INTRODUCTION

All eukaryotic mRNAs, with the exception of major histone mRNAs, have a poly(A) tail at their 3 end. The process generating the poly(A) tail, referred to as 3 end formation, involves endonucleolytic cleavage of the precursor RNA followed by the sequential addition of adenylate residues. 3 end formation is an essential step in the maturation of pre-mRNAs, and it appears to be coupled with other events in the nucleus, including transcription termination (1-3) and mRNA splicing (4-6). The resulting poly(A) tail has been suggested to play important roles in mRNA stability (reviewed in Ref. 7) and translation (reviewed in Ref. 8). Alternate usage of polyadenylation sites can regulate gene expression through changing the capacity of coding or noncoding sequences in the 3 region of mRNA (9-10) (reviewed in Ref. 11).

Although the cleavage and poly(A) addition reactions are tightly coupled in vivo, development of in vitro systems made it possible to study these reactions separately and to purify factors required for each step (for reviews, see Refs. 12 and 13). Cleavage can be assayed by blocking poly(A) polymerase (PAP)¹ activity with either EDTA or chain-terminating ATP analogs, such as 3dATP, whereas poly(A) addition independent of cleavage can be studied by using "precleaved" RNA substrates. In mammalian cells, two RNA sequence elements are essential for efficient 3 end formation. One is the highly conserved hexanucleotide AAUAAA located 10-30 nucleotides upstream of the cleavage site, and the other is a less conserved GU- or U-rich sequence that lies just downstream of the cleavage site (for review, see Ref. 14). Accurate and efficient cleavage requires five protein factors: cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), two cleavage factors (CFI and CFII), and, in many cases, PAP (15-18). CPSF consists of at least three subunits (160, 100, and 73) and likely a fourth (30 kDa) that is nonessential in vitro (19-22). CstF is composed of three subunits (77, 64, and 50 kDa) (23-26). CPSF and CstF can specifically and cooperatively interact with AAUAAA and the downstream GU-rich sequence in the precursor RNA, respectively (21, 22, 27-30), and thus they likely can specify the poly(A) site. CFI (16) has been purified from HeLa cells and appears also to be a multisubunit factor (31), although the roles of both CFI and CFII in cleavage have not been well defined. PAP, a single polypeptide of ~85 kDa (32, 33), is dispensable for cleavage of SV40 late pre-mRNA in vitro but is required for efficient cleavage of other substrates (Ref. 15). Following cleavage, CPSF, PAP, and poly(A) binding protein II participate in poly(A) addition (34-36). CstF, CFI, and CFII are not necessary for this reaction in vitro, although whether these factors dissociate from the complex at this stage is not clear.

Although the RNA sequences and protein factors responsible for 3 end formation have been well studied, cofactor requirements for the reaction have been examined only with relatively crude extracts (for review, see Refs. 37 and 38). Early experiments conducted with unfractionated nuclear extracts suggested that ATP is a necessary cofactor for the cleavage reaction (39, 40) and for formation of polyadenylation-specific complexes (40-43). However, several observations have confused the issue. First, the cleavage reaction was found to proceed in the absence of divalent cation and the presence of EDTA (39, 44), which would not be expected for an ATP-requiring reaction. Second, the addition of creatine phosphate (CP), commonly used to regenerate ATP by phosphate transfer from CP to ADP (catalyzed by creatine phosphokinase (CPK)), was shown to be sufficient for cleavage in the absence of exogenously added ATP and divalent cation (15). These observations could reflect an unusual mechanism of ATP regeneration from endogenous ATP/ADP in the crude protein fractions or indicate that ATP is not in fact necessary for cleavage. We therefore reexamined the cofactor requirement for in vitro cleavage using highly purified factors. Here we present evidence that CP can in fact function as a necessary and sufficient cofactor for efficient in vitro cleavage. Although ATP is not required, it can function as either an inhibitor or an activator of the reaction, depending on the specific substrate, and can also affect the precise cleavage site in PAP-dependent cleavage. Possible mechanisms of CP-activated cleavage are discussed.

EXPERIMENTAL PROCEDURES

Creatine phosphate was purchased from Sigma, Calbiochem, and Boehringer Mannheim. Arginine phosphate was from Sigma. ³²P-Labeled creatine phosphate (2.3 × 10⁷ cpm/µg) was enzymatically synthesized from [-³²P]ATP (4500 Ci/mmol; ICN) and creatine (Sigma) by creatine phosphokinase (Boehringer Mannheim) and then purified by MonoQ FPLC.

Partial purification of CPSF, CFI, and CFII from HeLa cell nuclear extracts was performed as described (16). CPSF (0.12 µg/µl) was further purified by heparin-5PW FPLC. CFI (0.4 µg/µl) and CFII (0.15 µg/µl) were MonoS fractions (16). CstF (0.15 µg/µl) was also a MonoS fraction (purity greater than 90%; Ref. 23). CFI was further purified by phenyl-Superose FPLC (PS-CFI; 0.12 µg/µl). Alternatively, the MonoQ low salt fraction (mixture of CFI and CFII; Ref. 16) was fractionated by heparin-5PW FPLC, MonoS FPLC, and heparin-5PW FPLC (HP-CFI; 0.15 µg/µl). CFII was further purified by MonoS (MSS-CFII; 50 ng/µl), or the MonoQ low salt fraction was fractionated by heparin-5PW FPLC (HP-CFH; 40 ng/ml). Histidine-tagged recombinant bovine PAP (50 ng/µl) was purified from Escherichia coli (gift of K. G. K. Murthy). All protein fractions were stored in Buffer D (20 mM HEPES-NaOH, pH 7.9, 20% glycerol, 50 mM (NH₄)₂SO₄, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride). Details of the CFI and CFII purification procedures will be described elsewhere.²

