Development 106, 367-373 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

367

Poly(A) shortening accompanies the activation of translation of five mRNAs

during spermiogenesis in the mouse

KENNETH C. KLEENE

Department of Biology, University of Massachusetts at Boston, Boston, Massachusetts 02125, USA

Summary

I have compared the quantity and the length of thepoly(A) tracts of five haploid-expressed mRNAs in thepolysomal and nonpolysomal fractions of round andelongating spermatids in mice: transition proteins 1 and2, protamines 1 and 2, and an unidentified niRNA ofabout 1050 bases. Postmitochondrial supernatants ofhighly enriched populations of round and elongatingspermatids (early and late haploid spermatogenic cells)were sedimented on sucrose gradients, and the size andamount of each mRNA in gradient fractions wereanalyzed in Northern blots. In round spermatids, all fivemRNAs are restricted to the postpolysomal fractions,but in elongating spermatids about 30-40% of eachmRNA is associated with the polysomes. The distri-bution of these mRNAs in sucrose gradients suggests

that all five mRNAs are stored in a translationallyrepressed state in round and early elongating sperma-tids, and that they become translationally active inmiddle and late elongating spermatids. The trans-lationally repressed forms of all five mRNAs are longand homogenous in size, whereas the polysomal formsare shorter and more heterogenous due to shortening oftheir poly (A) tracts. The relationship between trans-lational activity and poly(A) size exemplified by thesefive mRNAs may be typical of mRNAs which aretranslationally repressed in round spermatids and trans-lationally active in elongating spermatids.

Key words: poly(A), translational regulation, spermatids,protamines 1 and 2, transition proteins 1 and 2.

Introduction

The specialized organelles of the mammalian spermato-zoon are formed during the haploid (spermatid) phaseof spermatogenesis which is known as spermiogenesis(reviewed in Bellvf & O'Brien, 1982). Spermiogenesisin mice lasts approximately two weeks and is dividedroughly in half into round spermatids (steps 1-8) andelongating spermatids (steps 9-15). In round sperma-tids, the acrosome and flagellum start to differentiate,and the rate of RNA synthesis declines ceasing entirelyby the beginning of the elongating spermatid stage(Monesi et al. 1978; Kierszenbaum & Tres, 1975). Inelongating spermatids, the mitochondria becomearranged in a spiral around the base of the flagellum, afibrous sheath is deposited around the flagellar axon-eme, and the nuclei become elongate, dense, compactand resistant to disruption by sonication (Kierszenbaum& Tres, 1975; Meistrich et al. 1976). The differentiationof the spermatid nucleus is accompanied by the replace-ment of the histones in two stages, first by a group ofspermatid-specific basic nuclear proteins, called tran-sition proteins, which are replaced in turn by prota-mines, the sole basic chromosomal protein in the spermof most mammals (Kistler et al. 1973; Bellv6 et al. 1975;

Grimes et al. 1977; Balhorn etal. 1984; Mayer & Zirkin,1979; Mayer etal. 1981).

The absence of detectable transcription in elongatingspermatids in mammals necessitates post-transcrip-tional regulation of mRNA translation and degra-dation. Iatrou & Dixon (1977) found that protaminemRNAs in the trout are synthesized and stored in atranslationally inactive state in meiotic cells and aretranslated in spermatids. Subsequently, the mRNAs formouse protamine 1 (Kleene et al. 1984), rat transitionprotein 1 (Heidaran & Kistler, 1987; Heidaran et al.1988) and rat protamine 1 (Mali et al. 1988) weredemonstrated to be translationally regulated but ac-cording to a different schedule than the trout: themRNAs are synthesized and translationally repressedin round spermatids and are translationally active inelongating spermatids. The protamine and transitionprotein mRNAs must be very stable because theypersist at high levels for about 6-7 days after thecessation of transcription (Kleene et al. 1983, 1984;Heidaran & Kistler, 1987; Heidaran et al. 1988; Mali etal. 1988). In addition, there is reason to suspect thatsome spermatidal mRNAs may be degraded after therequisite amount of protein has been synthesized be-cause rat transition protein 1 mRNA disappears from

368 K. C. Kleene

spermatids as the transition proteins are replaced byprotamines (Heidaran et al. 1988).

