RNA synthesis. Labeled product should then accumulate while the putative precursor diminishes. Strictly speaking, less labeled nucleoside should be incorporated into total RNA than appears in the product. This condition is difficult to attain if the product is only a minor fraction of the RNA synthesised, since addition of unlabeled nucleoside is usually insufficient to completely prevent incorporation of labeled nucleoside triphosphates present in the intracellular pool or arising by turnover of short-lived RNA species. With complete cessation of RNA synthesis or incorporation into RNA, one should in fact observe a decrease in amount of labeled RNA, as over 90% newly synthesised RNA is degraded with a half-life of approx. 20 min. Though inhibition of RNA synthesis by actinomycin D is quite effective, the drug can cause other changes in RNA metabolism [48,49]. Bastos and Aviv [41] working with induced MEL cells and mouse anaemic cells, showed that the 15 S RNA species behaved as a precursor to mature 10 S /3-globin mRNA in a pulse-chase experiment, the estimated half-life of 15 S RNA was about 5 min. In a different approach, Curtis et al. [44,45] quantified the amounts of labeled 15 S globin RNA and 10 S/3-globin mRNA by labeling induced MEL cells for different time periods. The 15 S globin RNA had reached its steady-state level by the shortest labeling time, 5 min, while 10 S/3-globin mRNA accumulated for at least 1 h (Fig. 1). From these kinetic studies it was calculated that the 15 S RNA is synthesised at a rate of 6.6 molecules/min/cell, compared with 5.4 molecules/min/cell for 10 S/3-globin mRNA (Table I). Therefore, a precursor-product relationship is possible. The estimated half-life of the 15 S /3-globin RNA was 2.5 min or less and the steady-state level was calculated to be 24 molecules/min/cell. Very similar values have been obtained by Ross and Knecht [50] for the 16 S globin precursor in mouse foetal liver cell indicating that the induced MEL cell behaves very similarly in this respect to normal erythroid cells. In addition Lowenhaupt et al. [34] have shown that globin RNA precursors in uninduced cells are processed to mature mRNA in the same way as in induced cells, so that the increased level of globin RNA in induced cells is the result of increased transcription of the globin genes and not of changes in post-transcriptional processing. Further support for this conclusion has been obtained from the labeling of globin transcripts in isolated nuclei from uninduced and induced cells [33].

351 ._I

/

•

lOs

o.

500

d z z

,

100

0 .J (.9

/

7~

..1 hi "~

50,

_J

15s o

z

3'0 TIME ( m i n )

6'0

Fig. 1. Accumulation of 15 S and 10 S-labeled #-globin RNA in dimethylsulphoxide-induced MEL cells. The values were calculated from the data presented in Curtis et al. [42].

The results described above were obtained using sucrose gradients to resolve the different species. Utilization of 100% formamide gels to detect globin-specific sequences has greater resolution and allows a more accurate estimate of the molecular size of the RNA species. With mouse a- and/3-globin cDNA plasmids, Farace et al. [51 ] were able to detect B-globin-specific sequences with molecular weights of 0 . 6 - 1 0 6 (corresponding to 1 5 16 S species detected earlier), 0.34 • 106, 0.28 • 106, 0.26 • 106, and 0.2 • 106 in nuclear poly(A)-RNA from induced MEL cells. The last species is the size of mature /3-globin mRNA. t~-Globin-specific RNA was detected with molecular weight of 0.26 • 106 (presumably corresponds to 11 S species) and 0.21 • 106 which is the size of mature ~-globin mRNA. In poly(A)--RNA no globin-specific species were detected. The 15 S ~-globin precursor is largely polyadenylated [41,50]; in mouse foetal liver the p o l y ( A ) i s 150 nucleotides. Examination of the 5' terminus of the 15 S/3-globin precursor [45] revealed only the presence of a 'cap' structure which behaved identically to one of the 'cap' structures, 'cap' 1, isolated from mature/3-globin m R N A [52,53]. 'Cap' 1 TABLE I PARAMETERS OF B-GLOBIN RNA SYNTHESIS The values below were calculated from the data of Curtis et al. [42]

