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A model for nucleocytoplasmic transport of ribonucleoprotein particles
J. theor. Biol. (1982) 95,607-613
A Model for Nucleocytoplasmic Transport of Ribonucleoprotein Particles? GARY
A.
CLAWSON
AND
EDWARD
A.
SMUCKLER
Department of Pathology, University of California School Medicine, San Francisco, California 94143, U.S.A. (Received
of
4 May 1981)
Some aspects of the transport of ribonucleoprotein particles from nucleus to cytoplasm have been derived from in vitro assays employing isolated nuclei. The transport process has an apparent activation energy of 13 kcal/mol, shows an Arrhenius relationship without evidence of a transition between 35 and 0°C requires hydrolysis of one high-energy phosphate bond per nucleotide in transported RNA. All of these analyses of the process are independent of the type of ribonucleoprotein particle (ribosomal or messenger) transported. A serious conceptual difficulty arises when the size of the transported particles is considered. They must presumably travel through an aqueous channel in the nuclear envelope (since lipid phase transitions do not appear in the Arrhenius graphs), but all of the transported particles are too large to pass through the nuclear pore complexes. In reviewing what is known about ribonucleoprotein structure, we find common features which suggest the following model: (1) the RNA chain is exposed at the surface of the particles (2) small, local regions of particle structure “unfold” (3) these unfolded (linear) segments of RNA interact with a translocation mechanism containing a nucleoside triphosphatase (4) the RNA chain is linearly translocated through the pore channel the length of one nucleotide for each high-energy phosphate bond hydrolyzed (5) the particle then refolds outside of the nuclear envelope.
Introduction The mechanism by which RNA is transferred from the nuclear interior to the cytoplasm is a process integral to cellular function. Due to the inherent complexities of this transport in vivo, in vitro assays employing isolated nuclei, have been used to define the mechanisms involved; the process involves maturation of large nuclear RNA transcripts to much smaller molecules, and movement of cellular RNA complexed with protein in t Supported
by grant
CA21141
from
the NIH. 607
0022-5193/82/080607+07$03.00/0
@ 1982 Academic
Press Inc. (London)
Ltd.
608
G.
A.
CLAWSON
AND
E.
A.
SMUCKLER
ribonucleoprotein (RNP)S particles (Roy et al., 1979; Chisick, Brennessel & Biswas, 1979; Sato, Ishikawa & Ogata, 1977). Ribosomal, messenger, and transfer RNA maturation occurs with considerable fidelity with isolated nuclei in vitro. Transfer RNA (tRNA) is correctly processed in vitro (O’Farrell et al., 1978). When ribosomal proteins are present in the incubation brei, these proteins are rapidly taken up by isolated rat-liver nuclei (Bolla et al., 1977); they complex with 45s pre-ribosomal RNA to form 80s pre-ribosomal RNP particles in the nucleolus, analogous to formation of 80s pre-ribosomal RNP in the nucleolus in vivo (Matsuura et al., 1974). The RNA in these particles is then processed to mature 28s and 18s ribosomal RNA (rRNA) species, which are found in the nucleoplasm as components of 60s and 40s ribosomal subunits (Bolla et al., 1977). When ATP is included with cell sap in the incubation mixture, the 60s and 40s ribosomal subunits are the predominant form of RNP transported from the nucleus to surrogate cytoplasm (Sato et al., 1977; Racevskis & Webb, 1974). The 60s and 40s ribosomal subunits released in vitro have buoyant densities (respectively) of 1.61 and 1.56 g/cm3 (Sato et al., 1977), characteristics of cytoplasmic ribosomal subunits. Maturation of heterogeneous nuclear RNA (hnRNA) to messenger RNA (mRNA) has recently been demonstrated with specific probes. Goldenberg & Raskas (1980) and Yang et al. (1981) have demonstrated that correct “splicing” of adenovirus-2 hnRNA (to mRNA) occurs with isolated nuclei, a process which involves removal of an intervening sequence and subsequent ligation of two disjoint sequences. When isolated nuclei are incubated in mixtures containing ATP (but not cell sap) predominantly messenger RNP (mRNP) are released from them. They display many similarities with mRNP and pre-mRNP found in rho (Deimel, Louis & Sekeris, 1977; Howard, 1978; Jain, Pluskal & Sarkar, 1979). The mRNP released in vitro sediment through sucrose gradients at about 40-458 (Ishikawa, Kuroda & Ogata, 1969; Raskas, 1971; Smuckler & Koplitz, 1974), have buoyant density (Ishikawa et al., 1969; Raskas, 1971) characteristic of cytoplasmic mRNP (1.40 g/cm3), and can be incorporated into polysomes in reconstructed systems (Ishikawa et al., 1970u). They contain RNA similar in size and base composition (Ishikawa et al., 19706) to cytoplasmic RNA, and which can direct protein synthesis in vitro (Ishikawa et al., 1970b). Poly(A)-containing RNA released in vitro hybrid$ Abbreviations used are: RNP, ribonucleoprotein ribosomal RNA; hnRNA, heterogeneous nuclear messenger RNP; cDNA, complementary DNA; adenosine triphosphatase.