It is also known that the translation of trout prota-mine, mouse protamine 1 and rat transition protein 1mRNAs is paralleled by poly(A) shortening (Iatrou &Dixon, 1977; Kleene et al. 1984; Heidaran & Kistler,1987). The poly (A) tracts on translationally inactiveforms of mouse protamine 1 and rat transition protein 1mRNA are about 160 bases long and remarkablyhomogenous in size, whereas the poly(A) tracts on thetranslationally active mRNAs are heterogenous in sizevarying from about 30 to 160 bases (Kleene et al. 1984;Heidaran & Kistler, 1987). The shortening of poly(A)tracts on spermatidal mRNAs resembles the poly(A)shortening that normally starts when newly synthesizedmRNAs enter the cytoplasm (Sheiness & Darnell,1973). Poly (A) shortening may have a role in control-ling post-transcriptional gene expression during sper-miogenesis because mRNAs with poly(A) tracts lessthan about 30 residues long are thought to be degradedrapidly (Marbaix et al. 1975; Nudel et al. 1976) and/ortranslated inefficiently (Jacobson & Favreau, 1983;Palatnik et al 1984).

In this paper, I have compared the relationshipbetween poly(A) length and translational activity offive mRNAs which are synthesized after meiosis and arepresent at high levels in round and elongating sperma-tids in mice: transition protein 1, transition protein 2,protamine 1, the precursor for protamine 2 and a newunidentified haploid-expressed mRNA of about 1050bases, HEM1050 (Kleene et al. 1983, 1985, 1988,unpublished; Yelick et al. 1987; Kleene & Flynn, 1987).The experiments were designed to answer two ques-tions: First, does the distribution of these five mRNAsin the polysomal and nonpolysomal fractions of sucrosegradients change between round and elongating sper-matids? Second, are there differences in the lengths ofpoly(A) tracts on the polysomal and nonpolysomalforms of each mRNA? The results demonstrate strikingsimilarities in these mRNAs: all five mRNAs arepresent as nontranslated messenger ribonucleoproteinparticles (mRNPs) with homogenous poly(A) tractsabout 150 bases long in round spermatids, and 30-40 %of all five mRNAs are associated with the polysomes inelongating spermatids with heterogenous, shortenedpoly(A) tracts.

Thach, 1986) and cultured 15 min at 33 °C in an atmosphere of5 % CO2 in air to facilitate the recovery of polysomes aftercell separation (Kleene et al. 1984). The following operationswere carried out at 0-4 °C. The cells were collected bycentrifugation at 300g for 5 min. The cells were lysed in amotor-driven Teflon-glass homogenizer in 300/il 0-lM-NaCl,l-5mM-MgCl2, 20mM-Hepes (pH7-2), 0-5% Triton N-101and 0-1 % diethylpyrocarbonate. The nuclei and mitochon-dria were pelleted by centrifugation at 13 750 g for 2 min. Thesupernatants were adjusted to 0-5M-NaCl, 30mM-MgCl2 andcentrifuged again. 250 jA of the supernatant was layered over a3-7 ml linear 15—40% sucrose gradients inO-5M-NaCl, 30mM-MgCl2, 20mM-Hepes, pH7-2 (Weber et al. 1979). In someexperiments, the postmitochondrial supernatants were pre-pared and centrifuged on gradients in which 20mM-EDTAwas substituted for MgCl2. The gradients were centrifuged100 min at 50 000 revs min"1 in the Beckman SW60 rotor, anddeaccelerated to 8000 revs min"1 with the brake on. Thesecentifugation conditions are designed to separate nonpolyso-mal mRNAs from polysomes containing ^5 ribosomes, butpolysomes containing >5 ribosomes pellet. The gradientswere collected from the bottom'in a cold room, analyzed at254 run in a Beckman Model 153 flow cell (2 mm pathlength),SDS was added to 0-5 % to each fraction, and the fractionswere extracted once with 2 vol. of cold phenol: chloroform(1:1), and twice with chloroform. The pellets in the bottom ofthe ultracentrifuge tubes were dissolved in 0-5 ml sucrosegradient buffer containing 0-5 % SDS and extracted identi-cally. The RNA samples were precipitated with ethanol,digested with proteinase K, extracted with phenol and chloro-form again and precipitated with ethanol.