Rate of synthesis

15 S

10 S

6.6

5.4

(moleCules/min/cell) tl/2 Steady state level (molecules/cell)

2.5 rain 24

17 h 10 000

352 and 'cap' 2 have the structures mTGpppm6AmpC and mTGpppm6AmpCm-pN, respectively. Fingerprinting of the 15 S /~-globin precursor after RNAase T1 digestion revealed, in addition to oligonucleotides present in t3-globin mRNA, about 20 oligonucleotides not found in the mature mRNA; many migrated in a manner expected of U-rich oligonucleotides. Analysis of a-globin RNA purified from short-term labeled cells showed six oligonucleotides in addition to those found in mature a-globin mRNA [45]. The location of the additional sequences of the 15 S 13-globin precursor of MEL cells has been determined. The finding of intervening sequences in/3-globin gene implied either that RNA polymerase could jump across DNA sequences (and then the additional sequences would be found at either the 5' or 3' end) or the 15 S 13-globin RNA precursor was a transcript of the ~-globin gene including the intervening sequences. This latter possibility has been shown to be the explanation. The 15 S ~-globin RNA precursor was purified from induced MEL cells and hybridized to the cloned chromosomal/3-globin DNA under R loop-forming conditions [54]. The R loop showed a single continuous doublestranded structure compared with that seen with globin mRNA. The 3' end of the doublestranded region of the R loop mapped at the same position as the 3' end of globin mRNA Rloop, but the 5' end extended approx. 100 nucleotides beyond the 5' end of the mRNA R loop, the additional length corresponding in size to the small intervening sequence. The 15 S /3-globin RNA precursor from mouse foetal liver [55] and anaemic spleerL cell [56] also contained the two intervening sequences. From the sequence analysis of the cloned chromosomal t3-globin DNA [57] the 15 S 13-globinprecursor RNA including the poly(A) tail should be 1600 nucleotides long, assuming that the 5' and 3' ends are identical with the mature mRNA. This value is in agreement with the estimate of its size from sedimentation in a sucrose density gradient, 1500 nucleotides. A somewhat larger estimate of 1800 nucleotides has been obtained for the 15 S 13-globin precursor RNA derived from formamide gels [50,51]; such a result would imply the presence of extra sequences in addition to the two intervening sequences. Support for such extra sequences in the 15 S 13-globin RNA comes from the R loops formed between the 15 S 13-globin RNA (from mouse foetal liver ceils) and i3-globin cDNA-Ch3A XDNA [55]. In addition to the R loop structure there was a single-stranded loop (indicating the large intervening sequence) located in the middle of the RNA-DNA hybrid and also a tail extending from the 5' end. However, the size measurements are compatible with the possibility that the 5' end of the 15 S t3-globin RNA sequence was displaced by branch migration promoted by the presence of the small intervening sequence in this region. More recently, Weaver and Weissmann [58] have shown that the 5' termini of the 15 S/3-globin RNA and mature 10 S 13-globin mRNA map identically on the/3-globin genomic DNA, indicating that the 15 S species is processed to the mature mRNA without further modification of the 5' end. The evidence to date supports the model that the 15 S ~-globin RNA has only the two intervening sequences in addition to the mature mRNA sequences. In adenovirus 2 late mRNAs [13-15], SV40 late mRNAs [16,59] and ovalburnin mRNA [60] the 5' leader RNA originates from a DNA sequence distant from the coding sequences. This situation does not seem to apply to t3-globin mRNA.' The presence of a 'cap' rather than a triphosphate at the 5' end might be taken to suggest the 15 S ~-globin RNA is not the primary transcript. The precursor of adenovirus late mRNA also has been detected with a 5' terminal 'cap' structure when labeled in vivo and in isolated nuclei [61,62]. Transcription might begin at the 5' end of mRNA, and 'capping' could be extremely rapid; inhibitors of the 'capping' process might slow down