particles; tRNA, transfer RNA; rRNA, RNA; mRNA, messenger RNA; mRNP, poly(A), poly(adenylic acid); ATPase,
RNP
TRANSPORT
609
izes (under stringent conditions) to cDNA probes transcribed from cytoplasmic poly(A)-containing RNA to the same extent as the homologous cytoplasmic population (Clawson & Smuckler, in press), indicating a considerable sequence homology between these populations. Some aspects of the energetics of the transport process can be inferred from in vitro assays: (i) RNP transport displays an apparent activation energy of 13 kcal/mol (Clawson & Smuckler, 1978; see also Patterson, Lyerly & Stuart, 1981), a value which is equivalent to that for ATPase activity (Dean & Tanford, 1978; Clawson et al., 1980). (ii) Arrhenius analyses of initial linear rates of transport yield lines of continuous slopes over the temperature domain of 0-3S’C, and detergent treatment does not affect RNA transport (Clawson & Smuckler, 1978; Patterson et al., 1981); thus, the translocation mechanism is apparently exposed to an aqueous environment and not influenced by lipid. Since the function of translocation is to transport RNP (RNA) through a membranous envelope, we must assume an aqueous channel exists in the nuclear envelope, since all other exits would traverse lipid barriers. Whether or not this channel corresponds to the well-described nuclear pore complex is unknown. (iii) Approximately one high-energy phosphate bond is hydrolyzed in the facilitated transport of each nucleotide in RNA transported in vitro (Clawson et al., 1978). These high-energy phosphate bonds are hydrolyzed by a nucleoside triphosphatase activity associated with the nuclear envelope (Agutter, McCaldin & McArdle, 1979; Vorbrodt & Maul, 1980; Clawson el al., 1980). (iv) The activation energy for transport is unchanged by the presence or absence of cell sap (Clawson & Smuckler, 1978); however, predominantly 60s and 40s ribosomal subunits are transported in the presence of cell sap, whereas only mRNP are transported without cell sap (Sato et al., 1977). Since the RNA is transported with the same energy requirement per nucleotide in both types of mixture, the RNA component, not the protein component or three-dimensional particle structure, must dictate the energy requirement. The great differences in the RNA-protein ratios in the different types of RNP (the buoyant densities of the mRNP and the ribosomal RNP differ greatly) and the dissimilarities among the particles in three-dimensional structure necessitate this conclusion. Since the translocation mechanism is exposed to the aqueous phase and interacts with transportable RNA on a per nucleotide basis, it seems reasonable to suggest that the RNA in RNP particles must be exposed on the surface of the particles (see below). However, definition of RNP particles in the nucleus, in surrogate cytoplasm, and in viva, leads to significant conceptual difhculties concerning their transport. Although electron microscopic studies show nuclear pore
610
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A.
CLAWSON
AND
E.
A.