Deadenylation with RNase HTotal testis polysomal and nonpolysomal RNAs were pre-pared by centrifugation in sucrose gradients, extracted withphenol and chloroform, hybridized to oligo(dT) and digestedwith RNase H as described by Kleene et al. (1984).

RNA blotsRNA samples were denatured with glyoxal, electrophoresedthrough 25 cm 1-5-2% agarose gels for 24-26 h at 35 volts,and blotted to nitrocellulose (Thomas, 1980). About 60 ng ofcDNA inserts was labeled by random primer extension(Feinberg & Vogelstein, 1986), hybridized as described inKleene et al. (1984) except that the blots were washed in0-125 x SSPE 0-2% SDS at 65°C. For RNA dot blots, theRNAs were bound to nitrocellulose (Thomas, 1980), hybrid-ized to probes, and small pieces of nitrocellulose weredissolved in lml 2-methoxyethanol and counted in 10 mlAquasol (New England Nuclear).

Materials and methods

Isolation of polysomal and nonpolysomal RNA frompurified spermatogenic celbCD-I mice at least 60 days old were obtained from CharlesRiver Laboratories. Single cell suspensions from the testes of10 mice were fractionated by sedimentation at unit gravity onlinear 2-4% bovine serum albumin gradients in a StaputChamber (Meistrich et al. 1973; Romrell et al. 1976). Theround spermatids and nuclear and cytoplasmic fragments ofelongating spermatids from a cell separation were resus-pended in 3 ml RPMI 1640 medium containing 10% fetalbovine serum, 6mM-L-lactate, lmM-sodium pyruvate(O'Brien, 1987) and 0-2 jig ml"1 cycloheximide (Walden &

Results

Distribution of mRNAs in sucrose gradientsSince the rate of mRNA translation is usually regulatedat the level of initiation, changes in the rate of trans-lation of an mRNA are generally accompanied by ashift in the distribution of the mRNA between thepolysomal and nonpolysomal fractions. It follows thatchanges in the rate of translation of individual mRNAscan be detected by comparing the distribution ofmRNAs in sucrose gradients in Northern blots withcDNA probes (Rosenthal etal. 1983; Kleene etal. 1984;Heidaran & Kistler, 1987).

3-2

16

(A)RS

w(B)ES (C)» ES EDTA

lAll1 2 3 4 5 6 7 1 2 3 4 5 6 7

Fraction1 2 3 4 5 6 7

Fig. 1. Distribution of polysomes in sucrose gradients.Postmitochondrial supernatants were prepared frompurified round and elongating spermatids, centrifuged onsucrose gradients, and the absorbance at 254 nm wasmonitored during collection of gradient fractions. Theabsorbances were monitored using a flow cell with a 2 mmpathlength and have been converted to the absorbanceexpected for a lcm pathlength. (A) Round spermatids,gradient with Mg2"*"; (B) elongating spermatids, gradientwith Mg2"1"; (C) elongating spermatids, gradient with 20 mM-EDTA instead of M r .

Here, I have compared the size and translationallyactive and inactive fractions of five small mRNAs inround and elongating spermatids from mice: transitionproteins 1 and 2, protamines 1 and 2, and an unident-ified mRNA of about 1050 bases (HEM1050). Roundand elongating spermatids were purified on bovineserum albumin gradients. Postmitochondrial extractswere sedimented on sucrose gradients, and the size andlevels of mRNAs in gradient fractions were determinedin Northern blots. The sequence-of each cDNA probe isknown and each probe hybridizes to a single mRNA(Kleene et al. 1983, 1984, 1985, 1988, unpublished;Kleene & Flynn, 1987; Yelick et al. 1987).

