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direct neuralizing activity. Cell 77, 238295 18 Hansen C.S. et al. (1997) Direct neural induction and selective inhibition of mesoderm and epidermis inducers by Xnr3. Development 124, 483492 19 Zimmermann, L.B. et al. (1996) The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599606 20 Piccolo, S. et al. (1996) Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP4. Cell 86, 589598 21 Fainsod, A. et al. (1997) The dorsalizing and neural inducing gene follistatin is an antagonist of BMP4. Mech. Dev. 63, 3950 22 Biehs, B. et al. (1996) The Drosophila Short gastrulation gene prevents Dpp from autoactivating and suppressing neurogenesis in the neuroectoderm. Genes Dev. 10, 29222934 23 Dale, L. et al. (1992) Bone morphogenetic protein 4: A ventralizing factor in early Xenopus development. Development 115, 573585 24 Jones, C.M. et al. (1996) Bone morphogenetic protein-4 (BMP4) acts during gastrula stages to cause ventralization of Xenopus embryos. Development 122, 15451554 25 Smith, W.C. et al. (1993) Secreted noggin protein mimics Spemann organizer in dorsalising Xenopus mesoderm. Nature 361, 547549 26 Sasai, Y. et al. (1994) Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79, 779790 27 Smith, W.C. and Harland, R.M. (1992) Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70, 829840 28 Lamb, T.M. and Harland, R.M. (1995) Fibroblast growth factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern. Development 121, 36273636 29 Storey, K.G. et al. (1998) Early posterior neural tissue is induced by FGF in the chick embryo. Development 125, 473484 30 lvarez, I.S. et al. (1998) Neural induction in whole chick embryo cultures by FGF. Dev. Biol. 199, 4254 31 Launay, C. et al. (1996) A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers. Development 122, 869880 32 Sasai, Y. et al. (1996) Endoderm induction by the organizer secreted factors chordin and noggin in Xenopus animal caps. EMBO J. 15, 45474555 33 Kroll, K.L. and Amaya, E. (1996) Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 31733183 34 Bouwmeester, T. et al. (1996) Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemanns organizer. Nature 382, 595601 35 La Bonne, C. and Whitman, M. (1997) Localization of MAP kinase activity in early Xenopus embryos: implications for endogenous FGF signaling. Dev. Biol. 183, 920 36 Savage, R. and Phillips, C.R. (1989) Signals from the dorsal blastopore lip region during gastrulation bias the ectoderm toward a nonepidermal pathway of differentiation in Xenopus laevis. Dev. Biol. 133, 157168 37 Kroll, K.L. et al. (1998) Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation. Development 125, 32473258 38 Zhang, J. and Jacobson, A.G. (1993) Evidence that the border of the neural plate may be positioned by the interaction between signals that induce ventral and dorsal mesoderm. Dev. Dyn. 196, 7990 39 Sharpe, C.R. et al. (1987) A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction. Cell 58, 749758 40 Otte, A.P. and Moon, R.T. (1992) Protein kinase C isozymes have distinct roles in neural induction and competence in Xenopus. Cell 68, 10211029 41 Bradley, L. et al. (1996) Positive and negative signals modulate formation of the Xenopus cement gland. Development 122, 27392750 42 Streit, A. et al. (1998) Chordin regulates primitive streak development and the stability of induced neural cells, but is not sufficient for neural induction in the chick embryo. Development 125, 507519 43 George-Weinstein, M. et al. (1996) Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro. Dev. Biol. 173, 279291 44 Winnier G. et al. (1995) Bone morphogenetic protein-4 is required