SMUCKLER
complexes with an inner diameter of about 66 nm, a much smaller channel is generally observed (Gall, 1967), and pore complexes have a much smaller functional pore radius (Feldherr & Pomerantz, 1978). The most refined estimate of patent pore diameter is 9 nm, based on the isotopic tracer diffusion studies of Paine, Moore & Horowitz (1975). But Nonomura, Blobel & Sabatini (1971) have shown that ribosomal subunits (both large and small) are far too large to pass through 9 nm pore channels. Similarly, Lukanidin et al. (1973a) have shown that nuclear mRNP are about 20 nm in diameter and Dubochet et al. (1973) have demonstrated that cytoplasmic mRNP are also much greater than 9 nm in diameter. Analogous considerations hold for mRNP transported in vitro, which are dimensionally similar to the small ribosomal subunits. Further, measurements such as those of Pilz et al. (1970) clearly indicate that even mature tRNA would require from a few hours to a day to reach nucleocytoplasmic equilibrium (based on the data of Paine et al., 1975), although this is clearly not the case in viva. A Model The mechanisms we propose to explain these diverse data involves the following ideas: (1) the RNA chain is exposed at the surface of the RNP, (2) small, local regions of RNP structure “unfold”, (3) these unfolded (linear) segments of RNA interact with a translocation mechanism containing a nucleoside triphosphatase, (4) the RNA chain is linearly translocated through an aqueous channel the length of one nucleotide for each highenergy phosphate bond hydrolyzed, and (5) the RNP particle then refolds outside of the nuclear envelope. This sequence is consistent with the results of our book-keeping in the in vitro system we have employed, and with the structural features of RNP, some of which are discussed below. In the model of ribosome structure proposed by Cotter, McPhie & Gratxer (1967), the ribosomal surface is largely composed of RNA, with helical regions protruding outward and with spherical protein domains (about 3 nm in diameter) attached to nonhelical RNA segments inside the subunits. Cox (1969) along with Bonanou (Cox & Bonanou, 1969), later presented a more detailed “horseshoe and cap” model, which retained protein domains 3-4 nm in diameter, and which had hairpin loops (two loops per protein domain, each composed of about 7 base pairs and 9 unpaired residues) covering the surface of each ribosomal subunit. Studies of endonuclease digestion by Spencer & Walker (197 1) support the hairpinloop hypothesis, and further studies by Lind, Villems & Saarma (1975) suggest that the single-stranded hairpin loops consist of pyrimidine-rich clusters. Thus, the main features of the model of the ribosome, as it has
RNP
TRANSPORT
611
evolved, are that the RNA is coiled into hairpin loops and is located at the ribosomal surface, and that the protein subunits (which are tightly linked to single-stranded segments of rRNA) are 4 nm or less in diameter. The existence of small domains of similar secondary structure would have tremendous energetic advantages. The linearization of small hairpin segments would require much less energy than the unfolding or linearization of large portions of the rRNA molecules. In addition, the single-stranded hairpin-loop segments are composed of pyrimidines, which convey a great deal of flexibility to such structures. The demonstration by Matsuura et al. (1974) that nucleolar RNP particles (sedimenting at 80-100s) containing rRNA and precursors of rRNA also exist as pleomorphic, rod-like, and filamentous structures, with nodular thickenings, provides experimental support for the postulated unfolding. There are basic similarities between the structure of the ribosomal subunits and that of informofers. Hairpin-like structures have been found in pre-mRNA in nuclear RNP particles (Molnar, Besson & Samarina, 1975); apparently, these structures are formed directly after transcription and participate in mRNA processing. Lukanidin et al. (1973a) proposed that the RNA chains in nuclear RNP particles are wrapped around the outside of the particles and thus are available for interaction with a translocation mechanism. Lukanidin, Georgiev & Williamson (19736) found a 30s nuclear RNP composed of RNA associated with many copies of a homogeneous protein termed “informatin”. The particles were self-assembling in reconstitution experiments, providing direct evidence that RNP packaging is not an energy-requiring step. Billings & Martin (1978) have reported similar results. The hairpin loops in Cox’s model of the ribosome have the general features of those described by other workers (Pilz et al., 1970; Cramer, 1971) for tRNA structure. Therefore, as required by our model, in all transported material, the RNA chain is exposed to the aqueous phase and has single-stranded hairpin loops, whether it is free in solution (as in tRNA precursor) or localized on the surfaces of nuclear RNP particles (as in mRNP and ribosomal subunits). The translocation mechanism presumably recognizes specific conformational aspects of RNA or mature RNP particles, unfolding local regions of RNA (RNP) structure and translocating the RNA chain linearly with associated protein domains. On the basis of our model, the specificity of transport must lie in the processes occurring before interaction with the translocation mechanism, since it is doubtful that the selective mechanism could operate with suitable fidelity through interaction with short segments of many diverse RNA chains. An interesting possibility is that small RNA species (such as 5s ribosomal RNA and the small species
612
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A.