The populations of round spermatids used herecontain about 80% round spermatids, 10% spermato-gonia and 10% elongating spermatids and theelongating spermatid fraction contained >95 % intactelongating spermatids and nuclear and cytoplasmicfragments of elongating spermatids. Fig. 1 (A and B)shows the distribution of absorbance at 254 nm insucrose gradients prepared from purified populations ofround and elongating spermatids. Note the large ab-sorbances of ribosomal subunits and polysomes that arepresent in postmitochondrial extracts of purified roundand elongating spermatids. These sucrose gradientscontain a high concentration of NaCl (0-5 M) to mini-mize contamination of the polysomal regions of thesucrose gradients by nonpolysomal mRNPs (Weber etal. 1979). The peaks for the 40S (fractions 2 and 3) and60S (fractions 3 and 4) ribosomal subunits are muchlarger than the 80S single ribosomes (fraction 4) inFig. 1A and B, because the high salt dissociates thesubunits of translationally inactive single ribosomes(Weber et al. 1979). The peaks of polysomes (fractions5-8) sedimenting more rapidly than single ribosomesfrom elongating spermatids were dissociated to ribo-somal subunits by EDTA as expected (Fig. 1C).(EDTA was equally effective in dissociating polysomes

Polyadenylation of spermatidal mRNAs 369

from round spermatids - data not shown.) In sevenexperiments, the amounts of ribosomal subunits werealways much larger than the polysomes in both roundand elongating spermatids, and the proportion of poly-somes to ribosomal subunits in round spermatids wasgreater than in elongating spermatids as shown inFig. 1A and B. The preponderance of translationallyinactive ribosomes in spermatids resembles a variety ofcells in which the overall rate of protein synthesis islimited at the level of initiation (reviewed by Jackson,1982). These data also suggest that the fraction oftranslationally active ribosomes decreases betweenround and elongating spermatids.

The Northern blots demonstrate that in round sper-matids (Fig. 2, RS), the vast majority of all five mRNAssediments slower than single ribosomes in fractions 2and 3, so these mRNAs are translationally inactive.Furthermore, each nonpolysomal mRNA migrates as anarrow band in agarose gels implying that there is littlevariation in the size of the mRNA. In elongatingspermatids, a large fraction of the five mRNAs is alsofound in the nonpolysomal region of the gradient, andthese mRNAs are indistinguishable in size from thenonpolysomal mRNAs in round spermatids (Fig. 2,ES). Fig. 3 (lane 2) demonstrates the homogeneity ofeach nonpolysomal mRNA more clearly because theautoradiogram was exposed for a shorter period than inFig. 2.

Fig. 2 (ES) also demonstrates that in elongatingspermatids substantial quantities of all five mRNAssediment in the polysomal region, and that thesepolysomal mRNAs migrate as a broad band of hetero-genous-sized RNAs ranging from the size of nonpolyso-mal RNAs to shorter sizes. There are two additionalreasons for believing that the heterogenous-sizedmRNAs sedimenting in the polysomal regions aretranslationally active: First, mRNAs encoding largerprimary translation products - protamine 2 precursor,109 amino acids and transition protein 2, 117 aminoacids (Yelick et al. 1987; Kleene & Flynn, 1987),sediment with larger polysomes than mRNAs encodingsmaller primary translation products - protamine 1, 51amino acids and transition protein 1, 55 amino acids(Kleene et al. 1985, 1988). Although the size of theprimary translation product of the HEM1050 mRNA isnot known, it is reasonable that this mRNA sedimentswith the largest polysomes as shown in Fig. 2E (ES),because the mRNA is the largest studied here (Fig. 3).Second, to verify that the heterogenous forms of themRNAs are associated with polysomes, the postmito-chondrial supernatants from elongating spermatidswere sedimented on sucrose gradients in which theMg2+ was replaced by EDTA. EDTA dissociatesmRNA and ribosomes causing polysomal mRNA tosediment slower than single ribosomes (Penman et al.1968). Most of the polysomes are dissociated to sub-units by EDTA (cf. Fig. IB and C), and most of theheterogenous forms of all five mRNAs are shifted to thepostpolysomal region of the gradient (Fig. 2, ES-EDTA).

It should be pointed out that the exposure range of

370 K. C. Kleene

A p r o t a m l n * 11 » .1 A K R ;