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for mesoderm formation and patterning in the mouse. Genes Dev. 9, 21052116 Schultheiss T.M. et al. (1997) A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11, 451462 Albano, R.M. et al. (1994) Expression of inhibin subunits and follistatin during postimplantation mouse development: decidual expression of activin and expression of follistatin in primitive streak, somites and hindbrain. Development 120, 803813 Levin, H. (1998) The roles of activin and follistatin signaling in chick gastrulation. Int. J. Dev. Biol. 42, 553559 Connolly, D.J. et al. (1997) Chick noggin is expressed in the organizer and neural plate during axial development, but offers no evidence of involvement in primary axis formation. Int. J. Dev. Biol. 41, 389396 Storey, K.G. et al. (1992) Neural induction and regionalization in the chick embryo. Development 114, 729741 McMahon, J.A. et al. (1998) Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev. 12, 14381452 Matzuk M.M. et al. (1995) Multiple defects and perinatal death in mice deficient in follistatin. Nature 374, 360363 Dudley, A.T. et al. (1995) A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9, 27952807 Zhang H. and Bradley A. (1996) Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122, 29772986 Mishina Y. et al. (1995) Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9, 30273037 Streit, A. and Stern, C.D. (1999) More to neural induction than inhibition of BMPs. In Cell lineage and fate determination (Moody, S.M., ed.), pp. 435447, Academic Press Keller, R.E. (1975) Vital dye mapping of the gastrula and neurula of Xenopus laevis. I. Prospective areas and morphogenetic movements of the superficial layer. Dev. Biol. 42, 222241 Sive, H.L. et al. (1989) Progressive determination during formation of the anteroposterior axis in Xenopus laevis. Cell 58, 171180

mRNA degradation
a tale of poly(A) and multiprotein machines
Agamemnon J. Carpousis carpousi@ ibcg.biotoul.fr Nathalie F. Vanzo vanzo@ibcg.biotoul.fr Lelia C. Raynal raynal@ibcg.biotoul.fr
Laboratoire de Microbiologie et Gntique Molculaire, Centre National de la Recherche Scientifique, UPR 9007, 118, Route de Narbonne, 31062 Toulouse Cedex, France. 24

The Escherichia coli RNA degradosome is a multiprotein complex containing an endoribonuclease, polynucleotide phosphorylase and a DEAD-box RNA helicase. A related complex has been described in the spinach chloroplast. The exosome and the mtEXO complex have recently been described in yeast and it is likely that related complexes also exist in animal cells. This research suggests the widespread existence of sophisticated machines for the efficient degradation of messenger RNA. The DEAD-box helicase in the degradosome can unwind regions of RNA structure that interfere with 35 degradation. The polyadenylation of RNA 3 ends is also known to promote degradation by creating a toehold for the degradation machinery. Much remains to be learned about the regulation of mRNA stability. The complexity of the degradation process, both in the eubacteria and in the eukaryotes, suggests that many steps are possible points of control.

ll RNAs can be classified by their stability in the cell. The best known stable RNAs are the transfer and ribosomal RNAs. Messenger RNAs (mRNAs) are unstable with half-lives in Escherichia coli ranging from 30 seconds to 20 minutes. In eukaryotic cells, mRNA turnover is slower, but the half-lives are usually shorter than the generation time. Until recently, the degradation of mRNA was not generally TIG January 1999, volume 15, No. 1

considered in discussions of the regulation of gene expression. RNA degradation was mainly viewed as a nuisance in the laboratory, causing problems when trying to do a northern blot or construct a cDNA library. With the growing appreciation of the complexity of gene regulation, interest in the mechanism of the degradation of mRNA has rapidly increased during the past decade. The instability of
0168-9525/99/$ see front matter 1999 Elsevier Science All rights reserved. PII: S0168-9525(98)01627-8

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the exonuclease encoded by XRN1. The 35 pathway involves a recently discovered multiprotein complex, the exosome (described below). Curiously, E. coli does not have a 53 degradation pathway. The 5 ends of its mRNA are not capped and there are no known 53 exo-ribonucleases. This also appears to be the case for other eubacteria. Thus the 53 degradation pathway is apparently a specific feature of the eukaryotic cell.

mRNA is now known to be an important property permitting timely adjustments to changes in growth conditions or to genetically controlled programs of expression. The degradation of mRNA used to be viewed as an unsophisticated process in which a hodgepodge of ribonucleases attacked any accessible RNA. In contrast to mRNA, transfer and ribosomal RNAs were believed to be protected by their rapid folding into compact structures. This simplistic view has been made obsolete by the discovery of multiprotein machines with the capacity to unwind and degrade structured RNA. Another widely held preconception was that the enzymes involved in RNA processing and RNA degradation are different. With the discovery in E. coli and Saccharomyces cerevisiae that ribonucleases in the processing of ribosomal RNA are also important in the degradation of mRNA, it is now clear that the connection between processing and degradation is much closer than previously appreciated. This article describes recent advances in our understanding of the degradation of mRNA in E. coli. When feasible, we make the comparison with related developments in other organisms. For comprehensive reviews of the degradation of mRNA in the eubacteria and the eukaryotes, we recommend the articles published in 1996 by Nierlich and Murakawa, and by Jacobson and Peltz1,2.

