Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver

The present study is undertaken to investigate whether the phospholipase A(2) (PLA(2)) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA(2) (10(-5) approximately 10(-2)/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA(2), regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA(2)-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA(2)-treated group. Messenger RNA export rate in PLA(2) (10(-3) unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA(2) plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified.     

Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport.

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-promoting phorbol esters resulted in inhibition of nuclear-envelope NTPase activity and in a concomitant decrease of mRNA (actin) efflux rate from isolated rat liver nuclei. Protein kinase C, but not nuclear envelope NI-like or NII-like protein kinase, was found to be solubilized from the envelope by Triton X-100, whereas the presumable poly(A)-binding site [the 106 kDa polypeptide, representing the putative carrier for poly(A)+mRNA transport] remained bound to this structure. RNA efflux from detergent-treated nuclei lost its susceptibility to phorbol esters. Addition of purified protein kinase C to these nuclei restored the effect of the tumour promoters. Protein kinase C was found to bind also to isolated rat liver nuclear matrices in the absence but not in the presence of ATP. The NII-like nuclear-envelope protein kinase co-purified together with the 106 kDa polypeptide which specifically binds to poly(A) in an ATP-labile linkage.     

Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3''-deoxyadenosine (cordycepin).

The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.     

Differential effect of insulin and epidermal growth factor on the mRNA translocation system and transport of specific poly(A+) mRNA and poly(A-) mRNA in isolated nuclei

The efficiency of efflux of rapidly labeled poly(A)-containing mRNA from isolated rat liver nuclei was found to be modulated by insulin and epidermal growth factor (EGF) in a biphasic but opposite way. At physiological concentrations (10 pM insulin and 1 pM EGF), maximal stimulation of the transport rate by insulin (to 137%) and maximal inhibition by EGF (to 69%) were obtained; at higher concentrations (greater than 100 pM and greater than 10 pM, respectively), the amount of poly(A)-containing mRNA released into the postnuclear supernatant was nearly identical with the level found in untreated nuclei (= 100%). Using mRNA entrapped into closed nuclear envelope (NE) vesicles as a model system, it was found that the modulation of nuclear efflux of mRNA by the two growth factors occurs at the level of translocation through the nuclear pore. The NE nucleoside-triphosphatase (NTPase) activity, which is thought to mediate nucleocytoplasmic transport of at least some mRNAs, responded to insulin and EGF in the same manner as the mRNA transport rate. The increase in NTPase activity caused by insulin and the decrease in NTPase activity caused by EGF were found to be due to changes of the maximal catalytic rate; the Michaelis constant of the enzyme remained almost constant. Investigating the effect of the two growth factors on transport of specific mRNAs, poly(A)-containing actin mRNA was found to display the same alteration in efflux rate as rapidly labeled, total poly(A)-containing mRNA. In contrast, efflux of histone H4 mRNA, which lacks a 3'-poly(A) sequence, decreased in response to insulin and reached minimum levels at the same concentration at which maximum levels of actin mRNA transport rate were obtained. Studying the mechanism of action of insulin and EGF on NE mRNA translocation system, insulin was found to cause an enhancement of NE-associated phosphoprotein phosphatase activity, resulting in a dephosphorylation of the NE poly(A) binding site (= mRNA carrier) and, hence, in a decrease in its affinity to poly(A) [the poly(A) binding affinity of the poly(A)-recognizing mRNA carrier within the envelope is increased after phosphorylation]. EGF, on the other hand, stimulated the protein kinase, which phosphorylates the carrier, and, hence increased the NE poly(A) binding affinity. Because the stage of phosphorylation of the mRNA carrier (which is coupled with the NTPase within the intact NE structure) is inversely correlated with the activity of the NTPase, an enhancement of poly(A)-containing mRNA transport rate by insulin and an inhibition by EGF are observed.     

Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures

Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.     

The poly(A)-binding protein facilitates in vitro translation of poly(A)-rich mRNA

To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A). Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)-binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation.     

HeLa cell cytoplasmic mRNA contains three classes of sequences: predominantly poly(A)-free, predominantly poly(A)-containing and bimorphic

The mRNA species which exist in the HeLa cell polyribisomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)-free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)-containing RNA. If poly(A)-free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single-copy DNA. When a large excess of steady poly(A)-containing RNA is added before hybridization of labelled poly(A)-free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)-free mRNA with no poly(A)-containing counterpart. Some mRNA species can exist solely as poly(A)-containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitro of Kaufmann et al. [Proc. Natl Acad. Sci. U.S.A. 74, 4801--4805 (1977)]. In addition, if cDNA to total poly(A)-containing mRNA is fractionated into abundant and scarce classes, 47% of the scarce class cDNA can be readily hybridized with poly(A)-free mRNA. 10% of the abundant cDNA to poly(A)-containing mRNA will hybridize with poly(A)-free sequences very rapidly while the other 90% hybridize 160 times more slowly, indicating two very different frequency distributions. The cytoplasmic metabolism of these three distinct mRNA classes is discussed.     