CLAWSON
AND
E.
A.
SMUCKLER
shown to be present in mRNP structures, Deimel ef al., 1977; Howard, 1978) fulfill such a function, with the “reusable” small RNA species being shuttled back and forth. This idea is consistent with the observed shuttling behavior of some RNA species (Wise & Goldstein, 1973) and with the reentry of small RNA observed in rat-liver nuclei (Clawson & Smuckler, 1978). REFERENCES AGU?TER,P.,MCCALDIN,B.&MCARDLE,H.(~~~~).B~~~~~~.J. 182,811. BILLINGS.P.& MARTIN.T. (1978j.J. CellBiol. 79.375a (absrt.). BoLLA,~.,RoTH,H.,WEISSBAC&,H.& B~o~,fi.(197?). B.biol. Chem.252,721. CHISICK,M.,BRENNESSEL,B.& BISWAS,D.(~~~~). Biochem. biophys.Res. Commun. 91, 1109. CLAWSON,G.& SMUCKLER,E.(~~~~). Proc. natn. Acad.Sci. U.S,A.75,5400. CLAWSON, G., KOPLITZ,M., CASTLER-SCHECHTER, B. & SMUCKLER, E. (1978). Biochemistry
17,3747.
CLAWSON, G.,JAMEs,J., Woo, C., FRIEND,D., MOODY,D. & SMUCKLER, E. (1980). Biochemistry 19,2748. CO-I-IXR,R.,MCPHIE,P.& GRATZER,~.(~~~~). Nature,Lond. 216,864. Cox,R.(1969). Bi0chem.J. 114,753. COX,R.&BONANOU,S. (1969). Biochem.I.l14,769. CRAMER, F. (1971). Prog. Nucl. Acid Res. Molec. Biol, 11,391. DEAN,W.& TANFORD, C. (1978). Biochemistry 17,1683. DEIMEL,B.,LOUIS,C. & SEKERIS, C. (1977). FEBSLett. 73,80. DuBocHET,J.,MoREL,C.,LEBLEU,B.& MERZBERG,M. (1973). Eur. J. Biochem.36, 465.
FELDHERR,~.& PoMERANTz,J.(~~~~).J. CellEiol.78, 168. GALL, J. (1967). J. CellBiol. 32, 391. GoLDENBERG,C.&RASKAS,H.(~~~O). Biochemistry 19,2719. HOWARD.F. (1978). Biochemistrv 17.3228. ISHIKAW&K:,KU~ODA, C.& e~~T~,K.(1969). Biochim. biophys. Acta 179,316. IsHIKAwA,K.,UEKI,M.,NAGAI,K.& OGATA,K.(~~~O~). Biochim. biophys. Acta 213, 542.
ISHIKAWA, K., KURODA, C., UEKI, M. & OGATA, K. (1970b). Biochim. biophys. Acta 213,495. JAIN,S.,PLUSKAL,M.& SARKAR, S. (1979). FEBSLett. 97,84. LIND,A.,VILLEMS,R.& SAARMA, M. (1975). Eur. J. Biochem. 51,529. LUKANIDIN,E.,KUL'GUSKII,V.,AITKHOZHINA,N.,KOM~ROMI,L.,TIKHONENKO,A. & GEORGIEV, G. (1973a). Molekulyamaya Biologiya 7,360. LUKANIDIN,E.,GEORGIEV,G. & WILLIAMSON,R. (19736). Molekulyarnaya Biologiya 7,264.