FIGURE 1. mRNA degradation in eubacteria

Protected 3 end

5 Endonuclease 5 Exonuclease 5 3

Multiprotein machines in the degradation of mRNA

The degradation of mRNA in eubacteria and eukaryotes

In E. coli, the degradation of mRNA is mediated by the combined action of endo- and exo-ribonucleases1,3. Two enzymes, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase), degrade RNA in a 35 pathway. Enzymes related to RNase II and PNPase are widespread both in the eubacteria and in the eukaryotes4. RNase II is a hydrolytic enzyme producing nucleotide monophosphates (AMP, etc.). PNPase uses inorganic phosphate yielding nucleotide diphosphates (ADP, etc.). Although we sometimes speak of PNPase as an exonuclease, this is not strictly correct because the nucleases are hydrolytic enzymes by definition. A double mutant in the genes encoding RNase II and PNPase is conditionally lethal and mRNA decay intermediates, produced by endonucleolytic cleavage, accumulate in the cell5. The 3 ends of certain mRNAs, such as those formed by rho-independent termination, are sequestered in stemloop structures that protect them from degradation. An internal endonucleolytic cleavage can remove the stemloop, creating new single-stranded 3 ends that are accessible to the exonucleases (Fig. 1). The single-strandspecific endo-ribonuclease E (RNase E) is essential for 5S ribosomal RNA processing and conditional mutations in its gene also affect the stability of a large number of mRNAs. RNase E is now generally believed to be a major endonuclease in E. coli mRNA decay. The concept of the cooperation between endonucleases and exonucleases was an important step in advancing our understanding of the degradation of mRNA in E. coli. The endonucleolytic decay of mRNA appears to be less important in eukaryotes although it could be involved in the regulation of the stability of certain messages6. Two exonucleolytic pathways have been described in yeast: 53 and 35 pathways2,610. Eukaryotic mRNAs are protected by 5 cap structures and 3 poly(A) tails complexed with poly(A)-binding protein (PAB). Deadenylation and decapping are prerequisites for degradation (Fig. 2). In the 53 pathway of S. cerevisiae, decapping is deadenylation-dependent and the mRNA is degraded by

A large multiprotein complex, now called the RNA degradosome, was The 3 end of the mRNA is protected from discovered during the purification exonuclease attack by the stemloop. The of E. coli RNase E (Refs 11, 12). endonuclease (scissors) cleaves upstream of The major components of the RNA the stemloop creating a free 3 end that is degradosome include RNase E, then attacked by the exonuclease (yellow). PNPase and the DEAD-box RNA helicase, RhlB (Refs 1315). The association of RNase E and PNPase in a complex provides direct evidence for their cooperation in the degradation of mRNA. The complex also contains enolase, a glycolytic enzyme, as an integral component. Associated proteins, present in substoichiometric amounts, include DnaK, GroEL and polyphosphate kinase (PPK). The role of enolase, DnaK, GroEL and PPK in the degradation of mRNA remains to be clarified. RNase E is a multidomain protein whose endonuclease activity is in the N-terminal domain16,17. In the same domain,

FIGURE 2. mRNA degradation in eukaryotes

(a)
53 pathway PAB AAAA PAB AAAA PAB AAAA 3

5 Gppp

Deadenylation and decapping

(b)

35 pathway

PAB AAAA Deadenylation

PAB AAAA

PAB AAAA 3

5 Gppp

5 Gppp

The 53 decay pathway (a) requires the removal of the 5 cap that protects the mRNA from the 5 exonuclease (blue). Decapping is deadenylation-dependent. The 35 pathway (b) requires deadenylation removing the poly(A)/poly(A) binding protein (PAB) complex that protects the mRNA from the 3 exonuclease (yellow).

