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Cytoplasmic mRNA-protein interactions in eukaryotic gene expression
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~A=PROTE~N interactions are involved in most of the component steps of gene expression, including the splicing, nucieocytoplasmic transport, translation and degradation of messenger P~A (mP~A) and its precursors ~ig. ]). It is now apparent that all of these involve the mo~eoorqess spedfic recognition of sequences and structural elements in ~RNA molecules by an awe-inspiring va~~:etyof proteins. This is a relatively new field, generating many challenging questions. Of overriding importance is the necessity to identif3, the structural and functional principles underlying the known interactions and their roles in the cell. In the following we attempt to convey an impression of some of these emerging principles, focusing primarily on cytoplasmic events. For the sake of clarity, we have generally tried to distinguish between RNA-protein interactions intrinsic to the mechamsm and (constitutive) control of processes in the cell, and those involved in regulating these processes in response to environmental or developmental influences. This is analogous to the distinction between 'housekeeping' and regulatory genes. RNA.bindingproteins peffoml many essential functions in post-transcriptional gone expression mRNA transcribed from RNA polymerase II (Pol I1) promoters undergoes a long series of interactions with various proteins soon after synthesis has begun. Early events, of great significance for the stability and function of the transcript, are the capping of the 5'-terminal nucleotide and the polyadenylation of the 3'-end generated by cleavage at predetermined sites. PremRNA processing involves spliceosomal proteins and nuclear pre-messenget ribonucleoproteins (pre-mRNPs), also known as heteronuclear ribonucleoproteins (hnRNPs) (Fig. 1), many of which are, as yet, poorly characterized. Mature mRNA is accompanied by various proteins through the nuclear pores into the cytoplasm. Current knowledge of these processes is reviewed elsewhere and is not the main subject of this article ]-3. A whole new set of protein factors (eukaryotic translation initiation J. E. G. McCarthyand H. Ko,mus are at the Departmentof GeneExpression,National BiotechnologyResearchCentre(GBF), MascheroderWeg1, i)-38124 Braunschweig, Germany. © 1995, Elsevier Science Ltd 0968- 0004•95]$09.50
Cyto#a m NA ?rote n ateraetie ia e karyetie gene o
John E. G. McCarthy and Heike Kotimus Post-transcriptional mechanisms contribute in many important ways te the overalU control and regulation of gone expression, and in doing so employ a veritable army of proteins that bind a wide range of targets in messenger RNA (mRNA). The full range of these RNA-protein interactions is on;t just beginning to emerge, and much remains to be learned about the mechanisms underlying the rapidly increasing number of regulatory systems now being described. factoc% elFs, and the factors involved in elongation and termination) is subsequel:tly required for the cytoplasmic process of translation, although at least one of these (elF-4E) may be able to interact with mRNA and/or ribosomal components in both the cytoplasm and the nucleus 4,5.The most complex phase of translation is initiation. At least 12 different elFs are thought to be involved in this process 6,7, and those with known RNA-binding activities are included in Table I. A core issue concerns the pathway of translational initiation as well as the kinetics and thermodynamics of the component steps. A number of assumptions about the pathway have been made on the basis of studies of partial reactions involving isolated factors in vitro. Thus, many questions remain open regarding the functional roles of the various eIF-mRNA interactions believed to participate in initiation. Perhaps the most specifically targeted mRNA-binding elF is elF-4E (elF-4a), which is an essential component of the cytoplasmic cap-binding complex (elF417). A nuclear cap-binding complex also exists 8, which presumably has to be displaced from the cap at some point, and is eventually replaced by elF-4F, elF-4E has hit the news recently because its overproduction leads to transformation or abnormal growth in certain mammalian cells 9. Whether this is due to the suspected nuclear and/or known cytoplasmic function(s) of this factor remains unclear. The structural basis of this protein's affinity for the mRNA cap is unknown. So far, a number of con-
served tryptophan residues are suspected to be involved I°. Previously characterized RNA-binding motifs have been identified in other elF's (Table 1), although the functional significance of these motifs has yet to be defined.
Translationa]control via the 5'~'R of the mRNA Translational initiation can be controlled by intramolecular base-pairing within the 5'-untranslated region (UTR) of the mRNA. The mechanism has a bearing on the ways that translation can be regulated by RNA-binding proteins, and will be considered in some detail here. A hairpin seems to inhibit translation more effectively in mammalian systems when placed close to the 5'-end of the mRNA than an equivalent structure located further downstreamn. This could be due to the fact that in the 5'-position the structure blocks cap-binding by the 43 S proinitiation complex, whereas a more distal structure might only block the 'scanning' process, during which the ribosome is thought progressively to screen the 5'-UTR for potential start codons ~2 (see Fig. 2). Alternatively, a stem-loop near the 5'-end may inhibit a subsequent intermediate reaction, such as a transitional step towards 'scanning', rather than the initial binding of the 40 S ribosome. This could, in turn, allow reversible binding of the 40S ribosome to the cap or cap-binding complex, but prevent its further translocation along the mRNA. The significance of the position of specific sequences or structures
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Pre-mRNP Capping
/
Splicing (snRNPs)
Polyadenylation mRNP
/
Nuclear
Exchange of binding proteins(?) mRNA (localization?) RNAses
~:
elFs, eEFs, eRFs etc.
> Translation
l Regulatory factors
Decay Figure 1 Assumed pathway of RNA polymerase !1 (Pol II) transcripts in the eukaryotic cell. The initial product of transcription is pre-mRNA(or heteronuclearRNA,hnRNA),which is bound rapidlyby proteins forming ribonucleoprotein particles (pre-mRNP or hnRNP). The pre-mRNA is processedand spliced before being escorted out of the nucleus by as yet poorly characterized proteins. At least some of these must be replaced by cytoplasmic proteins that are involved in the translation and degradation of mature mRNA. These include the eukaryotic initiation factors (elFs), elongation factors (eEFs) and release factors (eRFs), as well as exo- and endoribonucleases. Other mRNA.binding proteins regulate the various steps of post-transcriptional gene expression or may be involved in the localization of the mRNA.
in the mRNA in terms of their functional roles is a recurrent theme in the control of gene expression. However, in vivo experiments performed with yeast show that not all eukaryotes need to follow the same pattern of positional modulation of translational control 13. Extreme cases of structural inhibition are observed in the 5'-leaders of a large number of mammalian mRNAs, many of which encode proteins of regulatory significance, such as oncoproteins and transcription factors 14. How does a scanning preinitiation complex negotiate inhibitory structures that stand between it and the start codon? At least in higher eukaryotes, elF-4A and elF-4B are thought to be required for the entry of 40 S ribosomes Into the initiation pathway via their interaction with mRNA. Since elF-4Ahas weak ATP-hydrolysis-dependent hellcase activity that is greatly enhanced by elF-4Bis, it has been proposed that these two factors, in combination with components of elF-4F, can catalyse the unwinding of otherwise inhibitory structures in the 5'-UTRT. However, it is not known whether these factors unwind the 5'-UTR independently of the 40 S ribosome in vioo ~2. Whatever the mechanism involved, it has to be asked
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whether the unfolding of a stem-loop structure occurs repeatedly during successive cycles of translation. One possibility, for example, Is that once the Initiation machinery Is loaded onto the 5'-UTR, the stem-loop is held in an 'open' conformation by repeatedly scanning ribosomes 16. Translational initiation at the start codon is also influenced by the context sequence of the AUGn. Here, recognition of the AUG by the 43 S preinitiation complex is dependent upon Watson-Crick base-pairing with the initiator transfer RNA (tRNA) in a process that is influenced by elF-2 (Ref. 17). While effectively controlling the fidelity of an RNA-RNA interaction, elF-2 is not thought to recognize the start codon directly itself. Although eukaxyotic translational initiation is usually a process involving caP
mammalian leaders bearinguORFshave been reported, but little is known about their functional significance|4. Eukaryotic ribosomes reinitiate poorly compared with their prokaryotic counterparts, which frequently and efficiently perform this task via a pathway that, in certain respects, resembles eukaryotic 'scanning'~9. The mechanistic details of eukaryotic reinitiation are no clearer than those of normal cap-dependent initiation, but the kinetics of the process can be regulated by the availa~)ility of active eIF-2~GTPwhich, in turn, is regulated by the phosphory|ation status of the elF-2~ subunit ~8. A radically different form of eukaryo otic translational initiation has been proposed, in which 'internal ribosome entry segments' (IRESs) allow cap-indepel~dent ribosome binding2°. Internal initiation is believed to occur on picomaviral mRNAs as well as on a few cellular mRNAs (such as that encoding the immunoglobulin heavy-chain-binding protein BiP2°). Various in vitro binding and crosslinking experiments have pointed to the possible involvement of a number of additional P,bJA-binding proteins in initiation on picomaviral mRNAs, including two that bind polypyrimidine tracts (PTB and the La-antigen; Table D. An IRES is thought to act, in some ways, analogously to a prokaryotic translational initiation region, but the mechanism by which IRES-binding proteins could mediate ribosomal binding is unknown. Finally, translation in eukaryotic organelles is more akin to that in prokaryotes, while at the same time being dependent on many nucleusencoded proteins2L A number of these are required for the translation of mitochondrial mRNAs (Table I).