MATSUURA,S.,MORIMOTO,T.,TASHIRO,Y.,HIGASHINAKAGAWA,T.&MURAMATSU, M. (1974). J. Cell Biol. 63,629. MOLNAR,J.,BESSON,J.& SAMARINA,~.(~~~~). Mol. Biol. Rprs 2, 11. NONOMURA,Y.,BLOBEL,G.& SABATINI,D.(~~~~). J. mol. Biol.60,303. O'FARRELL,P.,CORDELL,B.,VALENZUELA,P.,R~R,W.&GOODMAN,H. Nature,
Land.
(1978).
274,438.
PAINE,P.,MooRE,L.& HOROWITZ, S. (1975). Nature,Lond.254, 109. PA'ITERSON.R.,LYERLY,M.& STUART, S. (1981). CellBiol. Int. Rpts 5,27. PILZ, I., KRATKY, O., CRAMER, F., HARR, F. & VON DER SCHLIMME, E. (1970). Eur. J. B&hem.
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RACEVSKIS, .I. & WEBB, T. (1974). Eur. J. Biochem. 49,93. RASKAS, H. (1971). Nature New Biol. 233, 134. ROY, R.,LAu, A.,MuNRo,H.,BALIGA,B. & SARKAR,~.(~~~~).
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SATO,T.,ISHIKAWA,K.& OGATA,K.(~~~~). Biochim.biophys. Acru 474,536. SMUCKLER,E.& KoPLITz,M.(~~~~). Cancer Res.34,827. SPENCER, M. & WALKER, I. (1971). Eur. J. Biochem. 19,451. VORBRODT, A. & MAUL, G. (1980). J. Histochem. Cyrochem. 28,27. WISE,G.& GoLDsTEIN,L.(~~~~),J. Cell Biol.56, 129. YANG, V., LERNER, M., STEITZ, J. & FLINT, S. (1981). Proc. natn. Acad. Sci. U.S.A.
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A Model for Nucleocytoplasmic Transport of Ribonucleoprotein Particles? GARY
A.
CLAWSON
AND
EDWARD
A.
SMUCKLER
Department of Pathology, University of California School Medicine, San Francisco, California 94143, U.S.A. (Received
of
4 May 1981)
Some aspects of the transport of ribonucleoprotein particles from nucleus to cytoplasm have been derived from in vitro assays employing isolated nuclei. The transport process has an apparent activation energy of 13 kcal/mol, shows an Arrhenius relationship without evidence of a transition between 35 and 0°C requires hydrolysis of one high-energy phosphate bond per nucleotide in transported RNA. All of these analyses of the process are independent of the type of ribonucleoprotein particle (ribosomal or messenger) transported. A serious conceptual difficulty arises when the size of the transported particles is considered. They must presumably travel through an aqueous channel in the nuclear envelope (since lipid phase transitions do not appear in the Arrhenius graphs), but all of the transported particles are too large to pass through the nuclear pore complexes. In reviewing what is known about ribonucleoprotein structure, we find common features which suggest the following model: (1) the RNA chain is exposed at the surface of the particles (2) small, local regions of particle structure “unfold” (3) these unfolded (linear) segments of RNA interact with a translocation mechanism containing a nucleoside triphosphatase (4) the RNA chain is linearly translocated through the pore channel the length of one nucleotide for each high-energy phosphate bond hydrolyzed (5) the particle then refolds outside of the nuclear envelope.
Introduction The mechanism by which RNA is transferred from the nuclear interior to the cytoplasm is a process integral to cellular function. Due to the inherent complexities of this transport in vivo, in vitro assays employing isolated nuclei, have been used to define the mechanisms involved; the process involves maturation of large nuclear RNA transcripts to much smaller molecules, and movement of cellular RNA complexed with protein in t Supported
by grant
CA21141
from
the NIH. 607
0022-5193/82/080607+07$03.00/0
@ 1982 Academic
Press Inc. (London)
Ltd.