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FIGURE 3. The E. coli RNA degradosome
Endo- and poly(A)/(U)nucleases RNA unwinding ? 35 degradation

mRNA degradation

All the integral components of the exosome are essential for viability. The in vivo depletion of any one of the five components leads to impaired RNA degradation. These results suggest that the constituent enzymes in the exosome might act in a highly cooperative manner.

RhIB RNase E

Enolase

PNPase

RNA helicases in the degradation of mRNA

The identification of the DEAD-box protein, RhlB, in the E. coli degradosome was one of the first indications that RNA helicases could have an active role in the degradation of mRNA. The DEAD-box proteins are a family of putative ATP-dependent RNA helicases that have a conserved core sequence containing eight highly conserved motifs including the N-acids D-E-A-D (Refs 24, 25). Members of this family have been implicated in a variety of processes including ribosome assembly, translation initiation and RNA splicing. The advantage of having an RNA helicase in a complex was demonstrated in vitro with the RNA degradosome13. RNAs with internally structured regions often impede the progress of enzymes such as PNPase, forcing the enzyme to pause26,27. RhlB in the degradosome facilitates PNPase-mediated degradation of structured substrates in an ATP-dependent reaction that appears to involve the unwinding of RNA stemloops (Fig. 4). RhlB is strongly activated by its interaction with the C-terminal half of RNase E and polypeptides derived from this region can form a complex with RhlB that is capable of unwinding short RNA helices in vitro19. The yeast mtEXO complex contains Suv3p, a DExH-box protein that is essential for RNA degradation in vivo21. The mtEXO complex also requires ATP hydrolysis for exonucleolytic activity in vitro. Two DEvH-box proteins appear to be factors associated with the exosome10,28. The DExHbox and DEvH-box proteins, putative RNA helicases, belong to families related to the DEAD-box proteins.

N-terminal catalytic

C-terminal protein interactions

Diagram showing the integral components of the E. coli RNA degradosome and illustrating the multidomain composition of RNase E (blue), a dimer with an extended C-terminal half that serves as the scaffold for the interaction with RhlB (orange), enolase (green) and PNPase (yellow).

a novel activity that degrades 3 poly(A) or poly(U) tails has also been described18. Recent results have shown that the C-terminal half of RNase E interacts with the three other major components of the degradosome, RhlB, enolase and PNPase (Refs 19, 20). The description of RNase E now emerging is that of a complex multidomain protein containing N-terminal catalytic sites and a C-terminal region that serves as the scaffold upon which the other components of the RNA degradosome are assembled (Fig. 3). Several other multiprotein machines have recently been identified (Table 1). All of these complexes act in a 35 degradation pathway. A complex containing a homologue of PNPase and an endonuclease, apparently related to RNase E because it cross-reacts with antibodies against the E. coli enzyme, is implicated in the processing and degradation of chloroplast mRNA (Ref. 21). Two other complexes have been described in yeast. The mtEXO complex, containing an exonuclease possibly related to E. coli RNase II, degrades mRNA introns in the yeast mitochondria22. The exosome, involved in both the processing of ribosomal RNA and the degradation of mRNA, is a complex containing five 35 RNA degrading enzymes, four of which are related to E. coli RNase II or PNPase (Refs 10, 23). The human homologue of the yeast exonuclease, Rpr4p, has been detected in a large complex suggesting that animal cells also have an exosome.

Poly(A)-promoted RNA degradation in the eubacteria

E. coli poly(A) polymerase I (PAP I) is encoded by the pcnB gene that was originally identified because of its influence on ColE1 plasmid copy number29,30. PAP I is implicated in the degradation of the plasmid-encoded RNA I, a small regulatory RNA that interacts with RNA II, the primer initiating ColE1 DNA replication. RNA I, which has a short two-minute half-life, is initially cleaved by RNase E removing five nucleotides from its 5 end to form the RNA I5 decay intermediate. In pcnB mutants, which

TABLE 1. Composition of the multiprotein complexesa

RNA degradosome E. coli Integral components
RNase E (endonuclease) PNPase (35 phosphorylase) RhlB (DEAD-box protein) Enolase (glycolytic enzyme) p67 (endonuclease related to RNase E) 100RNP (35 phosphorylase) 33RNP (regulation?) p110 (35 exonuclease possibly Dss1p, a protein related to RNase II) Suv3p (p90) (DExH-box protein) p75 (?) Rrp4p (35 exonuclease related to RNase II) Rrp41p, Rrp42p, Rrp43p (35 phosphorylases related to PNPase) Rrp44p (35 exonuclease)

RNA degradosome spinach chloroplast

mtEXO S. cerevisiae mitochondria

Exosome S. cerevisiae nucleus (cytoplasm?)