B t r i m I t I on pro t a i n 1

RS

ES

ESEDTA

• t

RS

ES

E S

E D T A

C P r o t a m l n * 2

1 2 3 4 S S 7 Ft DD t r a n s i t i o n p r o t e i n 2

RS

ES

ES

E D T A

RS

•ES

EES

E HEM 1 0 5 0

1 2 3 4 5 8 7 A P

RS -

ES

ES

EDTA

Fig. 2. Distribution of mRNAs for protamine 1, protamine2, transition protein 2, and HEM1050 mRNAs in polysomegradients from purified round and elongating spermatids.Postmitochondrial supernatants from round and elongatingspermatids were sedimented on sucrose gradients, and thegradients were collected in 8 fractions as shown in Fig. 1,and the pellet. RNA was extracted from each fraction,denatured with glyoxal, electrophoresed through 1-5%agarose (HEM1050) or 2 % agarose (protamine andtransition protein mRNAs), blotted to nitrocellulose andhybridized to the indicated 32P-labeled cDNA probes. Forprotamine and transition protein mRNAs, each lanecontained 10% of RNA from a sucrose gradient fraction,and for HEM1050 mRNA each lane contained 40 % of theRNA from a single cell separation. Each probe washybridized to three sets of sucrose gradient fraction RNAs:RS, round spermatid, Mg2+ gradient; ES, elongatingspermatids, Mg2"1" gradient; ES-EDTA, elongatingspermatid, EDTA gradient. The exposures varied from 3hto 3 days. The apparent differences in rate of sedimentationof the various nontranslated mRNAs are due to slightdifferences in fractionating the gradients, since there is nodifference in the distribution of the various mRNPs in thesame gradients.

the autoradiograms in Fig. 2 is so great that a singleexposure cannot illustrate all of the information in theNorthern blots. Longer exposures reveal that low levelsof the heterogenous polysomal forms of all five mRNAsare also detectable in the nonpolysomal fractions ofelongating spermatids (not shown). I do not knowwhether the heterogenous mRNAs in the nonpolysomalfractions are an artifact of cell separation (Kleene et al.1984) or a normal part of the translation cycle.

In addition, RNA dot blots were used to estimate theproportion of protamine 1 and transition protein 2mRNA in each sucrose gradient fraction from roundand elongating spermatids (data not shown). In roundspermatids, about 93-95 % of the protamine 1 andtransition protein 2 mRNAs sediments in the nonpoly-somal region (fractions 1-4). The small amounts ofthese mRNAs sedimenting in the polysomal regions(fractions 5-8) from round spermatids (5-7%) arepresumably derived from contamination of the roundspermatids by elongating spermatids and contaminationof the polysomal fractions of the gradient by nonpolyso-mal mRNPs. In four experiments, the average fractionof protamine 1 and transition protein 2 mRNAs sedi-menting in the polysomal fractions of elongating sper-matids was 28 % and 37 %, respectively.

Measurement of poly (A) tracts on polysomal andnonpolysomal mRNAsThe lengths of the poly(A) tracts on the nonpolysomaland polysomal mRNAs for the transition proteins andprotamines were analyzed in Northern blots before andafter selective degradation of the poly(A) tracts byhybridization to oligo(dT) and digestion with RNase H.Fig. 3 shows the relative sizes in a 2 % agarose gel ofintact and deadenylated polysomal and nonpolysomalmRNAs encoding transition proteins 1 and 2 andprotamines 1 and 2. The final panel shows the same datafor the HEM1050 mRNA in a 1-5 % agarose gel. Notethat the sizes of the polysomal mRNAs (lane 1) aremuch more heterogenous than the nonpolysomalmRNAs (lane 2). After deadenylation with RNase H,the mRNAs from the polysomal and postpolysomalfractions are reduced to virtually the same length (lanes3 and 4), demonstrating that the differences in size ofthe nonpolysomal and polysomal mRNAs are primarilydue to differences in the length of poly(A) tracts. Theapproximate sizes of the intact and deadenylated non-polysomal mRNAs based on three experiments are,respectively: protamine 1, 550 and 400 bases; transitionprotein 1, 590 and 440 bases; transition protein 2, 690and 540 bases; and protamine 2, 760 and 610 bases;HEM1050 mRNA, 1050 and 900 bases. Estimates of thelengths of the poly(A) tracts on the five mRNAs variedbetween about 120 and 180 bases. The difference in sizebetween the shortest polysomal mRNAs and deadenyl-ated mRNAs indicates that the majority of polysomalmRNAs have poly(A) tracts at least 30 bases long.