608
G.
A.
CLAWSON
AND
E.
A.
SMUCKLER
ribonucleoprotein (RNP)S particles (Roy et al., 1979; Chisick, Brennessel & Biswas, 1979; Sato, Ishikawa & Ogata, 1977). Ribosomal, messenger, and transfer RNA maturation occurs with considerable fidelity with isolated nuclei in vitro. Transfer RNA (tRNA) is correctly processed in vitro (O’Farrell et al., 1978). When ribosomal proteins are present in the incubation brei, these proteins are rapidly taken up by isolated rat-liver nuclei (Bolla et al., 1977); they complex with 45s pre-ribosomal RNA to form 80s pre-ribosomal RNP particles in the nucleolus, analogous to formation of 80s pre-ribosomal RNP in the nucleolus in vivo (Matsuura et al., 1974). The RNA in these particles is then processed to mature 28s and 18s ribosomal RNA (rRNA) species, which are found in the nucleoplasm as components of 60s and 40s ribosomal subunits (Bolla et al., 1977). When ATP is included with cell sap in the incubation mixture, the 60s and 40s ribosomal subunits are the predominant form of RNP transported from the nucleus to surrogate cytoplasm (Sato et al., 1977; Racevskis & Webb, 1974). The 60s and 40s ribosomal subunits released in vitro have buoyant densities (respectively) of 1.61 and 1.56 g/cm3 (Sato et al., 1977), characteristics of cytoplasmic ribosomal subunits. Maturation of heterogeneous nuclear RNA (hnRNA) to messenger RNA (mRNA) has recently been demonstrated with specific probes. Goldenberg & Raskas (1980) and Yang et al. (1981) have demonstrated that correct “splicing” of adenovirus-2 hnRNA (to mRNA) occurs with isolated nuclei, a process which involves removal of an intervening sequence and subsequent ligation of two disjoint sequences. When isolated nuclei are incubated in mixtures containing ATP (but not cell sap) predominantly messenger RNP (mRNP) are released from them. They display many similarities with mRNP and pre-mRNP found in rho (Deimel, Louis & Sekeris, 1977; Howard, 1978; Jain, Pluskal & Sarkar, 1979). The mRNP released in vitro sediment through sucrose gradients at about 40-458 (Ishikawa, Kuroda & Ogata, 1969; Raskas, 1971; Smuckler & Koplitz, 1974), have buoyant density (Ishikawa et al., 1969; Raskas, 1971) characteristic of cytoplasmic mRNP (1.40 g/cm3), and can be incorporated into polysomes in reconstructed systems (Ishikawa et al., 1970u). They contain RNA similar in size and base composition (Ishikawa et al., 19706) to cytoplasmic RNA, and which can direct protein synthesis in vitro (Ishikawa et al., 1970b). Poly(A)-containing RNA released in vitro hybrid$ Abbreviations used are: RNP, ribonucleoprotein ribosomal RNA; hnRNA, heterogeneous nuclear messenger RNP; cDNA, complementary DNA; adenosine triphosphatase.