Intramolecularinteractionsbetweenthe 5; and 3'-UTRs Several lines of evidence point to the importance of 'long-range' interactions in many post-transcriptional processes. For example, biochemical and genetic evidence indicates that the 3" and 5"UTRs on the same mP~IA (see Fig. 2)
can interact with each other 13,16,22and that at least one protein binding to the poly(A) tail [the poly(A)-binding protein, PABP] might interact with the preinitiation complex, although not necessarily directly23,24. While the relationship between the 5'and 3'-UTRs remains to be clarified, the concept of 'communication' between the two would help explain
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System/functional category a
Protein: reJevant detai~s and proposed mechanism
RNA-bindin8 domain(s) ~ (protein)
RNA recognition element~
Posit~on of R~A eIement~
Heterotrimeric ~actor, involved in initiation-site recognition 46 kOa; ATP-dependent sin~Jestranded-RNA-binding protein; RNA heBicase; RNA-dependent ATPase 70 kDa (50 kDa in yeast); stimulates activities of eOF-4A and eOF-4F Cap-complex protein in higher/ ~owereukaryotes
Zinc(H)fingerin ~-subunit
tRNA/mRNA
?
TypicaOconserved regions of DEAD proteins
Nonspecific; minima~ size: 12-18 nucIeotJdes
1-3?
Amino-terminal RRM + Arg-rich region
Nonspecific
1-3?
RRM4ike + RNP-tike sequence elements Not defined Cap recognition via eOF-4E
Unknown
?
mZGpppX mZGppp×
1 1
?
SECJS
6
eRF1 unlikely to be only type of termination factor
?
Stop codon(s)
5
Nucleus-encoded proteins essential for translation of certain mitochondrial mRNAs
?
Sites in cox2/3 mRNAs
2?
Various protetins, including CPSF, poly(A) polymerase, PABPH
?
Poly(A), AAUAAAand others
6/7
AUF1, p37 AUF1
Two RRMs
6
Possibly stabilized by 47 kDa protein
?
One or more AREs in U-rich context ?
Binds poly(A) tail: stimulates translation and deadenylation Covalently binds to cad structure DNA/RNA binoing; transcription factor; binds maternal mRNA in Xenopus oocytes ATP-dependent helicase Cellular nut=antigen; binds U-rich transcript 3;ends; suspected internal 'initiation factor' Binds polypyrimidine tracts: suspected to act in pre-mRNA processing and internal initiation
Four RRMs
Poly(A)
7
Two in Gag-Pol form Basic/aromatic domains
mTGpppX Low specificity
1 ?
? RRM
Stem-loops Proposed: low-specificity binding to poiy(U), e.g iRES
2? 2/3?
Three RRMs
Suspected to bind to various iRES 2/3
TCans~onampathway Initiation factors (eIFs) eE-2 (Ref. 17)
' eE4A e
eOF4Bf p220/p150 (elF-4~/)~
eOF-4E(eOF-4~)"
ATP-independentcap binding
eOF-4P'~
Cap-binding complex; mammaOian: eOF-4A,eOF-4E,p220; yeast: eOF-4E(p24), p20, p150
Elongation factors Selenocysteine incorporation 2a Unknown factor (equivaUent of E. coil SeOB?) Termination f~ctors eRF1/sup4~ Mitochonddal 'activators' Nuclear PETgenes21
Nu©leat' po|yadeny]ation k 3'-end formation/poly(A) synthesis raRNA stability Rapid degradation of c-myc, c-los and GM-CSFmRNAsz Stabilization of chloroplast psbD mRNAm Other exantples Poly(A)-binding protein (PABP) L-A coat protein',° FRG Y2 (mRNP4)P
Yeast SSL2" La (p52)r,2° PT9 (p57; hnRNP I)s
2?
=Examples of the better-known cases, bOnly the known or suspected domains/motifs are given, see also Burd, C. G. and Dreyfuss, G. (1994) Science 265, 615-621. =Known or potential binding sites, dposition specified by numbers in Fig. 2. =Pause, A. et al. (1994) EMBO J. 13, 1205-1215. fNaranda, T. et al. (1994) J. Biol. Chem. 20, 14465-14472. ~Goyer, C. etal. (1993) Mol. Cell Biol. 13, 4860-4874. hLanker, S. et al. (1992) J. Biol. Chem. 267, 21167-21171. =Hershey, J. W. B. (1991) Annu. Rev. Biochem. 60, 717-755. JFrovola, L. et al. (1994) Nature 372, 701-703. kWahle, E. and Keller, W. (1992) Annu. Rev. Biochem. 61, 419-440. 'Zhang, W. et al. (1993) Mol. Cell. Biol. 13, 7652-7665. mNickelsen, J. et al. (1994) EMBO 1. 13, 3182-3191. "Blanc, A. et al. (1992) MoL Cell. Biol. 12, 3390-3398. °Ribas, J. C, et al. (1994) J. Biol. Chem. 45, 28420-28428. PMurray, T. M. (1994) Biochemistry 33, 13910--13917. qGulyas, K. D. and Donahue, T. F. (1992) Cell 69, 1031-1042. rGottlieb, E. and Steitz, J. A. (1989) EMBO J. 8, 841-850. =Hellen, C. U. T. et al. (1993) Proc. Nat/Acad. Sci. USA 90, 7642-7646. DEAD proteins belong to a family with a distinctive core region containing eight conserved domains, one of which includes the amino acid sequence DEAD; SelB is the protein in E. coil that binds tRNA[SerlSec;CPSF, cleavage and polyadenylation specificity factor; PABPII, nuclear poly(A)-binding protein II; GM-CSF, granulocyte-macrophage colony-stimulating factor; RRM, RNA recognition motif; RNP, ribonucleoprotein; PTB, polypyrimidine-tract-binding protein.
observations such as the positive influ- the 3'-UTR to mimic the function of the ence of poly(A) tails on translation and poly(A) tail in translational initiation 27. the link between deadenylation and Moreover, selenocysteine incorporation decapping deduced from the study of at in-frame UGA codons in a number of at least some mRNA degradation path- eukaryotic mRNAs is directed by a soways 25,26. It may even be possible for called SECIS element (Fig. 3) located in
the 3'-UTRzs. Clearly, the influence of many eukaryotic structural elements recognized by proteins can extend significantly beyond the immediate vicinity of their own location in the mRNA.
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mrGppp - - - ~ ~ 1
Coding region
3'-UTR
AUG
(UGA)
F---
3
4
5'-UTR F,--~'3
2
5
6
Poly(A) tail
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Figure 2 Schematic representation of a mature eukaryotic mRNA. The mRNA bears the cap structure and poly(A) tail at its respective 5'- and 3'-ends. The coding region (boxed) is flanked by 5" and 3'-untranslated regions (UTRs). The 5'-UTR contains more-or-less stable secondary structures, such as a stern-loop, at various positions relative to the 5'-end and start codon (AUG). A locally defined secondary structure can interact relatively nonspeciflcally with mRNA-binding proteins and, if stable enough, may inhibit translational initiation. Alternatively, the nucleotide sequence in this structured region may allow binding of a specific mRNA-binding protein. Binding sites can also be located in the coding region or the 3'-UTR. In a minority of cases, internal opal stop codons function as sites for the incorporation of selenocysteine into the encoded protein. The respective regions of the mRNA are numbered to facilitate specification of interaction sites in Tables I and I1. Not all of the features shown here are. present in any one mRNA.
Control of mRHAdecay mRNA decay is also a process thought to employ a sizable cast of proteins that catalyse and control the degradative pathway25,~6. A common principle of eukaryotic mRNA degradation seems to be the involvement of one or more destabilization elements. These can be located in the reading frame, as in the yeast mRNAs MATal, HIS3 and STE3, or in either of the UTRs, although they are most commonly 3' of the gene2s,26. A frequently occurring 3'-UTR determinant in higher eukaryotic cells Is the AU-rlch element (ARE). This has been shown to confer instability on a number of otherwise stable mRNAs2° (Table I and Fig. 3). A model proposed for mRNAdecay in yeast sees deadenylation as the Initial step, followed by decapplng and 5'-to-3' exonucleolytic degradation from the 5'-end2s. Thus both the cap structure and the poly(A) tail are regarded as stabilization elements, whose removal is critical to the rate control of the whole process. Alternatives to this pathway may include deadenylationindependent decapplng, or initiation of the decay process by endonucleolytic cleavage within the 3'-UTR (or reading frame) 2s. A key question is how the various mRNA elements dictate the course and the kinetics of the decay pathway. This is likely to involve various RNAbinding proteins. So far in yeast, the specific enzyme activities and/or the genes encoding them that have been identified include those involved in decapping, 5"to-3' degradation and poly(A) shortening2S,26 (Table I). Protein activities identified in higher eukaryotic cells include a number that bind moreor-less specifically to AREs29. Of these,
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only one (AUFI) has been isolated so far (Table I). In a different system, nucleus-encoded proteins of the alga Chlamydomonas reinhardtii have been proposed to control the stability of chloroplast mRNAs (Table 1"). The relationship between mRNA stability and translation is complexzg. While changes in translational initiation rate of up to 100-fold caused by leader structure do not affect mRNA stability22.3°, premature translational termination2° or uORFsal can markedly accelerate degradation. Indeed, translation is necessary for the function of certain destabilizing elements z~. Work in yeast has revealed the participation o[ at least one protein in the nonsensemediated degradation pathway32.