Despite the similarity in the sizes of the poly(A)tracts on the five spermatid-specific mRNAs, there areobvious differences in the size distribution of thepolysomal forms of these mRNAs. The polysomal

Polyadenylation of spermatidal mRNAs 371

protamine 1 mRNAs with the shortest poly(A) tractsare consistently much more abundant than the mRNAswith longer poly(A) tracts (Kleene et al. 1984). Bycomparison, the polysomal transition protein 1, tran-sition protein 2 and protamine 2 mRNAs usually showtwo distinct size classes, one corresponding to mRNAswith little or no poly(A) shortening and the other tomRNAs with shortened poly(A) tracts. The HEM1050mRNA differs from the transition protein and prota-mine mRNAs because the majority of its polysomalmRNAs shows no obvious poly(A) shortening (Fig. 2E,ES).

Discussion

Five haploid-specific mRNAs including the mRNAsencoding the four major basic nuclear proteins thatreplace the histones in elongating spermatids in themouse - transition proteins 1 and 2 and protamines 1and 2 - have been demonstrated here to undergo similarchanges in translational activity and poly(A) lengthduring spermiogenesis in the mouse. In round sperma-tids, all five mRNAs are present as translationallyinactive, nonpolysomal mRNPs bearing homogenous-sized poly(A) tracts about 150 bases long. In elongatingspermatids, approximately 30-40% of each mRNA isassociated with the polysomes bearing heterogenous-sized poly (A) tracts ranging from about 30 to 150 bases.These observations confirm and extend previous studiesof the mRNAs for trout protamines (Iatrou & Dixon,1977), mouse protamine 1 (Kleene et al. 1984) and rattransition protein 1 (Heidaran & Kistler, 1987). Sincethe mRNAs for transition proteins 1 and 2 and prota-mines 1 and 2 account for all of the abundant basicnuclear proteins in sonication-resistant spermatids inmice (Kleene, unpublished), the HEM1050 mRNAdoes not encode a spermatidal basic nuclear protein.Therefore, the correlation between active translationand poly(A) shortening in these five mRNAs mayextend to all mRNAs that are translationally repressedin round spermatids and translationally active inelongating spermatids.

The change in the distribution of mouse transitionprotein 1 and 2 and protamine 1 and 2 mRNAs between

the nonpolysomal and polysomal fractions in round andelongating spermatids parallels a lag between the syn-thesis of these mRNAs in round spermatids and thesynthesis of the transition proteins and protaminesduring steps 12-15 of elongating spermatids (Grimes etal. 1977; Mayer & Zirkin, 1979; Mayer et al. 1981;Kleene et al. 1983; Kleene & Flynn, 1987; Balhom et al.1984; Heidaran & Kistler, 1987; Heidaran et al. 1988).The large fraction (approximately 2/3) of the transitionprotein and protamine mRNAs that are translationallyinactive in elongating spermatids is at least partlyexplained by the repression of translation of the tran-sition protein and protamine mRNAs in earlyelongating spermatids (Bellve' et al. 1975; Mayer &Zirkin, 1979; Balhorn et al. 1984). However, it is alsopossible that protamine and transition protein mRNAsare totally active in translation for a very brief period inelongating spermatids.

The shortening of poly(A) on spermatidal mRNAs isanalogous to a general phenomenon of poly(A) short-ening in eukaryotic cells (reviewed in Brawerman,1981). Typically, long homogenous poly(A) tracts areadded post-transcriptionally to newly synthesizedmRNAs in the nucleus, and the poly(A) tracts graduallyshorten and become more heterogenous in the cyto-plasm. It seems more likely that poly(A) shortening inspermatids is a consequence of, rather than a prerequi-site for, translation. Poly (A) shortening is not sufficientfor translation because there is no obvious poly(A)shortening on a portion of each of the polysomal formsof the mRNAs studied here. In fact, most of thepolysomal HEM1050 mRNAs have undergone no de-tectable poly (A) shortening. Furthermore, nonhistonemRNAs with longer poly(A) tracts are translated moreefficiently than mRNAs with shorter or no poly(A)tracts (Jacobson & Favreau, 1983; Palatnik et al. 1984;Drummond et al. 1985; Galili et al. 1988). The alterna-tive that poly(A) shortening is a consequence of trans-lation explains why poly(A) tracts on polysomalmRNAs are shortened more rapidly than on nonpolyso-mal mRNAs in spermatids, mammalian tissue culturecells (Sheiness & Darnell, 1973; Merkel etal. 1976), andsea urchin embryos (Dworkin et ah 1977). Several linesof evidence also suggest that mRNA degradation andpoly(A) shortening are coupled to the interaction of