particles; tRNA, transfer RNA; rRNA, RNA; mRNA, messenger RNA; mRNP, poly(A), poly(adenylic acid); ATPase,
RNP
TRANSPORT
609
izes (under stringent conditions) to cDNA probes transcribed from cytoplasmic poly(A)-containing RNA to the same extent as the homologous cytoplasmic population (Clawson & Smuckler, in press), indicating a considerable sequence homology between these populations. Some aspects of the energetics of the transport process can be inferred from in vitro assays: (i) RNP transport displays an apparent activation energy of 13 kcal/mol (Clawson & Smuckler, 1978; see also Patterson, Lyerly & Stuart, 1981), a value which is equivalent to that for ATPase activity (Dean & Tanford, 1978; Clawson et al., 1980). (ii) Arrhenius analyses of initial linear rates of transport yield lines of continuous slopes over the temperature domain of 0-3S’C, and detergent treatment does not affect RNA transport (Clawson & Smuckler, 1978; Patterson et al., 1981); thus, the translocation mechanism is apparently exposed to an aqueous environment and not influenced by lipid. Since the function of translocation is to transport RNP (RNA) through a membranous envelope, we must assume an aqueous channel exists in the nuclear envelope, since all other exits would traverse lipid barriers. Whether or not this channel corresponds to the well-described nuclear pore complex is unknown. (iii) Approximately one high-energy phosphate bond is hydrolyzed in the facilitated transport of each nucleotide in RNA transported in vitro (Clawson et al., 1978). These high-energy phosphate bonds are hydrolyzed by a nucleoside triphosphatase activity associated with the nuclear envelope (Agutter, McCaldin & McArdle, 1979; Vorbrodt & Maul, 1980; Clawson el al., 1980). (iv) The activation energy for transport is unchanged by the presence or absence of cell sap (Clawson & Smuckler, 1978); however, predominantly 60s and 40s ribosomal subunits are transported in the presence of cell sap, whereas only mRNP are transported without cell sap (Sato et al., 1977). Since the RNA is transported with the same energy requirement per nucleotide in both types of mixture, the RNA component, not the protein component or three-dimensional particle structure, must dictate the energy requirement. The great differences in the RNA-protein ratios in the different types of RNP (the buoyant densities of the mRNP and the ribosomal RNP differ greatly) and the dissimilarities among the particles in three-dimensional structure necessitate this conclusion. Since the translocation mechanism is exposed to the aqueous phase and interacts with transportable RNA on a per nucleotide basis, it seems reasonable to suggest that the RNA in RNP particles must be exposed on the surface of the particles (see below). However, definition of RNP particles in the nucleus, in surrogate cytoplasm, and in viva, leads to significant conceptual difhculties concerning their transport. Although electron microscopic studies show nuclear pore
610
G.
A.
CLAWSON
AND
E.
A.
SMUCKLER
complexes with an inner diameter of about 66 nm, a much smaller channel is generally observed (Gall, 1967), and pore complexes have a much smaller functional pore radius (Feldherr & Pomerantz, 1978). The most refined estimate of patent pore diameter is 9 nm, based on the isotopic tracer diffusion studies of Paine, Moore & Horowitz (1975). But Nonomura, Blobel & Sabatini (1971) have shown that ribosomal subunits (both large and small) are far too large to pass through 9 nm pore channels. Similarly, Lukanidin et al. (1973a) have shown that nuclear mRNP are about 20 nm in diameter and Dubochet et al. (1973) have demonstrated that cytoplasmic mRNP are also much greater than 9 nm in diameter. Analogous considerations hold for mRNP transported in vitro, which are dimensionally similar to the small ribosomal subunits. Further, measurements such as those of Pilz et al. (1970) clearly indicate that even mature tRNA would require from a few hours to a day to reach nucleocytoplasmic equilibrium (based on the data of Paine et al., 1975), although this is clearly not the case in viva. A Model The mechanisms we propose to explain these diverse data involves the following ideas: (1) the RNA chain is exposed at the surface of the RNP, (2) small, local regions of RNP structure “unfold”, (3) these unfolded (linear) segments of RNA interact with a translocation mechanism containing a nucleoside triphosphatase, (4) the RNA chain is linearly translocated through an aqueous channel the length of one nucleotide for each highenergy phosphate bond hydrolyzed, and (5) the RNP particle then refolds outside of the nuclear envelope. This sequence is consistent with the results of our book-keeping in the in vitro system we have employed, and with