353

the reaction sufficiently to allow us to detect intermediates in its synthesis. Another possibility is that 'capping' is the initiating step, evidence for which has been found at least for the synthesis of reovirus mRNA [63]. It is worth noting that the 15 S/3-globin RNA contained only 'cap' 1, and no other 5' terminal structure that could have been an intermediate, even though this RNA is very rapidly processed [45]. Alternatively, transcription might begin at a position distant from the 5' end of/3-globin mRNA, but processing is so rapid that no intact primary transcript exists. There are two approaches that would define that site of initiation of the primary transcript. One method could use ultraviolet inactivation to define the transcriptional unit as Goldberg et al. [64] have defined the transcriptional unit of the late mRNAs of adenovirus 2 virus. Alternatively the cloned chromosomal /3-globin DNA could be used to probe for transcripts from the 5' end preceding the 5' end of the mRNA [65]. IV. Processing of the nuclear precursors

It is not known what processing steps, if any, precede the appearance of the 15 S /3-globin RNA, i.e., whether there are cleavage or splicing events removing sequences at the 5' and 3' end. In adenovirus, transcription proceeds beyond the sequence to which poly(A) is added. Nucleolytic cleavage and poly(A) addition occurs rapidly (within 1 min) after transcription passes a potential poly(A) site. Since many nuclear transcripts do not possess poly(A), the decision to add poly(A) could be a critical regulatory step in cell mRNA biosynthesis [66]. Removal of the intervening sequences presumably occurs by the process of splicing, as described for adenovirus 2 late mRNA [13-15], which requires cutting the RNA at two precise places, followed by ligation of two ends. A number of/%globin RNA species have been identified in the nucleus of induced MEL cells, corresponding in size to 1850, 1030, 850, and 770 nucleotides [51]. Some of these species have been identified also in mouse foetal liver cells [50] and presumably are intermediates of processing to mature mRNA. Only the largest species could contain the large intervening sequence, so we assume it to be the first to be removed (Fig. 2), followed by the small intervening sequence and any other sequence that may be present at or near the 5' end. In fact the results of Kinniburgh and Ross [67] indicate that the large intervening sequence is removed in at least two steps as they detected/3-globin RNA of 1030 nucleotides which still possessed two intervening sequences. COOH

NH 2

30

i l

31

pvsq

104

I

105

1

I

I

ivs2 t

AAAAAS'OH

5' COp I

-.~.---

5 ~COPl - -

5' cop!

~

l

A A AAA 3'0 H

AAAAA3'OH

Fig. 2. Synthesis and processing of #-globin mRNA. The initial steps of synthesis have not been identified. The first detectable product is 15 S #-globin RNA precursor. Removal of the large intervening sequence IVS2 from the 15 S precursor before IVS1 because the next largest nuclear globin species is not large enough to contain IVS2.

354 The original model for eukaryotic mRNA processing [68] proposed that the extra sequences determined the subsequent expression of an mRNA, implying an important rate limiting role for post-transcriptional processing, and the detection of globin RNA sequences in nonerythroid ceils has been taken to provide evidence supporting this role [69]. However, the ratio of labeled 15 S and 10 S /3-globin RNA in uninduced and induced MEL cells, labeled for a very short time remained unchanged [34]. No evidence has appeared to explain how splicing occurs in induced MEL cells. An activity has been detected in yeast capable of cleaving and rejoining yeast tRNA [70,71]. It has been suggested that splicing of RNA could involve the formation of base-paired structures between opposite ends of the intervening sequence and/or adjoining coding sequences to bring into close proximity the ends of the coding sequences with looping out of the intervening sequences occurring in one or several steps [72,73]. Undoubtedly the enzymes responsible for such a precise operation have specific substrate requirements. Though there may be more than one enzyme recognition system we assume that there is not one for each mRNA. Recognition of the signals appears to be a general property of animal cells, e.g., splicing signals appear to be recognised by Xenopus oocytes on SV40 RNA [74]. To search for common sequence features, we have available sequences of mouse and rabbit /3-globin DNA [12,57], as well as immunoglobulin [73], ovalbumin [75] and SV40 [76]. From the ovalburnin sequences Chambon [75] noted a sequence at the 5'.end TCAGGGTA and at the 3' end TXCAGG, with invariably GT at the 5' end and AG at the 3' end of the intervening sequences. The sequences for the two intervening sequences for mouse and rabbit fit this rule (Fig. 3) [12], the intronexon junction of yeast tRNA does not. For globin there is three and four base repeat at the junctions so that the exact site of excision cannot be unambiguously defined. There is six to nine base homology at the 5' end between mouse and rabbit, but beyond there is considerable divergence of sequence. There is no exact homology or significant secondary structures for recognition of the splicing enzyme, the sequences as defined by Chambon's rule clearly are not sufficient. Long range secondary and tertiary structure might, in combination with the nucleotide sequences defined by Chambon's rule, contribute to a recognition site [77]. In fact, it is striking that the mouse and rabbit DNA sequences show exact homology extending for approx. 50 nucleotides at the site of the intervening sequences. There is no other extensive homology between the mouse and rabbit ~-globin DNA sequences. Insertion of rabbit globin coding sequence into different sites in the SV40 late region demonstrated that SMALL IVS AIo