PI2 3 4

TP12 3 4

TP22 3 4

P22 3 4

HEM10502 1 3 4

7 80 -

530 -

400 -

• „ -1280

-780

Fig. 3. Effects of deadenylationwith RNase H on the sizes ofpolysomal and nonpolysomalmRNAs for protamine 1 (PI),protamine 2 (P2), transitionprotein 1 (TP1), transition protein2 (TP2), and HEM1050 mRNAs.Postmitochondrial supernatants

from total testes were sedimented on sucrose gradients and separated into fractions which sediment faster and slower thansingle ribosomes. (The region of the gradient containing single ribosomes was discarded.) A sample of polysomal andnonpolysomal RNA was hybridized to oligo(dT) and digested with RNase H to remove poly(A) tracts. Intact and RNaseH-treated RNAs were denatured with gjyoxal, electrophoresed through 1-5% agarose (HEM1050 mRNA) or 2% agarose(protamine and transition protein mRNAs), blotted to nitrocellulose, and hybridized to the indicated 32P-labeled DNAprobes. The RNA size markers were obtained from Bethesda Research Laboratories. Lane 1, 1-2 fig intact polysomal RNA;lane 2, 0-2 fig intact postpolysomal RNA; lane 3, 1-2 ^g RNase H-treated polysomal RNA; lane 4, 0-2 fig RNase H-treatedpostpolysomal RNA.

372 K. C. Kleene

mRNAs and ribosomes (Sheiness et al. 1975; Shaw &Kamen, 1986; Graves et al. 1987; Pachter et al. 1987;Brewer & Ross, 1988; Wilson & Treisman, 1988).

There are two implications of the idea that poly(A)shortening of spermatidal mRNAs is a consequence oftranslation. First, it raises the question whether themarked differences in the size distribution of poly(A)tracts on the polysomal forms of the five mRNAsstudied here are due to differences in the rate ofshortening of poly(A) tracts of different lengths orchanges in the rate of translation at different stages ofspermiogenesis. Second, it predicts that mRNAs thatare synthesized and translated efficiently in roundspermatids will be exclusively associated with poly-somes with shortened poly(A) tracts. Similarly,mRNAs that are synthesized and translated ineffi-ciently in round spermatids will be found in bothpolysomal and nonpolysomal fractions with shortenedpoly(A) tracts because the mRNAs interchange be-tween the polysomal and nonpolysomal compartments.The mRNA for the testis-specific isozyme of lactatedehydrogenase (LDH-X) is synthesized and translatedinefficiently in meiotic and haploid spermatogenic cells(Meistrich et al. 1977; Wieben, 1981; Fujimoto et al.1988). In accordance with these predictions, the LDH-X mRNA is found in the polysomal and nonpolysomalfractions of meiotic and haploid stages with shortened,heterogenous poly(A) tracts (Fujimoto et al. 1988).

Although the function of poly(A) shortening inmRNA metabolism is disputed, most workers agreethat the loss of poly(A) tracts from nonhistone mRNAsresults in loss of mRNA function. In some cases,nonadenylated mRNAs are degraded rapidly (Marbaixet al. 1975; Brewer & Ross, 1988; Wilson & Treisman,1988; Bernstein et al. 1989) and, in other cases, non-adenylated mRNAs are translated inefficiently (Jacob-son & Favreau, 1983; Drummond et al. 1985; Galili etal. 1988). The conclusion that spermatidal mRNAs withpoly(A) tracts 30-150 bases long are translationallyactive is consistent with this work because the loss ofmRNA function is observed only when shorteningreduces the poly(A) tracts to less than about 30 bases(Nudel et al. 1976; Jacobson & Favreau, 1983). It maybe possible to elucidate whether poly(A) shortening onspermatidal mRNAs is associated with degradation ortranslational inhibition by microinjecting nonadenyl-ated and polyadenylated mRNAs or by examining thesize and distribution of nonadenylated mRNAs insucrose gradients.

This work was supported by Grants DCB-8510350 andDCB-8710485 from the National Science Foundation.

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(Accepted 3 March 1989)