Post-tmscdptionalregulationoccursin manyforumin eukaffotes A variety of ml~lA-protein interactions that regulate gene expression have been identified at different levels, including the transport (localization), translation and degradation of mRNA. The negative regulation of translational initiation mediated by a specific repressor binding to the translational initiation region of a gene is an established principle in bacteria~, and Table If shows that there are comparable examples in eukaryotes. To date, the best-characterized translational regulatory system in eukaryotes acts upon the mRNAs encoding ferritin and erythroid 5-aminolevulinate synthase (eAlAS). It involve~, binding of the iron regulatory protein (lRP) to the iron responsive element (IRE; Fig. 3) in the 5"UTR. Inhibition is more pronounced when the IRE is nearer the cap than the start codon, reminiscent of the positional effect
for stem-loop structures described by Kozak H. In vitro experiments indicate that the binding of IRP to the IRE interfetes with the stable interaction of 43 S preinitiation complexes with the mRNA34. Further work suggests that steric hindrance o[ an early stage of the 40 S-ribosome-mRNA interaction pathway can be achieved by any protein that binds sufficiently tightly to a cap-proximal specific target 35. The fact that the IRE-IRP interaction is less inhibitory further away from the 5"end is likely to be due to an increased thermodynamic driving force coupled to the scanning stage of the ribosome's progression towards the start codon (see later). Other examples of 5'-UTRmediated translational regulation can be seen in Table II. Translational regulation of eukaryotic mRNAs can also be mediated via elements in the 3"UTR, for which there seems to be no known prokaryotic precedent. Thus, the regulation of 15-1ipoxygenase (LOX) translation may involve interaction between a reticulocyte RNA-binding protein and a repeated pyrimidine-rich motif in the 3'-UTR of the LOX mRNA (Table 11 and Fig. 3). There are also examples of the regulation of maternal mRNA translation in mammalian, amphibian, Caenorhabditis elegans and insect cells (Table ID. 'Masking' proteins have beea known for some time to 'silence' maternal mRNAs until their translation needs to be switched on at a particular step of development36; but the relationship between masking and the regulation of polyadenylatlon (see below) is not always clear. More recent investigations indicate that translation can be spatially and temporally regulated via specific protein interactions with elements in the 3"UTR. In Drosophila, translation of the nanos, bicoid and hunchback mRNAs is dependent on their position on the posterior-anterior axis of the embryo (Table 11). At the same time, 3'-UTRs can also determine the localization of specific mRNAs in both embryos and somatic cells3r. There are fascinating parallels to C. elegans, in which the 3"UTRs of the sexdetermination genes tra-2 and fern-3, and of a transmembrane receptor gene ~/&l), define sites necessary for embryonic translational regulation~. 3"UTR-mediated translational regulation also occurs during spermiogenesis (Table ll). These observations underline once again the vital role played by interactions between the 3'-region of
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eukaryotic raRNAs and proLOX 3'-UTF~ that presumably me diate interactions with other NRE - - ~ ~ Nx regions o~ tt,e mP~A, with the translational machinery, G G Poiy(A) signa0 and with proteins mediating U G N N other processes, such as po!yN N N N N C A G G ARE adenylation and deadenylation. A N N--N GC Post-transcriptional reguA N N-N A-U CPE A N N--N G--C lation can also be positive. Of N--N N--N U special interest, in the light of N--N /,N--NAu U C I the hypothesis that cellular N--N U G U N A--U N G A organelles derive from ancient N--N ~_ccAC G---C C prokaryotic cells, is the obserN--N N--N 3 N--N N--G vation that nucleus-encoded N--N N--G N--N N--N UG N proteins activate psbA transN--N N U R u A o N N \ N--N N lation ~n C. re~nhardtfi chloroN-N g N A-." G . N-N plasts (Table ~I). A more U-A C G-N UUAC_ J LN-NJs Y--R recent study of the multiiuncN--N U N N Y--R N N A--N N N N--N tional NS1 protein of influenza Y--R N- N N--N N-N N-N virus provides a new example G-C N-N N-N N--N N--N of a eukaryofic translational AAAG--CAC N-N N-- N N-- N mTGpppN-- N effector, in this case acting Histone stem-aoop BRE SECTS U1A pre-mRNA TAR upon a set of viral mRNAs. Another form of positive reguFigure 3 lation is mediated by polyExamples of protein-binding sites in (pre-)mRNA. Four (consensus) stem-loop structures invoNed in adenylafion during developspecific pest-transcfiptmnam processes are compared with the TAR sequence of HN4. Many (but not ment. Many mRNAs in oocytes all) of the (conserved) nucleotides (red) known to be essential for tight binoing m~the respective proare translationally inert until teins (see Tables I and II) are located in single-stranded regions of the structures or in ~egions unlikely to conform to an undistorted helix structure. Various other mRNA-bindingsites comprise sequence activated by means of cytoelements expected to have relatively tittle secondary structure [nvoMng intramolecular base-pairing. plasmic polyadenylation ~3,~. The similarities between a number of these recognition sites suggest that the discrimination between On the other hand, deadenylsites by binding proteins is likely to involve a number of subtle interactions. NXindicates that the conation can be used to switch off served sequence motifs can be separated by different lengths of RNA. R and Y are pudne and pyrimispecific mRNAs at pro-deterdine nudeotides, respectively. The minimal sequence for TAR-Tat binding is taken from Ref. 50, and mined times3~.These examples the structure predicted for the two UIA protein-binding sites in the 3'-UTR of the U1A pre-mRNA is from Ref. 51. The sources of the other structures are to be found in TaNes q and Ba. NRE, Nos demonstrate that positive response element; ARE, ALl-rich element. TAR, trans-activation response element; CPE, cytoplasmic translational regulation can polyadenylation element. also be mediated by signals in both 5'- and 3'-UTRs. Several conserved sequence moti|s in a number of cases, mRNA degra- Principlos of mRNA~rotein interaciion Any attempt to identify general prin- have been identified in RNA-binding dation is also regulated by proteins binding to specific elements in the 3"UTR or ciples of RNA recognition by proteins in proteins 42.43. These allow proteins to in the reading frame of the mRNAz~. A eukaryotes is hampered by the general bind RNA, but generally do not confer special case is IRP, since its high-affinity lack of structural information about high specificity. Variations in noncon(apoprotein-like) form not only re- RNA-protein interactions. However, cer- served regions of proteins belonging to presses ferritin translation (Table II), tain observations provide useful hints. a given binding-motif family are likely to but also binds to IREs in the 3'-UTR of RNA helices usually assume the A-form, contribute to the generation of a range the transferrin receptor mRNA, thus indicating that the major groove of of target specificities. Future structure probably inhibiting endonucleolytic duplex RNA is likely to be too narrow determinations will provide a more attack. Stem-loops in the 3'-UTRs of to allow recognition of nucleotide se- complete picture as to what extent replication-dependent histone mRNAs quences unless the helix is distorted 4°. specificity can be achieved via the are also required for the coordination of This view seems to be supported by the recognition of secondary and 'tertiary' degradation with DNA synthesis (Table fact that consensus sequences in target structure in RNA. For example, pseudo!1 and Fig. 3). The stability of a number elements for RNA-binding proteins are knots may offer a range of possibilities of early-response-gene mRNAs, such frequently found in loops, bulges or in- for creating highly specific target strucas c-los and c-lye, is regulated by terior loops, which offer single-stranded tures4! Moreover, conformational changes destabilizing elements in the coding regions for binding (Fig. 3). None of the in the RNA induced by protein binding region~6. These function in combination known double-stranded-RNA-binding pro- may also open up additional possibilities with AREs in the 3'-UTRs in a trans- teins possesses sequence specificity4L for establishing specificity42. Another lation-dependent fashion. Although RNA- Other recognition elements are unlikely aspect is that the activity of certain RNAbinding proteins are suspected to play to form stable helix structures, although binding proteins can be modulated by a role in the regulation of stability in they may form some other type of means of conformational changes many of these systems, IRP remains the secondary structure via, for example, induced by amino acid modification, such as phosphorylation 6'z'ms'~. only one characterized (partially) so far. stacking interactions. teins
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Table ll, F~ant~es of mRNA-binding proteins involved in i~st-transcfiptional eegulatJon System/functional category
Protein: ralevant details and proposedmechanism
traludaUelud ~lulation Iron regulatory systema Iron-dependent IRP binding inhibits initiation; IRP is also a cytoplasmic aconitase; IRP2 recently discovered° 15-Upexygenase(LOX)b LOX-BP(cytoplasmic protein, 48 kDa); represses LOXtranslation Thymidylatesynthase (TS)c Negativeautoregulation involvingTS protein Dihydrofolate reductase DHFRprotein; binding may (DHFR)d autoregulate translation Yeast (L32) (compare L2)e Drosophila Mst(3)CGP~enes encoding sperm-tall proteinsf Nos posterior-anterior gradlente Translational regulation of bc/ hb~ Caenorhabditis elegans gll>l (membrane receptor gene=) tra-2=(sex-determination Eene) Other examples e.g. fern,3
RNA-bindingdomain(s) (protein)
RNA recognition ~lement
Cleft between IRP amino and carboxyltermini'
IREs in 5'-UTRsof ferritin and eALAS mRNAs
2
?
Ten repeats of pyrimidine-rich 19-nucleotide motif in 3;UTR Possibly binds heptanucleotide in stem-loop with bulge ?
6
? ?
2/3/4 2 or 6?
Proposed structure at splice site 2
Ribosomalprotein L32 autoregulates own splicing/translation Proteinsrepress translation in spermatocytes Localization and translation regulated by various gene products Nos protein involved in regulation of bicoid and hunchback
Position of RNA element
?
12-nucleotide sequence
2
?
?
6
?
Nos response elements (NREs)
6
Temporallyand spatially regulated in ? early embryo; represser unknown Possiblebinding factor (DRF)identified ?
Spatial controa invoBvesNRE-like 6 motifs Two direct repeat elements 6 (DREs) ? 6
?
AAUA@,+ other (U-dch) signals
6/7
? ?
Viral mRNAs Imperfect stem-loop?