Modulation of poly(A)(+)mRNA-metabolizing and transporting systems under special consideration of microtubule protein and actin

At present, three enzymes are known which participate in regulation of polyadenylation and polydeadenylation of eukaryotic mRNA: poly(A) polymerase, endoribonuclease IV and 2',3'-exoribonuclease. Moreover, poly(A)-associated proteins as well as the cytoskeletal proteins actin and tubulin have been found to be involved in poly(A) metabolism of mRNA; their modulation effects on poly(A) metabolizing enzyme systems will be described in greater detail. Nucleo-cytoplasmic transport of poly(A)-containing mRNA is thought to be mediated by nuclear-envelope nucleoside triphosphatase. The stimulation of this enzyme by the poly(A) segment of mRNA and its modulation by microtubule protein are discussed in the second part of this review.     

Purification and characterization of the major nucleoside triphosphatase from rat liver nuclear envelopes

Nuclear envelopes contain a nucleoside triphosphatase. Hydrolysis of ATP or GTP by this enzyme parallels energy-dependent efflux of poly(A)-containing mRNA from nuclei in vitro. Nucleoside triphosphatase has been purified from highly purified preparations of nuclear envelopes from rat liver by three successive affinity steps. The essentially homogeneous enzyme has an apparent molecular weight of 40,000 as checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and displays a rather broad substrate specificity. ATP and GTP are hydrolyzed at nearly equal rates, whereas UTP and CTP are only half as active as substrates. For optimal activity, a one-to-one ratio of a divalent cation (Mg2+, Mn2+, or Ca2+) and the nucleoside triphosphate substrate, an alkaline pH and a temperature of 34 degrees C are required. In contrast to the enzyme associated with nuclear envelopes which is stimulated by synthetic poly(A) and the poly(A) segment of the natural poly(A)-containing mRNA, homogeneous nucleoside triphosphatase is unable to be modulated by this polynucleotide species.     

RNA transport from isolated nuclei of cultured Djungarian hamster cells

The in vitro system of RNA transport containing isolated nuclei of Djungarian hamster cells transformed by SV-40 virus was studied. A functional test for cytoplasmic contaminations of the nuclei was proposed. The release of the newly synthesized RNA was found to be dependent on the duration of incubation, temperature and pH of the incubation medium as well as on the presence of spermine, spermidine, dithiothreitol, Mg2+, EDTA, exogenous RNA, nucleoside triphosphates and cytosol. The results obtained indicate that RNA release is an active process with activation energy of 12 kcal/mol. ATP, GTP, CTP and UTP have equal ability to serve as energy sources for the release of RNA. The nucleoside triphosphatase activity of the nuclei was the same in the presence of these four nucleoside triphosphates.     

In vitro methylation of undermethylated yeast poly(A)-rich RNA using mRNA(guanine-7-)-methyltransferase purified from wheat germ or yeast

By crossing two strains of Saccharomyces cerevisiae deficient for each of the two methionine adenosyltransferase isoenzymes (ATP: L-methionine S-adenosyltransferase EC 2.5.1.6) respectively, we have constructed a strain strictly auxotrophic for S-adenosylmethionine and used it as a source of undermethylated mRNA suitable for in vitro transmethylation studies. RNA has been phenol-extracted from yeast cells shifted down to S-adenosylmethionine-free medium for 90 min and poly(A)-rich RNA has been prepared by oligo(dT)-cellulose chromatography. Upon incubation in vitro in the presence of methyl-labeled S-adenosylmethionine and mRNA (guanine-7-)-methyltransferase purified from wheat germ or yeast, undermethylated poly(A)-rich RNA became significantly labeled as compared to non-starved cells from the same strain, or from a wild-type control. Cap structures were resolved by paper chromatography afer T2 and P1 RNase digestion, and shown to be a mixture of m7G5'ppp5'G and m7G5'ppp5'A, irrespective of the enzyme source, in agreement with earlier in vivo studies in yeast mRNA capping and methylation.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

Biogenesis and cell cycle relationship of poly(A)- actin mRNA in mouse ascites cells

A variety of rapidly growing mammalian cells contain a substantial portion of their actin mRNA in a poly(A)- form. We have used DNA-driven hybridization of a cloned actin cDNA-containing plasmid with pulse-labeled RNA from mouse S-180 ascites cells to examine newly synthesized actin mRNA. Our results indicate that the same proportion of newly synthesized and steady-state actin mRNA (approx. 40%) exists in a poly(A)- deficient form. This suggests that the poly(A)- form arises by some process other than slow cytoplasmic de-adenylation of a poly(A)+ precursor. We have also examined cell cycle-enriched populations of S-180 ascites cells for the presence of poly(A)- actin mRNA. Results from these experiments indicate that cells in G1 phase of the cell cycle contain predominantly poly(A)+ actin mRNA, while the poly(A)- form is restricted to late-S and post-S phase cells.     

Cytochalasin B selectively releases ovalbumin mRNA precursors but not the mature ovalbumin mRNA from hen oviduct nuclear matrix

Hen oviduct nuclear matrix-bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP-dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP, but inhibited the ATP-induced detachment of mature mRNA. RNA release was also achieved with a monoclonal antibody against actin but not with monoclonal antibodies against tubulin and intermediate filaments. These results suggest that actin-containing filaments are involved in the restriction of immature messengers to the cell nucleus.     

Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.     

Translational regulation of human beta interferon mRNA: association of the 3'' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.     

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol P_i/min per 10⁸ ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the ‘carrier structure’ for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.     

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.