Cell, Vol. 48, 5-8, January 16, 1967, Copyright 0 1987 by Cell Press

Determinants of Messenger RNA Stability

George Brawerman Department of Biochemistry Tufts University Health Sciences Campus Boston, Massachusetts 02111

The steady-state levels of functional mRNAs are deter- mined in part by their rates of decay in the cytoplasm. Hence, this process plays an important role in the control of gene expression. Individual mRNA species differ widely with respect to metabolic stability. In mammalian cells, the mRNAs for transiently expressed genes such as c-fos and c-myc have half-life (tIh) values as low as 15 min, while others such as 8-globin mRNA appear to be fully stable. Bacterial mRNAs decay far more rapidly, with usual Lh values of 2-3 min, although some species are considerably more stable. The rates of decay of some spe- cies can be altered in response to physiological signals, such as hormone induction in animals (Guyette et al., Cell 77,1013-1023,1979; Brock and Shapiro, Cell 34,207-214, 1983) and changes in growth rate in bacteria (Nilsson et al., Nature 372, 75-77, 1984). Recent studies have provided some insight into the nature of the mRNA decay process, and have led to the identification of structural fea- tures of mRNA that determine its susceptibility to decay, both in prokaryotes and eukaryotes.

The basis for differential degradation of mRNA is a challenging biochemical problem. If mRNA decay were due to a random endonucleolytic process, some diversity could be provided by the target size and by the degree of accessibility of the internucleotide linkages. However, at least in prokaryotes there is no obvious relationship be- tween mRNA size and decay rate. Belasco et al. (Cell 46, 245-251,1988) have addressed this question by compar- ing the stability of transcripts derived from the E. coli b/a gene (a gene carried on the plasmid pBR322 that en- codes p-lactamase), and from a truncated form of the gene containing an internal in-frame deletion. The tran- scripts differed in size by 500/o, yet had similar decay rates.

The possibility that mRNA can be protected by ribo- somes has also been considered. Although this idea was supported by early studies of the frp operon (Morse and Yanofsky, Nature 224,329-331,1969), more recent experi- ments with other systems indicate that protection by ribo- somes may not be a significant factor in mRNA decay. Stanssens et al. (Cell 44,711-718,1986) compared the sta- bility of /acZ transcripts that differed in their ability to initi- ate translation-and hence in the number of r ibosomes covering their coding region-and found no differences in their decay rates. In addition, von Gabain et al. (PNAS 80, 653-657, 1983) have shown that the most stable portion of the E. coli ompA transcript (coding for the bacterial outer membrane protein) lies mainly in the 5’ noncoding region, which is not covered by ribosomes.

If mRNA chains are somehow shielded from non- specific endonucleases, but are susceptible to exo-

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nucleases, then particular configurations at the termini could influence the rate of decay. The rho-independent transcription termination signals in bacteria, which con- sist of sequences capable of folding into a hairpin struc- ture, appear to play some role in mRNA protection, pre- sumably by providing a barrier against the action of a 3’exonuclease. One example is the 3’terminator of the Ba- cillus thuringiensis crystal protein gene, which positively regulates expression of this gene. Fusion of the terminator sequence to the 3’ end of other genes confers stability to the resultant transcripts (Wong and Chang, PNAS 83, 3233-3237, 1988). Deletion analysis suggests that the significant feature of this sequence is its potential for forming a stem-loop structure. Such a sequence has also been identified in some (pX174 transcripts (Hayashi and Hayashi, NAR 73,5937-5946, 1985). In this case, most mu- tations that reduce the sequence’s potential for stem-loop formation also reduce its stabilizing effect.

Hairpin structures appear to impart stability to selected regions of polycistronic mRNAs. The E. coli gene coding for DNA primase is in the middle of an operon that begins with the gene for ribosomal protein S21 and ends with the gene for the sigma subunit of RNA polymerase. The primase region of the transcript decays more rapidly than the other two regions, which have the potential to form stem-loop structures at their 3’ termini. It has been sug- gested that the polycistronic transcript is first cleaved at the primase-sigma boundary, and that the exposed 3’ter- minus of the primase region is then degraded proces- sively in the 3-5’ direction until the stem-loop structure in the S21 protein region is reached (Burton et al., Cell 32, 335-349, 1983). A similar process appears to operate for the rxcA operon of Rhodopseudomonas capsulata, which encodes products involved in photosynthesis. Here, the 5’ region of the polycistronic transcript, corresponding to the first two genes in the operon, is more stable than the 3’ region (Belasco et al., Cell 40,171-181,1985). A sequence with the potential to form a stem-loop structure, the “repeti- tive extragenic palindromic” (REP) sequence, which oc- curs in a wide variety of E. coli operons, also causes stabilization of upstream mRNA segments (Newbury et al., Cell 48, in press). It is not known whether the postu- lated stem-loop configurations do in fact occur in vivo, and the precise nature of the protective terminator structure re- mains to be established. Not all terminator sequences with stem-loop potential can confer stability (Wong and Chang, op. cit.). Moreover, some of the (pX174 terminator mutations that destroy its potential stem-loop configura- tion do not affect its stabilizing activity (Hayashi and Hayashi, op. cit.).

Special structures at the 3’terminus also appear to pro- tect mRNA chains against exonucleolytic attack in eu- karyotic cells. The poly(A) sequence, in conjunction with bound protein, is resistant to the action of this kind of en- zyme (Bergmann and Brawerman, Biochemistry 76,259- 284, 1977). Removal of the poly(A) from some stable mRNAs causes them to be degraded rapidly (Huez et al.,

PNAS 78, 908-911, 1981). Some of the histone mRNAs, which have no poly(A) at their 3’ end, are terminated by a sequence with the potential to form a stem-loop struc- ture possibly resistant to 3’ exonuclease (Georgiev and Birnstiel, EMBO J. 4, 481-489, 1985).