1-3? 1/2
See above
Tf;~ mRNA; five IREs in 3'-U~R
6
Stem-loopbinding protein(s) ? stabilize mRNAduring DNA synthesis
3'-stem-loop
6
Stimulates nuclear export and/or stability of viral mRNAs
Rev-responsive element (RRE); >300 nucleotides
4/5
Represser(s) unknown
PNItlve tm~latioMI ~gulation Cytoplasmic polyadenylation39 Certain components shared with nuclear complexP; various genes regulatedq Influenza virus NS1 protein" StimulatesInitiation Light.regulated translation In Activatorcomplex; apparently Chlamydomonas re/nhar~li ~ regulatedby phosphorylation and redox potential
mnNAmM.~ Iron regulatory system/ transferrln receptorm Replication-dependent hlstone mRNAs=o
IRP
Nuel~Ntransport Revn
Arg-rich sequence
aKlausner, R. D. et al. (1993) Cell 72, 19-28. bOstareck-Lederer,A. et aL (1994) EMBO J. 13, 1476-1481. "Chu, E. et aL (1993) Prec. Natl Acad. ScL USA 90, 517-521, =Chu, E. et al. (1993) Biochemistry 32, 4756-4760. eDabena, M. D. and Warner, J. R. (1993) J. BioL Chem. 268, 19669-19674. ~Kempe, E. et aL (1993) Day. Genet, 14, 449-459. IGavls, E. R. and Lehmann, R. (1994) Nature 369, 315-318. hWharton, R. R and Struhl, G. (1991) Cell 67, 955-967. ~oodwin, E. B° et al. (1993) Cell 75, 329-339. IAhrlnger, J. and Kimble, J. (1991) Nature 349, 346-348. kDe La Luna, S. et aL J. ViroL (in press). ~Danon,A. and Mayfleld, S, P. (1994) Sclenct~266, 1717-1719. "Binder, R. et aL (1994) EMBO J. 13, 1969-1980. "Gait, M. J. and Kam, J. (1994) Trends Biochem. ScL 18, 255-259. QSamanlego, F. et al. (1994) J. Biol. Chem. 269, 30904-30910. PBilger, A. et aL (1994) Genes Dev. 8, 1106-1116. qJackson, R. J. and Standart, N. (1990) Cell 62,15-24. 'Hiding, H. et aL (1994) EMBOJ. 13, 453-461.
Many RNA-binding proteins showing relatively low affinity and specificity are expected to play an important role in the interactions of cukaryotic mRNAs during their lifetime in the cell. At least some, such as pre.RNP components, may ha~e relatively high affinity for a specific type of sequence (such as hnRNPA1), but lower general affinity for other regions of the (pre-)mRNA. Proteins interacting with relatively low affinity/specificity are more readily displaced by others that bind their
specific targets tightly. Both this and the local concentration of 'competing' RNA-binding proteins are relevant to the question of how, for example, a processed mRNA leaving the nucleus might become repopulated by cytoplasmic RNA-binding proteins that participate in, or regulate, translation and mRNA degradation (Fig. 1). Proteins that bind specific mRNA targets can have Kd values of approximately 10-9 or even lower (Table liD. None of the translation factors seems to
belong to this high-affinity group, thus providing no evidence for the existence of a general nuclear--cytoplasmic mRNAaffinity 'gradient' that would favour the binding of cytoplasmic proteins. This contrasts with the case of the nuclear poly(A)-binding protein nucleolin, whose affinity for poly(A) is lower than that of the cytoplasmic PABP (Tables I and liD. Another aspect is that even a high-af¢inity interaction has little functional impact if it is targeted to an inappropriate step in a biological
RV[WS
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process. This is reflected, for exam#e, in the positionaa modulation off the ~unction of ~RE sequences (see earlier). Thus, the evolutionary pathway of such control/regulatory systems is likely to be dictated by the necessity to optimize both the affinity/specifidty and the site of effector binding. A further consideration is that the RNA-binding proteins can be multifunct~onal, raising questions about the nature of their mPd'~]A-binding sites. In prokaryotes, the RNAobinding site may overlap with or be identical to the main binding/active site of the protein or, alternatively, can be entirely separate ss. The eukaryotic IRP is an interesting case of multifunctionality, since this repressor is also a cytoplasmic aconitase (Table ]I). Other eukaryotic RNAbinding proteins have been ~ound to function both in splicing and in translation (such as PTB) or in transcription and translation (such as FRG Y2, Laantigen and $SL2; Table l).
R~A recognition element
Dissoci~on constant, o, i 4-6 x :tg-~ ~ I-3 × !0 -~ 7 × 10-a M 3 × :!0-7 ~,t > 2 x 10 -7 ~ 4 x 10 ,8M~ < 1-3 x 10-9 ~
enF-4A
nRESfragment (poUiovirus) UAGGGA/U motif Naturan spOicesite sequences Nonspecific RNA Homopogymers aRESfragment (EMCV) RNA secondary structure with purina-rich bubble TAR mZGpppG mRNA cap RNA
eQF-4B
RNA
5 x I0 -z Mh
La Ip52) hnRNP A1 Po~ypydmidine-tract-bmding protein (PTB) Rev Tat eBF-4E(eOF-4~)
Poly(A)-binding protein (PABP) ~ron regulatory protein - Fe
3 x 10-9 M: -5 x 10-6 M~ ~5 x 10-7 M~ > 10~ M~'
STNV-RNA
10-7--10-8 Mi
Poly(A) 8 nuc~eotides 12 nucleotides IRE
6 x 10-~ 2 x 10"~ ~#
+ Fe
/ - 3 x 10 -~ ~ 2-5 x 10-~ M"
aEach of these values was obtained under different conditions, and this table is intended to serve ongy as a rough guide. Direct comparisons si~ould therefore be interpreted with caution, bSv[tkin, Y. V. et aL (!994~ J. ViroL 68, 1544-1550. ©Burd, C. G. and Dreyfuss, G. (1994) EMBO J. 13, 1197-!204. dWitherelL G. W. et al. (1993) Biochemistry 32, 8268-8275. eHeaphy, S. et aL (1991) Proc. Natl Acad. ScL USA 88, 7366-7370. fGait, M. J. and Kern, J. (1994) Trends. Biochem. ScL 18, 255-259. ~Goss, D. J. e~ aL (1990) Biochemistry 29, 5008-5012. bMSthet, N. et aL (1994) MoL Cell. BioL 14, 2307-2316. ~Butler, J. S. and Clark, J. M., Jr (1984) Biochemistry 23, 809-815. JSachs, A. B. et aL (1987) Mol. Cell. Biol. 7, 3268-3276; nucleolin has a lower affinity than PABP (H. Wormington, pets. commun), hHaile, D. J. et al. (1989) MoL Ceil. Biol. 9, 5055-5061.
Cenc~u~ingremarks The various examples of control and regulation at the mRNA level manifest both familiar and new functional prindples. One striking aspect is ~he participation of the 3CUTR as a regulatory site in such a diversity of systems. Not so new is the observation that a great deal o[ control and regulation is exercised via the initiation step of translation 33. Current knowledge of eukaryotic
mRNAobhlding proteins derives from studies of a wide range of genes and organisms, but only in a few cases is it more than superficial. Similarly, very few of the RNA-binding sites have been studied in detail. A major task will therefore be to obtain mere structural information about various mRNA-protein interactions in order to determine the structural basis of different levels of affinity and specificity for given binding motifs. However, an equally important objective will be the quantitative (thermodynamic and kinetic) characterization of these interactions in terms of their physiological functions. This will require a balanced combination of in vivo and in vitro experimental strategies. Comparisons also need to be made with other RNA-protein interactions relevant to gene expression involving, for example, snRNA~, tRNA4s, ribosomal RNA~, and the guide RNAs involved in editing~7. Apart from the expected revelations about post-transcriptional gene expression, the coming years may also
bring new insights into the molecular basis of diverse diseases ~s,49and perhaps even novel strategies for their treatment. References J. Rosbash, M. and Singer, R. H. (1993) Cell 75, 399-401 2 Guthrie, C. (1991) Science 253, 157-163 3 Dargemont, C. and KOhn, L. C. (1992) J. Cell. Biol. 118, 1-9 4 Leibkomiczh, F. et al. (1992) Proc. Natl Acad. Sci. USA 89, 9612-9616 5 Lang, V. et aL (1994) J. Biol. Chem. 269, 6117-6123 6 Altmann, M. and Trachsel, H. (1993) Trends Biochem. Sci. 18, 429-432 7 Merrick, W. C. (1992) MicrobioL Rev. 56, 291-315 8 Izaurralde, E. et aL (1994) Cell 78, 657-668 9 Lazaris-Karatzas, A. et aL (1990) Nature 345, 544-547 10 Altmann, M. et at. (1988) J. BioL Chem. 263, 17229-17232 11 Kozak, M. (1991) J. BioL Chem. 266, 19867-19870 12 Kozak, M. (1992) Crit. Rev. Biochem. MoL BioL 27, 385-402 13 Vega Laso, M. R. et al. (1993) J. BioL Chem. 268, 6453-6462 14 Kozak, M. (1991) J. Cell. Biol. 115, 887-903 15 Sonenberg, N. (1994) Curr. Opin. Goner. Dev. 4, 310-315 16 Sagliocco, F. A. et aL (1993) J. Biol. Chem. 268, 26522-26530 17 Donahue, T. F. et aL (1988) (;ell 54, 621-632 18 Hinnebusch, A. G. (1994) Trends Biochem. Sci. 19, 409-414 19 Rex, G. et aL (1994) J. BioL Chem. 269, 18118-18127 20 Jackson, R. J. et aL (1994) MoL BioL Repro. 19, 147-159 21 Brown, N. G. et aL (1994) MoL Cell. Biol. 14, 1045-1053 22 Sagliocco, F. A. et al. (1994) J. Biol. Chem. 269, 18630-18637 23 Sachs, A. and Wahle, E. (1993) J. Biol. Chem. 268, 22955-22958