Leu Gly

30 Arg

31 Leu Leu

Vol

M GCC CTG GGC AG~'-~TGGTATCC AGG......... CCCTTTTT['~G CTG CTG GTT I I I I R

GCC CTG GGC AGIGTITGGTATCC T T T ......... A T T T T C T C I A 6 1 6 CTG CTG GTT

LARGE IVS Glu

104 Ash Phe Arg

105 Leu Leu Gly

M

GAG AAC T TC AGG [ ~ ' ] G A G T C TGAT .......... TAT TCCCAC ~ - - ~ CTC CTG GGC

R

GAG AAC TTC AGG G[~.JGAGTTTTGG ......... TTTTCCTAC ~

CTC CTG GGC

Fig. 3. Comparison of the nucleotide sequences o f the introns of the mouse (M) and rabbit (R) chromosomal t3-globin genes at the junctions with the coding regions found in eukaryotic mRNAs as observed by Breathnach et al. [70]. The boxes indicate the invariable sequences.

355 expression of the cDNA gene into a stable translatable mRNA required the retention of splicing signals in the nuclear transcripts [78,79]. Insertion of the mouse chromosomal fl major giobin gene into the SV40 late region also resulted in the production offl-globin polypeptide in monkey kidney cells, suggesting that RNA splicing and polyadenylation signals are widely recognised and expression of globin genes is not dependent on the presence of enzymes that specifically process globin mRNA [80]. The strong evolutionary divergence of the large intervening sequence observed between mouse and rabbit [12], mouse fl major and fl minor [10], and human/3 and 6 [28], indicate that if the intervening sequences have any function, it is not dependent on a unique sequence. A number of suggestions have been made to explain their presence in terms of evolutionary significance. It has been proposed that intervening sequences may have increased recombination and the appearance of new proteins [81], possibly by bringing together domains that exist in a protein from different proteins [82]. Intervening sequences have been found in a wide range of eukaryotes and in a variety of genes in tRNA, rRNA and genes for specialised proteins. It will be interesting to know whether these sequences are found in all eukaryotes and all genes or if they are more restricted.

V. Structure and function of globin mRNA As was noted earlier, the MEL cells, upon induction, produce adult a- and fl-globin chains that are identical to those produced by the strain of mouse from which the cells were derived. We presume then that the globin mRNAs are identical (see Ref. 40 for partial sequence analysis of globin mRNA derived from MEL cells). For the mouse, the 5' noncoding regions of a, fl major and fl minor globin mRNA has been determined by RNA sequencing techniques [83], and the complete sequence for fl major and fl minor globin RNA has come from DNA sequencing of the cloned mouse chromosomal fl-globin gene [57] and cloned mouse fl-globin cDNA (Mantel, N. and Weissmann, C., unpublished data). In addition, we can compare these sequences with ones determined for rabbit aand fl-globin mRNA [84-88] and human fl-globin mRNA [89], to demonstrate where sequences are conserved and presumably under selective pressure and those sequences which have diverged.