The selectivity of mRNA decay is best explained by the action of specific factors that recognize unique sites on the mRNA chains. A rate-limiting endonucleolytic cut trig- gered by such an interaction would be followed by rapid destruction of the mRNA. Such a regulatory site has been identified next to the int gene of bacteriophage I.. This site, sib, located downstream of the int coding region, folds into the characteristic stem-loop structure that is recognized and cleaved by RNAase III. Destruction of the sib struc- ture by RNAase III leaves the transcript with an exposed 3’ terminus and leads to its rapid decay. In the absence of active enzyme, or in sib mutants no longer recognized by RNAase III, the transcript is more stable, apparently be- cause the intact stem-loop structure acts as a barrier against 3’ exonucleolytic cleavage (Guarneros et al., PNAS 79, 238-242, 1982; Schmeissner et al., JMB 776, 39-53,1984). A number of genes appear to be subject to this control by RNAase Ill, as indicated by alterations in patterns of protein synthesis in cells lacking this enzyme.

A specific sequence promoting mRNA decay has re- cently been identified in the 3’ noncoding region of a eu- karyotic gene. Shaw and Kamen (Cell 46,859-887,1986) have shown that insertion of an AU-rich sequence derived from the gene encoding the human lymphokine GM-CSF into the rabbit P-globin gene destabilizes the globin tran- script. A similar sequence is present in a variety of mRNAs from transiently expressed genes, such as c-myc, &OS, and interferon. These mRNAs are normally very un- stable, a feature that permits their rapid disappearance af- ter induction. The manner in which the AU-rich sequence promotes mRNA decay is not known. Possibly it is cleaved by a specific endonuclease, which would leave the mRNA susceptible to 3’ exonucleolytic attack. The detection of truncated P-globin mRNA chains in maturing reticulocytes, apparently generated by cleavages at AU sites in the 3 noncoding region, suggests that this type of cleavage may be a general feature of mRNA decay (Albrecht et al., JMB 778, 881-896, 1984).

Sequences at the 5’end of mRNA may also be involved in the decay process. Belasco et al. (op. cit.) have shown that the relatively long tl/, of the E. coli omp4 transcript is determined by a 5’ leader segment that includes the ribosomal binding site and the first few codons. The stabilization effect appears to involve ribosome interac- tion, as suggested by the fact that a hybrid transcript with a termination codon at the end of the 5’ omp4 segment is not stabilized. The relative stability of some bacteriophage T4 transcripts can also be attributed to a 5’ leader se- quence, as shown by gene fusion experiments using the 5’ terminal sequence of gene 32 (Gorski et al., Cell 43, 481-469, 1985). The active sequence appears to consist

of m40 nucleotides located close to the translation initia- tion codon. The gene 32 transcript and the hybrid tran- scripts are stable only in T4-infected cells, implying that stabilization requires interaction of the leader sequence with a factor produced only in the infected cells.

A 5’ leader sequence is involved in the regulation of de- cay of the human histone H3 mRNA. The histone mRNAs are unstable in the absence of DNA synthesis, and are sta- bilized in S phase cells, when there is a high demand for new histones. A hybrid P-globin mRNA containing the his- tone leader sequence was destabilized when DNA syn- thesis was blocked (Morris et al., PNAS 83, 981-985, 1988). Conversely, the histone mRNA remained stable in the absence of DNA synthesis when its leader sequence was replaced by that from Drosophila hsp70 mRNA. In the histone H4 mRNA species, the sequence that links stabil- ity to DNA replication appears to be located in the 3’termi- nal region (Liischer et al., PNAS 82, 4389-4393, 1985).

A destabilizing 5’terminal sequence seems to occur in c-myc mRNA, as suggested by the observation that trun- cated transcripts lacking most of the leader sequence are more stable (Piechaczyk et al., Cell 42, 589-597, 1985; Rabbitts et al., EMBO J. 4,3727-3733, 1985). However, fu- sion of exon I of the c-myc gene, which carries the long 5’ leader, to the chloramphenicol acetyltransferase (CAT) gene did not affect the stability of the CAT mRNA (Piechaczyk et al., Curr: Topics Microbial. Immunol., in press). Possibly, the destabilizing 5’ terminal segment must interact with some other c-myc sequence (see Saito et al., PNAS 80, 7478-7480, 1983). In any event, the trun- cated c-myc mRNAs are still rather unstable by mam- malian cell standards, and the major determinant of stabil- ity in this RNA is likely to be the 3’ terminal AU-rich sequence.

Information on the enzymes and other factors involved in the mRNA decay process is still very limited. Evidence provided by Donovan and Kushner (PNAS 83, 120-124, 1986) suggests that bacterial mRNA is normally degraded by two 3’ exonucleases, RNAase II and polynucleotide phosphorylase. Their observation that mRNA fragments accumulate in the absence of these enzymatic activities further suggests that no endonuclease is available in the cells for extensive mRNA degradation. These features could account for the apparent protection of mRNA by 3’ terminal structures that act as barriers against exonucleo- lytic attack, and for the loss of protection when a site- specific incision in the 3’ noncoding region generates an exposed 3’ terminus. RNAase III appears to function as such an endonuclease in bacteria. An enzyme specific for the mammalian AU-rich sequence, yet to be identified, may be the eukaryotic equivalent. Novel approaches in- volving the use of cell-free systems, and the identification of cleavage sites that trigger mRNA decay, will be re- quired to achieve further understanding of this important aspect of gene expression.