24 Munroe, D. and Jacobson, A. (1990) MoL CelL BioL 10, 3441-3455 25 Decker, C. J. and Parker, R. (1994) Trends Biochem. Sci. 19, 336-340 26 Belasco, J. G. and Brawerman, G. (1993) Control of Messenger RNA Stability, Academic Press 27 Leathers, V. et al. (1993) Mol. Cell. BioL 13, 5331-5347 28 Berry, M. J. and Larsen, P. R. (1993) Biochem. Soc. Trans. 21, 827-832 29 Sachs, A. B. (1993) Ceil 74, 413-421 30 Beelman, C. A. and Parker, R. (1994) J. Biol. Chem. 269, 9687-9692. 31 Oliveira, C. and McCarthy, J. E. G. i1995) J. Biol. Chem. 270, 8936-8943 32 Leeds, P. et aL (1992) MoL CelL Biol. 12, 2165-2177 33 McCarthy, J. E. G. and Gualerzi, C. (1990) Trends Genet. 6, 78-85 34 Gray, N. K. and Hentze, M. W. (1994) EMBO J. 13, 3882-3891 35 Stripecke, R. et aL (1994) MoL Cell. BioL 14, 5898-5909 36 Standart, N. (1992) Sere. Dev. BioL 3, 367-379 37 Singer, R. H. (1993) Curt. BioL 3, 719-721 38 Evans, T. C. et aL (1994) Cell 77, 183-194 39 Wickens, M. (1992) Sere. Day. BioL 3, 399-412 40 Steitz, T. A. (1993) in The Ri~JAWorld (Gesteland, R. F. and Atkins, J. F., eds), pp. 219-237, Cold Spring Harbor Laboratory Press 41 Bass, B. L. etal. (1994) Curt. Biol. 4, 301-314 42 Mattaj, I. W. (1993) Cell 73, 837-840 43 Kenan, D. J. et aL (1991) Trends Biochem. ScL 16, 214-220 44 Pleij, C. W. A. (1994) Curr. Opin. Struct. Biol. 4, 337-344 45 Gieg6, R. et al. (1993) Prog. Nucleic Acid Res. Mol. BioL 45, 129-206 46 Rau(~,H. A. and Planta, R. J. (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 89-129 47 G6ringei', H. U. et aL (1994) Proc. Natl Acad. Sci. USA 91,1776-1780 48 Siomi, H. et aL (1993) Cell 74, 291-298 49 Ma, K. et aL (1993) Cell 75,1287-1295 50 Klaver, B. and Berkhout, B. (1994) EMBO J. 13, 2650-2659 51 Oubridge, C. etal. (1994) Nature 372, 432-438
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~A=PROTE~N interactions are involved in most of the component steps of gene expression, including the splicing, nucieocytoplasmic transport, translation and degradation of messenger P~A (mP~A) and its precursors ~ig. ]). It is now apparent that all of these involve the mo~eoorqess spedfic recognition of sequences and structural elements in ~RNA molecules by an awe-inspiring va~~:etyof proteins. This is a relatively new field, generating many challenging questions. Of overriding importance is the necessity to identif3, the structural and functional principles underlying the known interactions and their roles in the cell. In the following we attempt to convey an impression of some of these emerging principles, focusing primarily on cytoplasmic events. For the sake of clarity, we have generally tried to distinguish between RNA-protein interactions intrinsic to the mechamsm and (constitutive) control of processes in the cell, and those involved in regulating these processes in response to environmental or developmental influences. This is analogous to the distinction between 'housekeeping' and regulatory genes. RNA.bindingproteins peffoml many essential functions in post-transcriptional gone expression mRNA transcribed from RNA polymerase II (Pol I1) promoters undergoes a long series of interactions with various proteins soon after synthesis has begun. Early events, of great significance for the stability and function of the transcript, are the capping of the 5'-terminal nucleotide and the polyadenylation of the 3'-end generated by cleavage at predetermined sites. PremRNA processing involves spliceosomal proteins and nuclear pre-messenget ribonucleoproteins (pre-mRNPs), also known as heteronuclear ribonucleoproteins (hnRNPs) (Fig. 1), many of which are, as yet, poorly characterized. Mature mRNA is accompanied by various proteins through the nuclear pores into the cytoplasm. Current knowledge of these processes is reviewed elsewhere and is not the main subject of this article ]-3. A whole new set of protein factors (eukaryotic translation initiation J. E. G. McCarthyand H. Ko,mus are at the Departmentof GeneExpression,National BiotechnologyResearchCentre(GBF), MascheroderWeg1, i)-38124 Braunschweig, Germany. © 1995, Elsevier Science Ltd 0968- 0004•95]$09.50
Cyto#a m NA ?rote n ateraetie ia e karyetie gene o
John E. G. McCarthy and Heike Kotimus Post-transcriptional mechanisms contribute in many important ways te the overalU control and regulation of gone expression, and in doing so employ a veritable army of proteins that bind a wide range of targets in messenger RNA (mRNA). The full range of these RNA-protein interactions is on;t just beginning to emerge, and much remains to be learned about the mechanisms underlying the rapidly increasing number of regulatory systems now being described. factoc% elFs, and the factors involved in elongation and termination) is subsequel:tly required for the cytoplasmic process of translation, although at least one of these (elF-4E) may be able to interact with mRNA and/or ribosomal components in both the cytoplasm and the nucleus 4,5.The most complex phase of translation is initiation. At least 12 different elFs are thought to be involved in this process 6,7, and those with known RNA-binding activities are included in Table I. A core issue concerns the pathway of translational initiation as well as the kinetics and thermodynamics of the component steps. A number of assumptions about the pathway have been made on the basis of studies of partial reactions involving isolated factors in vitro. Thus, many questions remain open regarding the functional roles of the various eIF-mRNA interactions believed to participate in initiation. Perhaps the most specifically targeted mRNA-binding elF is elF-4E (elF-4a), which is an essential component of the cytoplasmic cap-binding complex (elF417). A nuclear cap-binding complex also exists 8, which presumably has to be displaced from the cap at some point, and is eventually replaced by elF-4F, elF-4E has hit the news recently because its overproduction leads to transformation or abnormal growth in certain mammalian cells 9. Whether this is due to the suspected nuclear and/or known cytoplasmic function(s) of this factor remains unclear. The structural basis of this protein's affinity for the mRNA cap is unknown. So far, a number of con-
served tryptophan residues are suspected to be involved I°. Previously characterized RNA-binding motifs have been identified in other elF's (Table 1), although the functional significance of these motifs has yet to be defined.
Translationa]control via the 5'~'R of the mRNA Translational initiation can be controlled by intramolecular base-pairing within the 5'-untranslated region (UTR) of the mRNA. The mechanism has a bearing on the ways that translation can be regulated by RNA-binding proteins, and will be considered in some detail here. A hairpin seems to inhibit translation more effectively in mammalian systems when placed close to the 5'-end of the mRNA than an equivalent structure located further downstreamn. This could be due to the fact that in the 5'-position the structure blocks cap-binding by the 43 S proinitiation complex, whereas a more distal structure might only block the 'scanning' process, during which the ribosome is thought progressively to screen the 5'-UTR for potential start codons ~2 (see Fig. 2). Alternatively, a stem-loop near the 5'-end may inhibit a subsequent intermediate reaction, such as a transitional step towards 'scanning', rather than the initial binding of the 40 S ribosome. This could, in turn, allow reversible binding of the 40S ribosome to the cap or cap-binding complex, but prevent its further translocation along the mRNA. The significance of the position of specific sequences or structures
191
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Pre-mRNP Capping
/
Splicing (snRNPs)
Polyadenylation mRNP
/
Nuclear
Exchange of binding proteins(?) mRNA (localization?) RNAses
~:
elFs, eEFs, eRFs etc.
> Translation
l Regulatory factors
Decay Figure 1 Assumed pathway of RNA polymerase !1 (Pol II) transcripts in the eukaryotic cell. The initial product of transcription is pre-mRNA(or heteronuclearRNA,hnRNA),which is bound rapidlyby proteins forming ribonucleoprotein particles (pre-mRNP or hnRNP). The pre-mRNA is processedand spliced before being escorted out of the nucleus by as yet poorly characterized proteins. At least some of these must be replaced by cytoplasmic proteins that are involved in the translation and degradation of mature mRNA. These include the eukaryotic initiation factors (elFs), elongation factors (eEFs) and release factors (eRFs), as well as exo- and endoribonucleases. Other mRNA.binding proteins regulate the various steps of post-transcriptional gene expression or may be involved in the localization of the mRNA.
