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Trna Transport from the Nucleus in a Eukaryotic Cell

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Trna Transport from the Nucleus in a Eukaryotic Cell Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6436-6440, November 1983 Biochemistry tRNA transport from the nucleus in a eukaryotic cell: Carrier-mediated translocation process (RNA transport/nuclear transport/Xenopw laei oocyte/tRNAMet) MICHAEL ZASLOFF Human Genetics Branch, National Institute of Child Health and Human Development, Building 10, Room 8C-429, National Institutes of Health, Bethesda, MD 20205 Communicated byJ. E. Rall, June 22, 1983 ABSTRACT The mechanism by which a tRNA molecule is de- process rather than simple diffusion through a pore or channel. livered from the nucleus of a cell to the cytoplasm has been stud- I believe this data to reveal explicitly the functioning of a tRNA ied in the Xenopus lovis oocyte utilizing nuclear microinjection nuclear transport mechanism in a living cell. and manual microdissection techniques. tRNA nuclear transport in this cell resembles acarrier-mediated translocation process rather METHODS than diffusion through a simple pore or channel. tRNA transport Intranuclear Injections. X. laevis oocytes (stages 5 and 6) were is saturable by tRNA, with a maximal-rate measured to be about scraped from surgically excised ovaries with a 2-mm platinum 190 x 1 molecules per min per nucleus (210C) in the mature oo- loop. Individual oocytes were placed animal-pole-up in single cyte. Competitive inhibition between two different tRNA species wells of a 96-well microtiter tray with conically shaped wells, can be demonstrated, suggesting that many tRNA species share each well containing 0.15 ml of OR2 buffer (5) (82.5 mM NaCl/ a common carrier system. tRNA nuclear transport is sharply de- HCl/ pendent on temperature, with an optimal rate observed at 31C. 2.5 mM KCI/1 mM MgCl2/1 mM CaCl2/5 mM Hepes A single C-to-U substitution atposition 57 in the vertebrate tRNAMet 1 mM sodium phosphate, final pH 7.8). The trays were spun molecule reduces the transport rate of this tRNA by a factor of in a Beckman TJ-6 centrifuge at about 1,000 X g for 10 min at about 20, implicating this highly conserved region of the tRNA 18'C (6). Under optimal conditions, the germinal vesicle is vi- molecule (loop IV) as critical for recognition by the transport sualized in the animal pole as a distinct target-like structure. mechanism. On morphologic grounds Ipropose that ribosome-like Within 5 min of centrifugation, the oocytes were transferred to components surrounding the nuclear pore may function as the tRNA a Petri dish (60 x 15 mm containing about 20 ml of OR2 buff- translocation "motor." The tRNA nuclear transport mechanism er), on the bottom of which a nylon grid had been fixed to as- represents a distinctly eukaryotic process and a site of potential sure stability of the oocyte during microinjection. The oocytes control over cell growth and proliferation. were oriented animal-pole-up, and 20 nl of a solution was de- livered into the visualized germinal vesicle with a beveled glass Transfer RNA, in common with other cytoplasmic RNA spe- needle (20- to 25-,um o.d.). The oocytes were maintained in cies, is synthesized within the nucleus through a complex series OR2 buffer until further analysis. of reactions. Ultimately, the RNA molecule must cross the Oocyte Dissection. Two dissection techniques were utilized, boundary separating the nucleus from the cytoplasm. By what one using trichloroacetic acid and the other using an aqueous mechanism does this escape occur? Do RNA molecules, es- medium described previously (4). In the CC13COOH method, pecially tRNAs, the smallest cellular RNA species, simply dif- oocytes were transferred from OR2 buffer into ice-cold 1% fuse across the nuclear envelope once they have been processed CCl3COOH and then gently rocked at 40C for 30 min. tRNA or is the transport event more complex? My interest in this transport ceases at 40C, and the contents of the oocyte were problem arose recently in studies of the expression of a cloned effectively fixed at this concentration of CCl3(1OOH. The oo- human variant tRNA et gene (1-4). By direct microinjection of cytes were manually dissected in 0.3 ml of 1% CC13COOH on the cloned gene into the germinal vesicle of the Xenopus laevis a siliconized microscope slide at room temperature under a oocyte, the primary transcript of the variant gene was pro- standard binocular dissecting microscope with watchmakers' cessed slowly but accurately to a mature variant tRNAMet (4). forceps. The oocytes were opened with a single tear in the veg- A strking property of this species was its accumulation within etal pole. The germinal vesicle appeared as a greyish-white ball the oocyte nucleus in marked contrast to the normal surrounded by slightly yellow-white cytoplasm and separated tRNAMet species, which was found exclusively in the oocyte cy- from the cytoplasm in much the same way as the pit from a fruit. toplasm (4). The variant tRNA)et