the structural features of RNP, some of which are discussed below. In the model of ribosome structure proposed by Cotter, McPhie & Gratxer (1967), the ribosomal surface is largely composed of RNA, with helical regions protruding outward and with spherical protein domains (about 3 nm in diameter) attached to nonhelical RNA segments inside the subunits. Cox (1969) along with Bonanou (Cox & Bonanou, 1969), later presented a more detailed “horseshoe and cap” model, which retained protein domains 3-4 nm in diameter, and which had hairpin loops (two loops per protein domain, each composed of about 7 base pairs and 9 unpaired residues) covering the surface of each ribosomal subunit. Studies of endonuclease digestion by Spencer & Walker (197 1) support the hairpinloop hypothesis, and further studies by Lind, Villems & Saarma (1975) suggest that the single-stranded hairpin loops consist of pyrimidine-rich clusters. Thus, the main features of the model of the ribosome, as it has
RNP
TRANSPORT
611
evolved, are that the RNA is coiled into hairpin loops and is located at the ribosomal surface, and that the protein subunits (which are tightly linked to single-stranded segments of rRNA) are 4 nm or less in diameter. The existence of small domains of similar secondary structure would have tremendous energetic advantages. The linearization of small hairpin segments would require much less energy than the unfolding or linearization of large portions of the rRNA molecules. In addition, the single-stranded hairpin-loop segments are composed of pyrimidines, which convey a great deal of flexibility to such structures. The demonstration by Matsuura et al. (1974) that nucleolar RNP particles (sedimenting at 80-100s) containing rRNA and precursors of rRNA also exist as pleomorphic, rod-like, and filamentous structures, with nodular thickenings, provides experimental support for the postulated unfolding. There are basic similarities between the structure of the ribosomal subunits and that of informofers. Hairpin-like structures have been found in pre-mRNA in nuclear RNP particles (Molnar, Besson & Samarina, 1975); apparently, these structures are formed directly after transcription and participate in mRNA processing. Lukanidin et al. (1973a) proposed that the RNA chains in nuclear RNP particles are wrapped around the outside of the particles and thus are available for interaction with a translocation mechanism. Lukanidin, Georgiev & Williamson (19736) found a 30s nuclear RNP composed of RNA associated with many copies of a homogeneous protein termed “informatin”. The particles were self-assembling in reconstitution experiments, providing direct evidence that RNP packaging is not an energy-requiring step. Billings & Martin (1978) have reported similar results. The hairpin loops in Cox’s model of the ribosome have the general features of those described by other workers (Pilz et al., 1970; Cramer, 1971) for tRNA structure. Therefore, as required by our model, in all transported material, the RNA chain is exposed to the aqueous phase and has single-stranded hairpin loops, whether it is free in solution (as in tRNA precursor) or localized on the surfaces of nuclear RNP particles (as in mRNP and ribosomal subunits). The translocation mechanism presumably recognizes specific conformational aspects of RNA or mature RNP particles, unfolding local regions of RNA (RNP) structure and translocating the RNA chain linearly with associated protein domains. On the basis of our model, the specificity of transport must lie in the processes occurring before interaction with the translocation mechanism, since it is doubtful that the selective mechanism could operate with suitable fidelity through interaction with short segments of many diverse RNA chains. An interesting possibility is that small RNA species (such as 5s ribosomal RNA and the small species
612
G.
A.
CLAWSON
AND
E.
A.
SMUCKLER
shown to be present in mRNP structures, Deimel ef al., 1977; Howard, 1978) fulfill such a function, with the “reusable” small RNA species being shuttled back and forth. This idea is consistent with the observed shuttling behavior of some RNA species (Wise & Goldstein, 1973) and with the reentry of small RNA observed in rat-liver nuclei (Clawson & Smuckler, 1978). REFERENCES AGU?TER,P.,MCCALDIN,B.&MCARDLE,H.(~~~~).B~~~~~~.J. 182,811. BILLINGS.P.& MARTIN.T. (1978j.J. CellBiol. 79.375a (absrt.). BoLLA,~.,RoTH,H.,WEISSBAC&,H.& B~o~,fi.(197?). B.biol. Chem.252,721. CHISICK,M.,BRENNESSEL,B.& BISWAS,D.(~~~~). Biochem. biophys.Res. Commun. 91, 1109. CLAWSON,G.& SMUCKLER,E.(~~~~). Proc. natn. Acad.Sci. U.S,A.75,5400. CLAWSON, G., KOPLITZ,M., CASTLER-SCHECHTER, B. & SMUCKLER, E. (1978). Biochemistry
17,3747.
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