VA. 5' Noncoding region A comparison of these regions of mouse, rabbit and human shows that the sequences are to a large extent conserved. Lengths vary little, for fl-globin of mouse, rabbit and human they are 53, 54, 51 nucleotides, and there is a smaller size for the a-globins, range 3 2 - 3 7 . However, for different mRNAs from various sources, there is a wide range of lengths from nine nucleotides in BMV to 94 in TMV genome RNA [83]. These regions in a- and fl-globin contain some homology in particular a sequence CUUCUG and AGAAACAGA, but which is not found in a number of other sequenced mRNA except that the purine rich sequence is found in ovalbumin mRNA and the pyrimidine sequence in SV40 VPI and TYMV genome RNA. Conservation of sequence by fl-globin mRNA from different species suggests considerable selective pressure. It has been noted that this region can potentially form extensive secondary structure which may be strictly conserved because of a requirement in initiation of protein synthesis [87,88]. The rules derived by Tinoco et al. [90] are normally used to predict secondary structure. Since we expect secondary structure to play an important role in the functioning of

356 mRNA, it is necessary to determine by direct experiments the exact structure present in mRNA and in its corresponding mRNP, as binding proteins might affect considerably the structures. Shine and Dalgarno [91] have suggested that the 5' noncoding region of eukaryotic mRNA might interact with the 3' end of 18 S rRNA, as in prokaryotes (see Taniguchi and Weissmann [92] for evidence of a role for such an interaction in prokaryotes). There would appear not to be a conserved sequence in the 5' noncoding sequence, except for the initiation codon where the AUG might bind to 3' UAC 5' at the 3' end of 18 S rRNA. An alternative model could involve a purine sequence, eight nucleotides from the 3' end, 3' GAAGGC 5' which could base pair well with 5' CUUPyUG 3'. However, there are many mRNAs that do not possess this sequence. Binding studies of mRNA to ribosomes followed by RNAase digestion have shown that the AUG is protected together with 2 0 30 nucleotides to its 5' end [93-95]. The 'cap' is not protected by ribosome binding in the case of rabbit/3-globin mRNA though data suggest that the 'cap' is a requirement for initiation of translation in vitro [96,97].

VB. 3' Noncoding region This region shows more diversity in size on comparing mouse, rabbit and human /3-globin mRNA (92, 130 and 131 nucleotides, respectively). Earlier it had been noted that this region could be divided into two halves; the proximal half showed extensive divergence, while the distal half was quite homologous [86,87,98]. The sequence derived from mouse /3major globin DNA also fits this pattern and contains the sequence AAUAAA so far found in most eukaryotic 3' mRNA regions sequenced to date, except sea urchin H2A, H3 histone mRNA [99], suggesting that this sequence is a signal for polyadenylation. The highly divergent proximal half presumably has no specific function; one is uncertain how to explain the conservation found at the 3' end. Rabbit globin RNA, which lacks the 3' noncoding region, is translated efficiently [100]. No evidence exists to tell us where termination of RNA synthesis occurs; the characterised precursors exist only in the polyadenylated state [51 ], except transcription of adenovirus late mRNA continued beyond the polyadenylation site [66]. It is possible that a splicing or cleavage event might be necessary. Van den Berg et al. [12] have noted that the sequence surrounding a splicing junction is highly conserved. At least 90% of globin mRNA contains poly(A) at its 3' end in MEL cells. Poly(A) addition occurs very rapidly after transcription to give a rather uniform size of 150 [50], which undergoes stepwise degradation in the cytoplasm [101,102]. The fact that cordycepin (3' deoxyadenosine) blocks both polyadenylation and transport to cytoplasmic polyribosomes suggested that poly(A) played an essential role in processing [103]. The poly(A) is not essential for translation as deadenylated globin mRNA functions equally well as globin mRNA [104,105]. However, upon injection into frog oocytes, deadenylated globin mRNA is destroyed more rapidly [106], indicating that poly(A) may have a role in stabilising mRNA. These results, however, contrast with the result that polyadenylation made no difference to translation of interferon mRNA in frog oocytes [107].