Associated proteins
DnaK, GroEL (protein chaperons) PPK (regulation?) 24RNP, 28RNP, 55RNP (specificity?) ? Dob1p, Ski2p (DEvH-box proteins) Ski3p, Ski8p (specificity?)

a The associated proteins have been identified by copurification in substoichiometric amounts (E. coli RNA degradosome), as cofactors in biochemical assays (chloroplast RNA degradosome), or by genetic tests for functional interactions (S. cerevisiae exosome).

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degradation similar to that in E. coli. are deficient in PAP I activity, RNA I5 FIGURE 4. Helicase-facilitated Recent work in the plant chloroplast, is stabilized tenfold31,32. A similar role degradation an organelle of eubacterial origin, has for polyadenylation has recently been also implicated polyadenylation in dedescribed for the small RNAs that Internal stabilizing mRNA (Refs 44, 45). Thus, regulate the replication and partition stemloop poly(A)-promoted degradation of of R1 plasmids33,34. mRNA appears to be a common propRecent research suggests that E. 5 3 erty of the eubacteria. Because of the coli has a second poly(A) polymerase stabilizing effect of the long poly(A) tails (PAP II, Ref. 35). Strains with a disPausing in eukaryotes, the role of 3 polyadenylruption of the two genes encoding the ation in the degradation of eubacterial PAPs are not viable. However, single 5 3 mRNA was a surprise. However, in the gene disruptions are viable, suggesting eukaryotes, deadenylation precedes a possible functional redundancy. The ATP RNA degradation and mRNAs with short role of PAP II in RNA degradation helicase poly(A) tails are very unstable. Perhaps, remains to be clarified. There is no ADP like the eubacteria, short poly(A) tails obvious sequence homology between Unwinding are destabilizing and the poly(A)/PAB the two E. coli polymerases. PAP II is complex is a eukaryotic innovation not related to any other known pro5 3 regulating the accessibility of the mRNA tein, but PAP I is a member of the X 3 end to the exonucleolytic machinery. polymerase family that includes the eukaryotic poly(A) polymerases and all of the known tRNA nucleotidylPerspective Helicase-facilitated degradation of an internal transferases36,37. The tRNA nucleotidylIn E. coli, 3 ends that protect mRNA RNA stemloop structure. Without RNA helicase transferases specifically add CCA to can be removed by endonucleases or activity, progression of the exonuclease (yellow) the 3 end of tRNA substrates, but are unblocked by 3 polyadenylation. is blocked by the stemloop structure(pausing). The RNA helicase (orange) hydrolyzes ATP to unnot known to have poly(A)-adding Internal regions of RNA structure wind the stemloop, facilitating RNA degradation activity. The PAP I of E. coli, the that impede 35 degradation can be by the exonuclease. eukaryotic poly(A) polymerases and unwound by the helicase in the RNA the tRNA CCA-adding enzymes degradosome. It seems likely that the might all be derived from a common ancestral enzyme. main points for the control of degradation are in the RNA Polyadenylation has been implicated in the degradation structures that influence endonucleolytic cleavage, 3 polyof mRNA in E. coli3840. The demonstration that bacterial adenylation and 35 degradation. A better understandmessages are polyadenylated was difficult because the poly(A) ing of the RNA degradosome and the other enzymes tails are short, mRNA turnover is rapid, and only a small involved in the degradation of mRNA is now emerging. fraction of the total mRNA population is polyadenylated in The study of the cooperation between the constituents of wild-type E. coli. In double mutants (PNPase/ RNase II) the degradosome and their regulation is likely to be an important area of future research. that are exo deficient, the polyadenylRNase II and PNPase, both involved ation of intermediates in the degraFIGURE 5. Poly(A)-promoted in the 35 degradation of mRNA, dation of mRNA is easily detected. degradation seem to have redundant functions. Although the polyadenylation in wildNevertheless, related proteins are widetype cells is low, it seems likely that it Protected spread in both the eubacteria and the has a role in the degradation of inter3 end eukaryotes, suggesting that there must mediates that arise by pausing during be some advantage in having both exonucleolytic digestion or by endonutypes of enzyme. A plausible explacleolytic cleavage41,42. Poly(A)-addition 3 5 nation is that the efficient degrapromotes the degradation of RNAs dation of mRNA under various conwhose 3 ends are sequestered in secditions of growth requires a panoply ondary structure (Fig. 5). PNPase and of enzymes. This could also explain RNase II need a single-stranded toePAP the apparent overlapping functions hold to bind the 3 end of the RNA of PAP I and RhlB, both of which and initiate degradation. The polyaid the degradation of structured adenylation of the 3 end creates a bindRNA. It has been suggested that there ing site for the exonucleases. It has been Toehold is a hierarchy of RNA structures suggested that repeated polyadenylation with: (1) RNase II or PNPase acting and exonucleolytic attack could be alone to degrade single-stranded required to degrade certain intermedi5 A A A A A A A A 3 regions; (2) PAP I promoting the ates and recent in vitro experiments degradation of moderately stable RNA using purified PAP I, PNPase and ATP structures; and (3) RhlB facilitating have demonstrated the degradation of Poly(A)-promoted degradation of an RNA with a the degradation of highly stable RNA a structured RNA in a reaction involv3 end protected by a stemloop structure. The structures43. This hypothesis, based ing multiple cycles of polyadenylation43. exonuclease (yellow) cannot attack 3 ends that In the eubacteria, mRNAs with short on limited data, needs to be extended are sequestered in double-stranded structure. poly(A) tails have been detected in both by the analysis of other mRNAs Poly(A) polymerase (PAP) adds a single-stranded Gram-positive and Gram-negative orbefore it can be accepted as generextension giving a toehold to the exonucleases. ganisms suggesting poly(A)-promoted ally correct. TIG January 1999, volume 15, No. 1
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mRNA degradation