in the mRNA in terms of their functional roles is a recurrent theme in the control of gene expression. However, in vivo experiments performed with yeast show that not all eukaryotes need to follow the same pattern of positional modulation of translational control 13. Extreme cases of structural inhibition are observed in the 5'-leaders of a large number of mammalian mRNAs, many of which encode proteins of regulatory significance, such as oncoproteins and transcription factors 14. How does a scanning preinitiation complex negotiate inhibitory structures that stand between it and the start codon? At least in higher eukaryotes, elF-4A and elF-4B are thought to be required for the entry of 40 S ribosomes Into the initiation pathway via their interaction with mRNA. Since elF-4Ahas weak ATP-hydrolysis-dependent hellcase activity that is greatly enhanced by elF-4Bis, it has been proposed that these two factors, in combination with components of elF-4F, can catalyse the unwinding of otherwise inhibitory structures in the 5'-UTRT. However, it is not known whether these factors unwind the 5'-UTR independently of the 40 S ribosome in vioo ~2. Whatever the mechanism involved, it has to be asked
192
whether the unfolding of a stem-loop structure occurs repeatedly during successive cycles of translation. One possibility, for example, Is that once the Initiation machinery Is loaded onto the 5'-UTR, the stem-loop is held in an 'open' conformation by repeatedly scanning ribosomes 16. Translational initiation at the start codon is also influenced by the context sequence of the AUGn. Here, recognition of the AUG by the 43 S preinitiation complex is dependent upon Watson-Crick base-pairing with the initiator transfer RNA (tRNA) in a process that is influenced by elF-2 (Ref. 17). While effectively controlling the fidelity of an RNA-RNA interaction, elF-2 is not thought to recognize the start codon directly itself. Although eukaxyotic translational initiation is usually a process involving caP
mammalian leaders bearinguORFshave been reported, but little is known about their functional significance|4. Eukaryotic ribosomes reinitiate poorly compared with their prokaryotic counterparts, which frequently and efficiently perform this task via a pathway that, in certain respects, resembles eukaryotic 'scanning'~9. The mechanistic details of eukaryotic reinitiation are no clearer than those of normal cap-dependent initiation, but the kinetics of the process can be regulated by the availa~)ility of active eIF-2~GTPwhich, in turn, is regulated by the phosphory|ation status of the elF-2~ subunit ~8. A radically different form of eukaryo otic translational initiation has been proposed, in which 'internal ribosome entry segments' (IRESs) allow cap-indepel~dent ribosome binding2°. Internal initiation is believed to occur on picomaviral mRNAs as well as on a few cellular mRNAs (such as that encoding the immunoglobulin heavy-chain-binding protein BiP2°). Various in vitro binding and crosslinking experiments have pointed to the possible involvement of a number of additional P,bJA-binding proteins in initiation on picomaviral mRNAs, including two that bind polypyrimidine tracts (PTB and the La-antigen; Table D. An IRES is thought to act, in some ways, analogously to a prokaryotic translational initiation region, but the mechanism by which IRES-binding proteins could mediate ribosomal binding is unknown. Finally, translation in eukaryotic organelles is more akin to that in prokaryotes, while at the same time being dependent on many nucleusencoded proteins2L A number of these are required for the translation of mitochondrial mRNAs (Table I).
Intramolecularinteractionsbetweenthe 5; and 3'-UTRs Several lines of evidence point to the importance of 'long-range' interactions in many post-transcriptional processes. For example, biochemical and genetic evidence indicates that the 3" and 5"UTRs on the same mP~IA (see Fig. 2)
can interact with each other 13,16,22and that at least one protein binding to the poly(A) tail [the poly(A)-binding protein, PABP] might interact with the preinitiation complex, although not necessarily directly23,24. While the relationship between the 5'and 3'-UTRs remains to be clarified, the concept of 'communication' between the two would help explain
TIBS 2 0 -
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System/functional category a
Protein: reJevant detai~s and proposed mechanism
RNA-bindin8 domain(s) ~ (protein)
RNA recognition element~
Posit~on of R~A eIement~
Heterotrimeric ~actor, involved in initiation-site recognition 46 kOa; ATP-dependent sin~Jestranded-RNA-binding protein; RNA heBicase; RNA-dependent ATPase 70 kDa (50 kDa in yeast); stimulates activities of eOF-4A and eOF-4F Cap-complex protein in higher/ ~owereukaryotes
Zinc(H)fingerin ~-subunit
tRNA/mRNA
?
TypicaOconserved regions of DEAD proteins
Nonspecific; minima~ size: 12-18 nucIeotJdes
1-3?
Amino-terminal RRM + Arg-rich region
Nonspecific
1-3?
RRM4ike + RNP-tike sequence elements Not defined Cap recognition via eOF-4E
Unknown
?
mZGpppX mZGppp×
1 1
?
SECJS
6
eRF1 unlikely to be only type of termination factor
?
Stop codon(s)
5
Nucleus-encoded proteins essential for translation of certain mitochondrial mRNAs
?
Sites in cox2/3 mRNAs
2?
Various protetins, including CPSF, poly(A) polymerase, PABPH
?
Poly(A), AAUAAAand others
6/7
AUF1, p37 AUF1
Two RRMs
6
Possibly stabilized by 47 kDa protein
?
One or more AREs in U-rich context ?
Binds poly(A) tail: stimulates translation and deadenylation Covalently binds to cad structure DNA/RNA binoing; transcription factor; binds maternal mRNA in Xenopus oocytes ATP-dependent helicase Cellular nut=antigen; binds U-rich transcript 3;ends; suspected internal 'initiation factor' Binds polypyrimidine tracts: suspected to act in pre-mRNA processing and internal initiation
Four RRMs
Poly(A)
7
Two in Gag-Pol form Basic/aromatic domains
mTGpppX Low specificity
1 ?
? RRM
Stem-loops Proposed: low-specificity binding to poiy(U), e.g iRES
2? 2/3?
Three RRMs
Suspected to bind to various iRES 2/3
TCans~onampathway Initiation factors (eIFs) eE-2 (Ref. 17)
' eE4A e
eOF4Bf p220/p150 (elF-4~/)~
eOF-4E(eOF-4~)"
ATP-independentcap binding
eOF-4P'~
Cap-binding complex; mammaOian: eOF-4A,eOF-4E,p220; yeast: eOF-4E(p24), p20, p150
Elongation factors Selenocysteine incorporation 2a Unknown factor (equivaUent of E. coil SeOB?) Termination f~ctors eRF1/sup4~ Mitochonddal 'activators' Nuclear PETgenes21
Nu©leat' po|yadeny]ation k 3'-end formation/poly(A) synthesis raRNA stability Rapid degradation of c-myc, c-los and GM-CSFmRNAsz Stabilization of chloroplast psbD mRNAm Other exantples Poly(A)-binding protein (PABP) L-A coat protein',° FRG Y2 (mRNP4)P
Yeast SSL2" La (p52)r,2° PT9 (p57; hnRNP I)s
2?
=Examples of the better-known cases, bOnly the known or suspected domains/motifs are given, see also Burd, C. G. and Dreyfuss, G. (1994) Science 265, 615-621. =Known or potential binding sites, dposition specified by numbers in Fig. 2. =Pause, A. et al. (1994) EMBO J. 13, 1205-1215. fNaranda, T. et al. (1994) J. Biol. Chem. 20, 14465-14472. ~Goyer, C. etal. (1993) Mol. Cell Biol. 13, 4860-4874. hLanker, S. et al. (1992) J. Biol. Chem. 267, 21167-21171. =Hershey, J. W. B. (1991) Annu. Rev. Biochem. 60, 717-755. JFrovola, L. et al. (1994) Nature 372, 701-703. kWahle, E. and Keller, W. (1992) Annu. Rev. Biochem. 61, 419-440. 'Zhang, W. et al. (1993) Mol. Cell. Biol. 13, 7652-7665. mNickelsen, J. et al. (1994) EMBO 1. 13, 3182-3191. "Blanc, A. et al. (1992) MoL Cell. Biol. 12, 3390-3398. °Ribas, J. C, et al. (1994) J. Biol. Chem. 45, 28420-28428. PMurray, T. M. (1994) Biochemistry 33, 13910--13917. qGulyas, K. D. and Donahue, T. F. (1992) Cell 69, 1031-1042. rGottlieb, E. and Steitz, J. A. (1989) EMBO J. 8, 841-850. =Hellen, C. U. T. et al. (1993) Proc. Nat/Acad. Sci. USA 90, 7642-7646. DEAD proteins belong to a family with a distinctive core region containing eight conserved domains, one of which includes the amino acid sequence DEAD; SelB is the protein in E. coil that binds tRNA[SerlSec;CPSF, cleavage and polyadenylation specificity factor; PABPII, nuclear poly(A)-binding protein II; GM-CSF, granulocyte-macrophage colony-stimulating factor; RRM, RNA recognition motif; RNP, ribonucleoprotein; PTB, polypyrimidine-tract-binding protein.
observations such as the positive influ- the 3'-UTR to mimic the function of the ence of poly(A) tails on translation and poly(A) tail in translational initiation 27. the link between deadenylation and Moreover, selenocysteine incorporation decapping deduced from the study of at in-frame UGA codons in a number of at least some mRNA degradation path- eukaryotic mRNAs is directed by a soways 25,26. It may even be possible for called SECIS element (Fig. 3) located in
the 3'-UTRzs. Clearly, the influence of many eukaryotic structural elements recognized by proteins can extend significantly beyond the immediate vicinity of their own location in the mRNA.
~3
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TaBS 20 - MAY 1995 .
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.
.
.
.
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.
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.
.
i
Cap
mrGppp - - - ~ ~ 1
Coding region
3'-UTR
AUG
(UGA)
F---
3
4
5'-UTR F,--~'3
2
5
6
Poly(A) tail
7
Figure 2 Schematic representation of a mature eukaryotic mRNA. The mRNA bears the cap structure and poly(A) tail at its respective 5'- and 3'-ends. The coding region (boxed) is flanked by 5" and 3'-untranslated regions (UTRs). The 5'-UTR contains more-or-less stable secondary structures, such as a stern-loop, at various positions relative to the 5'-end and start codon (AUG). A locally defined secondary structure can interact relatively nonspeciflcally with mRNA-binding proteins and, if stable enough, may inhibit translational initiation. Alternatively, the nucleotide sequence in this structured region may allow binding of a specific mRNA-binding protein. Binding sites can also be located in the coding region or the 3'-UTR. In a minority of cases, internal opal stop codons function as sites for the incorporation of selenocysteine into the encoded protein. The respective regions of the mRNA are numbered to facilitate specification of interaction sites in Tables I and I1. Not all of the features shown here are. present in any one mRNA.