VC. Coding region The complete sequences of mouse, rabbit and human j3-globin mRNA have been determined and comparisons have been made [57,87,88]. The overall conclusions are that the

357 coding sequences remain closely related in these three species, corresponding approximately to the amino acid homology. There are variations noted within this region; in particular, the sequences coding for those amino acids considered to be the functionally most important and evolutionarily most conserved, i.e., those involved in haem interactions, etc., are significantly deficient in silent substitutions. This conservatism must result from, in addition to the constraint imposed by coding for functionally important amino acids, requirements for secondary structure of the globin mRNA. The longest blocks of conserved amino acids also partially overlap the junctions where splicing occurs. In both cases homology extends beyond the sequences coding for the functionally important amino acids, suggesting that a specific secondary structure may be required for the splicing events. The mouse mRNA coding sequence shows the nonrandom use of synonymous codons, which has been noted for prokaryotic mRNAs as well as several eukaryotic mRNAs. The distribution in mouse ~-globin corresponds closely to that found in rabbit and human ~-globin [ 108], but is significantly different in rabbit a-globin, immunoglobulin, insulin (see Ref. 88). A number of explanations have been offered. Selective translation would occur by the availability of tRNA [109], though such control cannot be absolute since mRNAs from diverse sources can be translated by a particular in vitro protein synthesising system [110]. Alternatively differentiated cells might selectively adjust their rates of production of tRNAs to acquire a tRNA complement appropriate for the synthesis of specific proteins [111]. The abundance of various tRNAs in rabbit reticulocytes correlates with the ratios of the corresponding amino acids in globin [ 112].

VD. Secondary structure of globin mRNA Globin mRNA from mouse, rabbit and duck erythroid cells has been examined for content of secondary structure [113-115]. Thermal denaturation and fluorimetric titration with ethidium bromide have shown that globin mRNA contains approx. 50% of its bases in helical structures. The evidence to date suggests that globin mRNA possesses a somewhat unique arrangement that is recovered after heat denaturation. However, no direct studies have been made to characterise further structures that may be present in solution, i.e., by partial digestion and mapping of exposed sequences, or chemical crosslinking. From the determined sequences of rabbit a- and ~-globin mRNA, secondary structures have been proposed [87,88], based mainly on the rules of Tinoco et al. [90]. Direct experiments are required to determine the validity of the proposed structures. Ribonucleoprotein particles containing globin mRNA from rabbit reticulocytes and duck erythrocytes have been characterised [116-118]. These mRNP particles contained only two or three polypeptides with mol. wt. 78 000 and 52 000 in rabbit and duck, in a phosphorylated state to give a structure sedimenting at 15 s. The poly(A) of globin mRNA appears to bind the 78 000 polypeptide. Two roles have been proposed for the RNP, (a) to confer stability on the mRNA, implying that the polypeptides would be responsible for the half-life of mRNA, (b) to complement translation initiation. Civelli et al. [119] have shown that chick erythrocyte 15 S RNP, which can be isolated from polyribosomes is as active as 9 S globin mRNA in an in vitro protein synthesising system, while 20 S RNP, which is found free in the cytoplasm, is completely inactive and is, in fact, inhibitory for other mRNAs added to the same incubation. Such results suggest a role of proteins providing translational control for mRNAs.