The degradation of messenger RNA: a tale of poly(A) and multiprotein machines

A recently published paper reports that E. coli RNase E is a 5-end-dependent endonuclease1. It was demonstrated that rpsT mRNA and 9S rRNA, when circularized by ligation of the 3 end to the 5 end, are resistant to endonucleolytic cleavage by RNase E. In control experiments where the RNA circles were converted back to linears, the capacity to cut the substrate with RNase E was restored demonstrating that the circularization protocol did not damage the substrate. In a further series of experiments, it was demonstrated that determinants at the 5 end of the molecule significantly affect the rate of endonucleolytic cleavage by RNase E. The initial cleavage by RNase E is much faster on substrates with 5 monophosphate ends than substrates with 5 triphosphate ends. Thus the 5 triphosphate may act as a cap structure protecting a nascent transcript. Hybridization of an oligonucleotide to the 5 end also protects against RNase E. These results may explain previous work, which showed that RNA stemloops that sequester the 5 end in a double-stranded structure impede RNase E-mediated degradation2. RNase E makes a product with a 5 monophosphate. Because RNAs with 5 monophosphates are the preferred substrate of RNase E, the initial cleavage of a protected substrate (5 triphosphate or a stemloop) is predicted to lead to rapid cleavage at downstream sites. This could explain why the decay of many mRNAs in E. coli appears to be in the 53 direction, as well as account for the phenomenon of all-or-none decay in which the initial cleavage is followed by the rapid decay of the entire message. 1 Mackie, G.A. (1998) Ribonuclease E is a 5-end-dependent endonuclease. Nature 395, 720723 2 Bouvet, P. and Belasco, J.G. (1992) Control of RNase E-mediated RNA degradation by 5-terminal base pairing in E. coli. Nature 360, 488491

Acknowledgements
We thank H.M. Krisch and L.G. Poljak for helpful suggestions to improve this manuscript. N.F.V. and L.C.R., graduate students in the Biology and Health Sciences Program of the Paul Sabatier University, are supported by the MENRT (N.F.V.) and the ARC (L.C.R). A.J.C. is a Research Director in the CNRS.

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TIG January 1999, volume 15, No. 1