species, furthermore, ap- The nucleus was transferred to a scintillation vial containing 0.1 peared to be prevented from free diffusion by the nuclear ml of water. The cytoplasmic contents remaining on the mi- membrane, and its behavior suggested that a specific transport croscope slide were transferred to a separate vial; 15 ml of a process between the nucleus and cytoplasm might exist for tRNA scintillant cocktail was added (Aquafluor, New England Nu- species (4). I report here the direct demonstration of a tRNA clear), and the radioactivity present in both nucleus and cy- nuclear transport mechanism in the living X. laevis oocyte. This toplasm was determined. For determination of transport rates, transport system can utilize mature tRNA efficiently as a sub- at least six oocytes were analyzed for each time point. strate and discriminates between the normal and variant The aqueous microdissection technique has been utilized for tRNAMet species. I show that tRNA transport from the nucleus gel electrophoretic analysis of tRNA species introduced into can be saturated by tRNA and resembles a carrier-mediated the oocyte (4). This method requires considerably greater tech- nical skill because the germinal vesicle is a delicate structure The publication costs of this article were defrayed in part by page charge easily torn during manual dissection. payment. This article must therefore be hereby marked "advertise- tRNA Labeled with 32P at the 5' End. tRNA labeled with ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 32P at the 5' end was prepared by the method of Wurst et al. 6436 Downloaded by guest on October 1, 2021 Biochemistry: Zasloff Proc. Natl. Acad. Sci. USA 80 (1983) 6437 (7) by utilizing T4 polynucleotide Idnase (P-L Biochemicals) and 123456789 [t- P]ATP (New England Nuclear). Labeled tRNA was puri- A B fied by preparative acrylamide gel electrophoresis (3). tRNA was extracted overnight from the crushed acrylamide gel in a small volume of 2.5 M ammonium acetate and recovered bX -ethanol precipitation. Specific radioactivities of about 2 x 107 cpm/,ug of tRNA were routinely achieved. The samples were 12 3456 resuspended in a small volume of 20 mM Tris HCI, pH 8.0/88 mM NaCl. Pre-tRNA ET in the X. lwvis Oocyte. -' Synthesis of 32PLabeled tRNAiet (gene 1) tRNAMET 32P-Labeled mature variant or normal tRNAMet was prepared tRNA1ME by injection of the corresponding gene and [a-32P]GTP into the Normal species UW variant_ . as About (gene 1) germinal vesicle of the X. laevis oocyte described (4). (gene 2) 1I 2 103 cpm of labeled tRNA!Met was recovered per injected oocyte. Cell fraction. o o o oo 2 L1J55 L5315 300 15 401i20 RESULTS Time after nuclear injection, min tRNA Nuclear Transport Can Be Studied with Mature tRNA. The human variant tRNAMet accumulates within the nucleus of FIG. 1. Intracellular distribution of 32P-labeled normal and vari- of either the variant ant human tRNAiet species in X. laevis oocytes after nuclear microin- the X. laevis oocyte after nuclear injection jection. 32P-LabeledtRNAmiet species were synthesized in intactX. Iae- gene or the purified primary transcript synthesized in vitro (4). vis oocytes by the injection of [a-32PIGTP and the corresponding gene. The sequence of this variant tRNA is identical to the normal 32P-Labeled primary transcript ofthe variant gene was synthesized in human tRNAret species save for a G-to-U substitution at po- a cell-free-transcription system from KB cells and was purified by gel sition 57 and has a mature 3' C-C-A terminus (4). Of almost 200 electrophoresis as described (3). Approximately 0.1 ng (200 cpm) of prokaryotic and eukaryotic species with sequences determined each species in 20 nl of 88 mM NaCl/20 mM Tris-HCl, pH 8.0, was mature se- injected into the germinal vesicles of stage 6 X. laevis oocytes. The oo- to date, no other contains a U at this position in the cytes were maintained at 210C in OR2 buffer (5) and microdissected in quence, occupied exclusively by a purine (usually C) (8). The J buffer at indicated times as described (4). RNA was extracted from defective transport behavior of the variant tRNAiM thus ap- both cytoplasm and germinal vesicle ofindividual oocytes, analyzed by peared to result from a single-base substitution within the highly electrophoresis in a 10% acrylamide gel containing 7 M urea, and au- conserved loop IV region. I wished to determine whether the toradiographed (4). (A) Transport of normal tRNA?. Normal tRNAPMF difference in transport behavior between the normal and vari- and variant pre-tRNAM' (gene 1 primary transcript) were coinjected (volume, 20 nl). Lanes: 9, RNA extracted from the whole oocyte im- ant tRNAiMet species could be observed after nuclear injection mediately after nuclear microinjection; 1, 3, 5, and 7, species present of the mature, processed species, thereby functionally dis- in the nucleus at indicated times after injection; 2, 4, 6, and 8, species secting the transport step from more proximal events in the presentinthecorrespondingcytoplasmicfractions.
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