358 VI. Stability of globin mRNA The way in which accumulation of globin mRNA in induced MEL cells occurs can help us to understand the specific enrichment of a mRNA in a differentiated cell. We have already described that stimulation of globin gene transcription occurs coupled with depression of total cell RNA synthesis. The level of globin mRNA synthesis, however, is still very low approx. 0.01% [45]. Post-transcriptional processing of newly transcribed globin RNA occurs at a much faster rate, tl/2 of the 15 S precursor globin RNA approx. 2 rain [44,50] compared with total nuclear RNA with tl/2 approx. 20 rain [120] of which less than 10% leaves the nucleus. Clearly selection occurs, but Lowenhaupt et al. [34] have shown that this is not the rate determining step as the ratio of precursor: mRNA does not change during the process of induction. Analysis of the poly(A) RNA population in dimethylsulphoxide (DMSO)-induced MEL cells [ 121 ] shows that there are two major populations when studying newly synthesised mRNA, mRNA1, with tl/2 approx. 3 h accounting for 85-90% of total poly(A) RNA, mRNA2, tv2 3 5 - 4 0 h, 1 0 15% of total poly(A) RNA, while globin mRNA accounts for 1.5% tv2 17 h, a value similar to that of globin mRNA in reticulocytes. With a long-term label the ratios change to mRNA~ 50%, mRNA2 40% and globin RNA 10%. The tv2 of globin RNA accounts for this accumulation; however, with a large population of stable mRNA in mRNA2, it is difficult to explain how globin mRNA can accumulate to become 90% poly(A) RNA as it does in reticulocytes except by postulating that the stable mRNA2 is specifically destabilized. These results are similar to those from anaemic spleen [122] and a similar conclusion was reached by Lodish and Small [123] who examined mRNA in reticulocytes by in vitro translation. Such destabilization would provide an additional step for globin mRNA accumulation, but at present there is no explanation of how this could occur. During the process of induction with DMSO there is an indication that the tl/2 of globin mRNA changes [124]. On induction of MEL cells globin mRNA begins to accumulate after 2 days, but after 4 days the amount of globin mRNA per cell decreases even though synthesis continues. Analysis of poly(A) RNA by pulse-chase experiments again indicated the presence of two main groups of mRNA with tl/2 of approx. 6 h and approx. 35 h. However, in uninduced cells and cells exposed to DMSO for 2 days, globin mRNA had a tx/2 > 50 h, while in cells in DMSO for 4 days globin mRNA had a tl/2 approx. 17 h as reported by Aviv et al. [121]. The initial long half-life of globin mRNA would aid its accumulation. Labeling of cells early in induction followed by chasing for 48 h showed that the labeled globin mRNA which had been synthesised behaved in the same way as globin mRNA synthesised late in induction. This behavior is not shared with all RNA molecules, ribosomal RNA remained stable throughout the induction period and cell death would not appear to be the explanation. As noted above, the 17 h half-life has been found for normal erythroid cells. Another example of the half-life of an mRNA changing during differentiation is myosin mRNA [125-127]. The mRNA is present in dividing myoblast cells at a time when very little myosin heavy chain polypeptide is being synthesised. At this time the mRNA has a tl/2 approx. 10 h and appears in the form of RNP particles. After fusion myosin mRNA is actively translated, mainly on polyribosomes and now has a tt/2 approx. 50 h. The explanation given for this change in tu2 is that there are different proteins binding to myosin mRNA that influence the tl/2; alternatively, active translation of a mRNA might increase its half-life. However, this change in tl/2 of myosin is the reverse of that observed by Lowenhaupt and Lingrel [ 124].