Control of mRHAdecay mRNA decay is also a process thought to employ a sizable cast of proteins that catalyse and control the degradative pathway25,~6. A common principle of eukaryotic mRNA degradation seems to be the involvement of one or more destabilization elements. These can be located in the reading frame, as in the yeast mRNAs MATal, HIS3 and STE3, or in either of the UTRs, although they are most commonly 3' of the gene2s,26. A frequently occurring 3'-UTR determinant in higher eukaryotic cells Is the AU-rlch element (ARE). This has been shown to confer instability on a number of otherwise stable mRNAs2° (Table I and Fig. 3). A model proposed for mRNAdecay in yeast sees deadenylation as the Initial step, followed by decapplng and 5'-to-3' exonucleolytic degradation from the 5'-end2s. Thus both the cap structure and the poly(A) tail are regarded as stabilization elements, whose removal is critical to the rate control of the whole process. Alternatives to this pathway may include deadenylationindependent decapplng, or initiation of the decay process by endonucleolytic cleavage within the 3'-UTR (or reading frame) 2s. A key question is how the various mRNA elements dictate the course and the kinetics of the decay pathway. This is likely to involve various RNAbinding proteins. So far in yeast, the specific enzyme activities and/or the genes encoding them that have been identified include those involved in decapping, 5"to-3' degradation and poly(A) shortening2S,26 (Table I). Protein activities identified in higher eukaryotic cells include a number that bind moreor-less specifically to AREs29. Of these,
194
only one (AUFI) has been isolated so far (Table I). In a different system, nucleus-encoded proteins of the alga Chlamydomonas reinhardtii have been proposed to control the stability of chloroplast mRNAs (Table 1"). The relationship between mRNA stability and translation is complexzg. While changes in translational initiation rate of up to 100-fold caused by leader structure do not affect mRNA stability22.3°, premature translational termination2° or uORFsal can markedly accelerate degradation. Indeed, translation is necessary for the function of certain destabilizing elements z~. Work in yeast has revealed the participation o[ at least one protein in the nonsensemediated degradation pathway32.
Post-tmscdptionalregulationoccursin manyforumin eukaffotes A variety of ml~lA-protein interactions that regulate gene expression have been identified at different levels, including the transport (localization), translation and degradation of mRNA. The negative regulation of translational initiation mediated by a specific repressor binding to the translational initiation region of a gene is an established principle in bacteria~, and Table If shows that there are comparable examples in eukaryotes. To date, the best-characterized translational regulatory system in eukaryotes acts upon the mRNAs encoding ferritin and erythroid 5-aminolevulinate synthase (eAlAS). It involve~, binding of the iron regulatory protein (lRP) to the iron responsive element (IRE; Fig. 3) in the 5"UTR. Inhibition is more pronounced when the IRE is nearer the cap than the start codon, reminiscent of the positional effect
for stem-loop structures described by Kozak H. In vitro experiments indicate that the binding of IRP to the IRE interfetes with the stable interaction of 43 S preinitiation complexes with the mRNA34. Further work suggests that steric hindrance o[ an early stage of the 40 S-ribosome-mRNA interaction pathway can be achieved by any protein that binds sufficiently tightly to a cap-proximal specific target 35. The fact that the IRE-IRP interaction is less inhibitory further away from the 5"end is likely to be due to an increased thermodynamic driving force coupled to the scanning stage of the ribosome's progression towards the start codon (see later). Other examples of 5'-UTRmediated translational regulation can be seen in Table II. Translational regulation of eukaryotic mRNAs can also be mediated via elements in the 3"UTR, for which there seems to be no known prokaryotic precedent. Thus, the regulation of 15-1ipoxygenase (LOX) translation may involve interaction between a reticulocyte RNA-binding protein and a repeated pyrimidine-rich motif in the 3'-UTR of the LOX mRNA (Table 11 and Fig. 3). There are also examples of the regulation of maternal mRNA translation in mammalian, amphibian, Caenorhabditis elegans and insect cells (Table ID. 'Masking' proteins have beea known for some time to 'silence' maternal mRNAs until their translation needs to be switched on at a particular step of development36; but the relationship between masking and the regulation of polyadenylatlon (see below) is not always clear. More recent investigations indicate that translation can be spatially and temporally regulated via specific protein interactions with elements in the 3"UTR. In Drosophila, translation of the nanos, bicoid and hunchback mRNAs is dependent on their position on the posterior-anterior axis of the embryo (Table 11). At the same time, 3'-UTRs can also determine the localization of specific mRNAs in both embryos and somatic cells3r. There are fascinating parallels to C. elegans, in which the 3"UTRs of the sexdetermination genes tra-2 and fern-3, and of a transmembrane receptor gene ~/&l), define sites necessary for embryonic translational regulation~. 3"UTR-mediated translational regulation also occurs during spermiogenesis (Table ll). These observations underline once again the vital role played by interactions between the 3'-region of
T~BS 2 0 -
MAY 1995
eukaryotic raRNAs and proLOX 3'-UTF~ that presumably me diate interactions with other NRE - - ~ ~ Nx regions o~ tt,e mP~A, with the translational machinery, G G Poiy(A) signa0 and with proteins mediating U G N N other processes, such as po!yN N N N N C A G G ARE adenylation and deadenylation. A N N--N GC Post-transcriptional reguA N N-N A-U CPE A N N--N G--C lation can also be positive. Of N--N N--N U special interest, in the light of N--N /,N--NAu U C I the hypothesis that cellular N--N U G U N A--U N G A organelles derive from ancient N--N ~_ccAC G---C C prokaryotic cells, is the obserN--N N--N 3 N--N N--G vation that nucleus-encoded N--N N--G N--N N--N UG N proteins activate psbA transN--N N U R u A o N N \ N--N N lation ~n C. re~nhardtfi chloroN-N g N A-." G . N-N plasts (Table ~I). A more U-A C G-N UUAC_ J LN-NJs Y--R recent study of the multiiuncN--N U N N Y--R N N A--N N N N--N tional NS1 protein of influenza Y--R N- N N--N N-N N-N virus provides a new example G-C N-N N-N N--N N--N of a eukaryofic translational AAAG--CAC N-N N-- N N-- N mTGpppN-- N effector, in this case acting Histone stem-aoop BRE SECTS U1A pre-mRNA TAR upon a set of viral mRNAs. Another form of positive reguFigure 3 lation is mediated by polyExamples of protein-binding sites in (pre-)mRNA. Four (consensus) stem-loop structures invoNed in adenylafion during developspecific pest-transcfiptmnam processes are compared with the TAR sequence of HN4. Many (but not ment. Many mRNAs in oocytes all) of the (conserved) nucleotides (red) known to be essential for tight binoing m~the respective proare translationally inert until teins (see Tables I and II) are located in single-stranded regions of the structures or in ~egions unlikely to conform to an undistorted helix structure. Various other mRNA-bindingsites comprise sequence activated by means of cytoelements expected to have relatively tittle secondary structure [nvoMng intramolecular base-pairing. plasmic polyadenylation ~3,~. The similarities between a number of these recognition sites suggest that the discrimination between On the other hand, deadenylsites by binding proteins is likely to involve a number of subtle interactions. NXindicates that the conation can be used to switch off served sequence motifs can be separated by different lengths of RNA. R and Y are pudne and pyrimispecific mRNAs at pro-deterdine nudeotides, respectively. The minimal sequence for TAR-Tat binding is taken from Ref. 50, and mined times3~.These examples the structure predicted for the two UIA protein-binding sites in the 3'-UTR of the U1A pre-mRNA is from Ref. 51. The sources of the other structures are to be found in TaNes q and Ba. NRE, Nos demonstrate that positive response element; ARE, ALl-rich element. TAR, trans-activation response element; CPE, cytoplasmic translational regulation can polyadenylation element. also be mediated by signals in both 5'- and 3'-UTRs. Several conserved sequence moti|s in a number of cases, mRNA degra- Principlos of mRNA~rotein interaciion Any attempt to identify general prin- have been identified in RNA-binding dation is also regulated by proteins binding to specific elements in the 3"UTR or ciples of RNA recognition by proteins in proteins 42.43. These allow proteins to in the reading frame of the mRNAz~. A eukaryotes is hampered by the general bind RNA, but generally do not confer special case is IRP, since its high-affinity lack of structural information about high specificity. Variations in noncon(apoprotein-like) form not only re- RNA-protein interactions. However, cer- served regions of proteins belonging to presses ferritin translation (Table II), tain observations provide useful hints. a given binding-motif family are likely to but also binds to IREs in the 3'-UTR of RNA helices usually assume the A-form, contribute to the generation of a range the transferrin receptor mRNA, thus indicating that the major groove of of target specificities. Future structure probably inhibiting endonucleolytic duplex RNA is likely to be too narrow determinations will provide a more attack. Stem-loops in the 3'-UTRs of to allow recognition of nucleotide se- complete picture as to what extent replication-dependent histone mRNAs quences unless the helix is distorted 4°. specificity can be achieved via the are also required for the coordination of This view seems to be supported by the recognition of secondary and 'tertiary' degradation with DNA synthesis (Table fact that consensus sequences in target structure in RNA. For example, pseudo!1 and Fig. 3). The stability of a number elements for RNA-binding proteins are knots may offer a range of possibilities of early-response-gene mRNAs, such frequently found in loops, bulges or in- for creating highly specific target strucas c-los and c-lye, is regulated by terior loops, which offer single-stranded tures4! Moreover, conformational changes destabilizing elements in the coding regions for binding (Fig. 3). None of the in the RNA induced by protein binding region~6. These function in combination known double-stranded-RNA-binding pro- may also open up additional possibilities with AREs in the 3'-UTRs in a trans- teins possesses sequence specificity4L for establishing specificity42. Another lation-dependent fashion. Although RNA- Other recognition elements are unlikely aspect is that the activity of certain RNAbinding proteins are suspected to play to form stable helix structures, although binding proteins can be modulated by a role in the regulation of stability in they may form some other type of means of conformational changes many of these systems, IRP remains the secondary structure via, for example, induced by amino acid modification, such as phosphorylation 6'z'ms'~. only one characterized (partially) so far. stacking interactions. teins
195
. . . . .
~
........