359

VII. Kinetics of globin mRNA induction Upon addition of an inducer to MEL cells, there is an early decrease in RNA synthesis, which affects primarily 4 S RNA and rRNA [128]. A number of studies have indicated that DNA synthesis in the presence of the inducer is necessary for commitment to differentiate [129-131]. However, there is also considerable evidence to the contrary [132, 133]. Induction of MEL cells with DMSO, though with no effect on the initial S, G2 and M phases of the cell cycle, results in a prolongation of the G1 phase by 4 - 1 0 h [134, 135]. Other inducing agents such as hypoxanthine and actinomycin D did not cause any lengthening of the G1 phase implying that the prolongation observed with several inducers is not essential for differentiation [ 136]. Appearance of globin mRNA and commitment occur in the prolonged G1 phase when the inducer is added no later than the preceding early S or G1 phase of the cell cycle [137,138]. Addition at a later stage such as S/G2 phase resulted in the appearance of globin mRNA at the second G1 phase. Thereafter, synthesis of globin mRNA continues throughout the cell cycle so that globin mRNA reaches a maximal concentration by 3 days after which it begins to decrease [124, 139-141]. Haem and globin polypeptides are closely coordinated with globin mRNA [142-144]; there is no evidence for the appearance of untranslated globin mRNA and little or no free haem. The maximal level of globin mRNA corresponds to about 10% of the poly(A)-RNA in induced MEL cells [ 121 ]. Accumulation of a- and/3-globin mRNA does not always occur coordinately, as determined by hybridization analysis with a- and/3-globin cDNA probes, the time of accumulation of each mRNA varying somewhat with the inducer used [145,146]. Increase of a-globin mRNA is detected at 16 h with DMSO, 12 h with HMBA, and 8 h with butyric acid. With these inducers, 13-globin mRNA accumulation begins at 2 0 - 2 4 h. Induction with haemin results in accumulation of both a- and/3-globin mRNA after 6 h. The ratio of a- to/3-globin mRNA varies also with inducers, approx. 1 with DMSO and HMBA, 0.66 with butyric acid and 0.3-0.5 with haemin. Erythrocytes of adult DBA/2 mice, from which MEL cell lines 745,707, TELC and FSD were derived, contain /3 major and/3 minor globin polypeptides in a ratio of 4 : 1 [147]. Uninduced MEL cells (lines 745, 707, TELC) contain predominantly/3 minor, while FSD contains/3 major. The other characterised MEL cell lines, T3C12 and 5000 from DDD mice, do not contain any/3 minor under any circumstances. Upon induction with DMSO, the different cell lines make mainly 13major (ratio approx. 4 : 1). Haemin, however, induces the appearance of almost exclusively/3 minor in lines 745, 707 and TELC, while FSD shows somewhat more /3 major than 13minor, but still more/3 minor than when DMSO is the inducer [143,148-152]. Poly(A)-RNA extracted from these induced cell lines and translated in a cell-free wheat germ system gave rise to about the same ratio of/3 major :/3 minor as observed in the intact cells, so that the changing ratio is not due to variation in efficiency of translation but to transcription, processing or stability of mRNA. Promotion of the growth of cells destined to synthesise a specific globin chain would be an alternative explanation.

VIII. Summary Accumulation of haemoglobin in induced MEL cells begins with the activation of transcription of the globin genes. Though much has been learned of cellular events affect-

360

ing expression of the globin genes, e.g., from noninducible variants of MEL cells and cell fusion between MEL cells and other cell types, there is at present no in vitro system available that would permit more detailed study of the molecular events leading to transcription of the globin genes. Presumably, with the availability of cloned chromosomal genes, such systems will soon be found. The products of transcription detected in induced MEL cells are 15 S and 11 S species which are precursor forms of/3- and a-globin mRNA, respectively. An unmodified primary transcript has not been detected. The 15 S species possesses a fully methylated 'cap' 1 structure at the 5' end and poly(A) at the 3' end. Conceivably, there could be cleaving or splicing events preceding the 'capping' and polyadenylation, but all these reactions must occur extremely rapidly, since with a tl/2 approx. 2 min a large proportion of the 15 S/3-globin RNA must be newly synthesised. It also contains the two intervening sequences found in the chromosomal genes. Selective processing occurs since from pulse and pulse-chase experiments most if not all of the 15 S/3-globin RNA is processed to mature 10 S/3-globin RNA very rapidly, whereas less than 10% of newly synthesised nuclear RNA (HnRNA) leaves the nucleus, the remainder being hydrolysed in the nucleus with a tl/2 approx. 20 rain. Perhaps rapid processing permits efficient transport to the cytoplasm. Further processing occurs in steps; apparently the large intervening sequence is removed first followed by the small intervening sequence. These steps do not appear to be rate limiting events and these sequences have not been detected separately from the 15 S /3-globin RNA. Such results and the wide divergency of intervening sequences, suggest that the intervening sequences per se play no essential function in the cell, though their presence in the nuclear transcript appears to be necessary for processing to the mRNA. The selective processing accounts for more increase of the globin RNA in MEL cells, and further accumulation occurs by virtue of the stability of globin mRNA (tl/2 approx. 17 h) compared with the bulk of poly(A)-RNA (tl/z approx. 3 h). It would appear, however, that specific destabllization of a class of stable mRNA (tl/2 approx. 35 h) is necessary to allow globin mRNA to account for 90% of the mRNA population in reticulocytes.

Acknowledgments The author is indebted to Dr. C. Weissmann and Dr. G. Rovera for their help in preparing this manuscript.

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