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MAY 1995
!l
Table ll, F~ant~es of mRNA-binding proteins involved in i~st-transcfiptional eegulatJon System/functional category
Protein: ralevant details and proposedmechanism
traludaUelud ~lulation Iron regulatory systema Iron-dependent IRP binding inhibits initiation; IRP is also a cytoplasmic aconitase; IRP2 recently discovered° 15-Upexygenase(LOX)b LOX-BP(cytoplasmic protein, 48 kDa); represses LOXtranslation Thymidylatesynthase (TS)c Negativeautoregulation involvingTS protein Dihydrofolate reductase DHFRprotein; binding may (DHFR)d autoregulate translation Yeast (L32) (compare L2)e Drosophila Mst(3)CGP~enes encoding sperm-tall proteinsf Nos posterior-anterior gradlente Translational regulation of bc/ hb~ Caenorhabditis elegans gll>l (membrane receptor gene=) tra-2=(sex-determination Eene) Other examples e.g. fern,3
RNA-bindingdomain(s) (protein)
RNA recognition ~lement
Cleft between IRP amino and carboxyltermini'
IREs in 5'-UTRsof ferritin and eALAS mRNAs
2
?
Ten repeats of pyrimidine-rich 19-nucleotide motif in 3;UTR Possibly binds heptanucleotide in stem-loop with bulge ?
6
? ?
2/3/4 2 or 6?
Proposed structure at splice site 2
Ribosomalprotein L32 autoregulates own splicing/translation Proteinsrepress translation in spermatocytes Localization and translation regulated by various gene products Nos protein involved in regulation of bicoid and hunchback
Position of RNA element
?
12-nucleotide sequence
2
?
?
6
?
Nos response elements (NREs)
6
Temporallyand spatially regulated in ? early embryo; represser unknown Possiblebinding factor (DRF)identified ?
Spatial controa invoBvesNRE-like 6 motifs Two direct repeat elements 6 (DREs) ? 6
?
AAUA@,+ other (U-dch) signals
6/7
? ?
Viral mRNAs Imperfect stem-loop?
1-3? 1/2
See above
Tf;~ mRNA; five IREs in 3'-U~R
6
Stem-loopbinding protein(s) ? stabilize mRNAduring DNA synthesis
3'-stem-loop
6
Stimulates nuclear export and/or stability of viral mRNAs
Rev-responsive element (RRE); >300 nucleotides
4/5
Represser(s) unknown
PNItlve tm~latioMI ~gulation Cytoplasmic polyadenylation39 Certain components shared with nuclear complexP; various genes regulatedq Influenza virus NS1 protein" StimulatesInitiation Light.regulated translation In Activatorcomplex; apparently Chlamydomonas re/nhar~li ~ regulatedby phosphorylation and redox potential
mnNAmM.~ Iron regulatory system/ transferrln receptorm Replication-dependent hlstone mRNAs=o
IRP
Nuel~Ntransport Revn
Arg-rich sequence
aKlausner, R. D. et al. (1993) Cell 72, 19-28. bOstareck-Lederer,A. et aL (1994) EMBO J. 13, 1476-1481. "Chu, E. et aL (1993) Prec. Natl Acad. ScL USA 90, 517-521, =Chu, E. et al. (1993) Biochemistry 32, 4756-4760. eDabena, M. D. and Warner, J. R. (1993) J. BioL Chem. 268, 19669-19674. ~Kempe, E. et aL (1993) Day. Genet, 14, 449-459. IGavls, E. R. and Lehmann, R. (1994) Nature 369, 315-318. hWharton, R. R and Struhl, G. (1991) Cell 67, 955-967. ~oodwin, E. B° et al. (1993) Cell 75, 329-339. IAhrlnger, J. and Kimble, J. (1991) Nature 349, 346-348. kDe La Luna, S. et aL J. ViroL (in press). ~Danon,A. and Mayfleld, S, P. (1994) Sclenct~266, 1717-1719. "Binder, R. et aL (1994) EMBO J. 13, 1969-1980. "Gait, M. J. and Kam, J. (1994) Trends Biochem. ScL 18, 255-259. QSamanlego, F. et al. (1994) J. Biol. Chem. 269, 30904-30910. PBilger, A. et aL (1994) Genes Dev. 8, 1106-1116. qJackson, R. J. and Standart, N. (1990) Cell 62,15-24. 'Hiding, H. et aL (1994) EMBOJ. 13, 453-461.
Many RNA-binding proteins showing relatively low affinity and specificity are expected to play an important role in the interactions of cukaryotic mRNAs during their lifetime in the cell. At least some, such as pre.RNP components, may ha~e relatively high affinity for a specific type of sequence (such as hnRNPA1), but lower general affinity for other regions of the (pre-)mRNA. Proteins interacting with relatively low affinity/specificity are more readily displaced by others that bind their
specific targets tightly. Both this and the local concentration of 'competing' RNA-binding proteins are relevant to the question of how, for example, a processed mRNA leaving the nucleus might become repopulated by cytoplasmic RNA-binding proteins that participate in, or regulate, translation and mRNA degradation (Fig. 1). Proteins that bind specific mRNA targets can have Kd values of approximately 10-9 or even lower (Table liD. None of the translation factors seems to
belong to this high-affinity group, thus providing no evidence for the existence of a general nuclear--cytoplasmic mRNAaffinity 'gradient' that would favour the binding of cytoplasmic proteins. This contrasts with the case of the nuclear poly(A)-binding protein nucleolin, whose affinity for poly(A) is lower than that of the cytoplasmic PABP (Tables I and liD. Another aspect is that even a high-af¢inity interaction has little functional impact if it is targeted to an inappropriate step in a biological
RV[WS
T~BS 2 0 - MAY 19~5
process. This is reflected, for exam#e, in the positionaa modulation off the ~unction of ~RE sequences (see earlier). Thus, the evolutionary pathway of such control/regulatory systems is likely to be dictated by the necessity to optimize both the affinity/specifidty and the site of effector binding. A further consideration is that the RNA-binding proteins can be multifunct~onal, raising questions about the nature of their mPd'~]A-binding sites. In prokaryotes, the RNAobinding site may overlap with or be identical to the main binding/active site of the protein or, alternatively, can be entirely separate ss. The eukaryotic IRP is an interesting case of multifunctionality, since this repressor is also a cytoplasmic aconitase (Table ]I). Other eukaryotic RNAbinding proteins have been ~ound to function both in splicing and in translation (such as PTB) or in transcription and translation (such as FRG Y2, Laantigen and $SL2; Table l).
R~A recognition element
Dissoci~on constant, o, i 4-6 x :tg-~ ~ I-3 × !0 -~ 7 × 10-a M 3 × :!0-7 ~,t > 2 x 10 -7 ~ 4 x 10 ,8M~ < 1-3 x 10-9 ~
enF-4A
nRESfragment (poUiovirus) UAGGGA/U motif Naturan spOicesite sequences Nonspecific RNA Homopogymers aRESfragment (EMCV) RNA secondary structure with purina-rich bubble TAR mZGpppG mRNA cap RNA
eQF-4B
RNA
5 x I0 -z Mh
La Ip52) hnRNP A1 Po~ypydmidine-tract-bmding protein (PTB) Rev Tat eBF-4E(eOF-4~)
Poly(A)-binding protein (PABP) ~ron regulatory protein - Fe
3 x 10-9 M: -5 x 10-6 M~ ~5 x 10-7 M~ > 10~ M~'
STNV-RNA
10-7--10-8 Mi
Poly(A) 8 nuc~eotides 12 nucleotides IRE
6 x 10-~ 2 x 10"~ ~#
+ Fe
/ - 3 x 10 -~ ~ 2-5 x 10-~ M"
aEach of these values was obtained under different conditions, and this table is intended to serve ongy as a rough guide. Direct comparisons si~ould therefore be interpreted with caution, bSv[tkin, Y. V. et aL (!994~ J. ViroL 68, 1544-1550. ©Burd, C. G. and Dreyfuss, G. (1994) EMBO J. 13, 1197-!204. dWitherelL G. W. et al. (1993) Biochemistry 32, 8268-8275. eHeaphy, S. et aL (1991) Proc. Natl Acad. ScL USA 88, 7366-7370. fGait, M. J. and Kern, J. (1994) Trends. Biochem. ScL 18, 255-259. ~Goss, D. J. e~ aL (1990) Biochemistry 29, 5008-5012. bMSthet, N. et aL (1994) MoL Cell. BioL 14, 2307-2316. ~Butler, J. S. and Clark, J. M., Jr (1984) Biochemistry 23, 809-815. JSachs, A. B. et aL (1987) Mol. Cell. Biol. 7, 3268-3276; nucleolin has a lower affinity than PABP (H. Wormington, pets. commun), hHaile, D. J. et al. (1989) MoL Ceil. Biol. 9, 5055-5061.
Cenc~u~ingremarks The various examples of control and regulation at the mRNA level manifest both familiar and new functional prindples. One striking aspect is ~he participation of the 3CUTR as a regulatory site in such a diversity of systems. Not so new is the observation that a great deal o[ control and regulation is exercised via the initiation step of translation 33. Current knowledge of eukaryotic
mRNAobhlding proteins derives from studies of a wide range of genes and organisms, but only in a few cases is it more than superficial. Similarly, very few of the RNA-binding sites have been studied in detail. A major task will therefore be to obtain mere structural information about various mRNA-protein interactions in order to determine the structural basis of different levels of affinity and specificity for given binding motifs. However, an equally important objective will be the quantitative (thermodynamic and kinetic) characterization of these interactions in terms of their physiological functions. This will require a balanced combination of in vivo and in vitro experimental strategies. Comparisons also need to be made with other RNA-protein interactions relevant to gene expression involving, for example, snRNA~, tRNA4s, ribosomal RNA~, and the guide RNAs involved in editing~7. Apart from the expected revelations about post-transcriptional gene expression, the coming years may also
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