³²P-labeled pre-mRNAs were prepared by SP6 RNA polymerase (Promega) from linearized plasmids pG3SVL-A and pG3L3-A, which contain the SV40 late site and adenovirus 2 L3 poly(A) site, respectively (15). A standard 12.5-µl cleavage reaction mixture contained 0.25 µl of CstF; 2.0 µl of CPSF; 1.75 µl of CFI; 2.0 µl of CFII; 0.25 µl of either Buffer D (for SV40 pre-mRNA) or recombinant bovine PAP (for L3 pre-mRNA); 10 mM HEPES-NaOH, pH 7.9; 10% glycerol; 25 mM (NH₄)₂SO₄; 0.1 mM EDTA; 0.25 mM dithiothreitol; 0.25 mM phenylmethylsulfonyl fluoride; 2.5% polyvinyl alcohol; 0.2-0.5 ng of substrate RNA; 500 ng of E. coli tRNA; and 2 mM EDTA, 0.5 mM MgCl₂, or no divalent cation as indicated, plus the indicated amounts of CP, ATP, or other potential cofactors. Reaction mixtures were incubated for 90-120 min at 30 °C, and purified RNAs were fractionated on 5% polyacrylamide, 8.3 M urea gels.

Reaction mixtures (30 µl) containing 25 µl of protein mixture (8 µl of CPSF, 1 µl of CstF, 7 µl of CFI, 8 µl of CFII, and 1 µl of Buffer D), 2.5 mM MgCl_2,, 2 mM glucose, 0.6 unit of hexokinase (Boehringer Mannheim), and 0.6 µCi of [-³²P]ATP were incubated for 10 min at 30 °C. Polyethyleneimine-cellulose TLC plates (Selecto Scientific) were washed by elution with deionized water and dried before use. Aliquots (1 µl) taken from reaction mixtures at various time points were diluted with 20 mM EDTA to 20 µl and frozen on dry ice until use. After thawing, diluted samples were spotted on the prepared polyethyleneimine-cellulose TLC plates and developed in 1 M formic acid and 0.5 M LiCl (Figs. 5A and 6C) or in 0.25 M LiCl (Fig. 6B). ³²P-Labeled compounds were visualized by autoradiography.

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RESULTS

Our previous experiments using a relatively crude preparation of 3 processing factors demonstrated that efficient cleavage could be detected in the presence of CP without added ATP, whereas addition of ATP alone did not allow cleavage (15). This observation suggested either that the protein fraction employed contained high ATPase levels, as well as sufficient CPK to regenerate ATP, or that CP functioned as a necessary cofactor for cleavage. We therefore reinvestigated the requirement of CP and/or ATP for 3 cleavage using protein factors that were more purified than those used in the previous experiments. We first assayed in vitro cleavage reconstituted with an RNA precursor containing the SV40 late poly(A) signal (SV40 pre-mRNA) with highly purified CstF and CPSF and partially purified CFI and CFII prepared from HeLa cell nuclear extracts under standard cleavage conditions, which included 2 mM EDTA (see "Experimental Procedures"). Importantly, these factors all lacked detectable CPK (measured as in Ref. 45) and ATPase (measured by hydrolysis of [-³²P]ATP) activities when assayed under cleavage reaction conditions, whereas in the presence of Mg²⁺, CPK activity was again undetectable, and only trace levels of ATPase were observed (data not shown). Fig. 1A shows that, as observed previously with less purified factors, addition of 20 mM CP alone was sufficient to allow efficient cleavage (lane 3), whereas no phosphate compound (lane 1) or ATP alone (1 mM) (lane 4) did not result in detectable cleavage. In fact, the presence of both ATP and CP in the reaction mixture reduced cleavage efficiency (compare Fig. 1, lanes 2 and 3, and see below). Fig. 1B shows that cleavage efficiency increased according to the concentration of CP. Cleavage was greatly reduced at concentrations below 10 and above 60 mM CP (data not shown).

Creatine phosphate (CP) can be a sufficient cofactor for efficient 3 end cleavage.

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We next examined whether a number of other compounds could function as cofactors in the cleavage reaction. Creatine, NaCl, sodium pyrophosphate, and a number of nucleotide tri-, di- or monophosphates were unable to allow cleavage over a wide range of concentrations (Fig. 2A and data not shown; note that GTP reproducibly caused degradation of the substrate). On the other hand, NaPO₄ and to a somewhat lesser extent KPO₄ did allow cleavage (Fig. 2B). However, the optimal efficiency of cleavage observed was lower by a factor of six than that observed with CP, as determined by PhosphorImager analysis. Importantly, the same high concentrations of these compounds were required to induce cleavage as was observed with CP. This is significant because NaPO₄ can be a trace contaminant of CP, but since such high amounts were necessary, it cannot be the active component in CP (concentrations lower than those shown in Fig. 2B were inactive; data not shown).

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Given the high concentration of CP required for cleavage, we were concerned about the possibility that a contaminant in the CP preparation other than inorganic phosphate might be the actual cofactor (see also below). To address this, we tested four different preparations of CP obtained from three different sources (one was prepared synthetically, whereas the others were prepared enzymatically; see "Experimental Procedures") and found that all four preparations of CP behaved identically (data not shown). The Tris and sodium salts of CP also gave indistinguishable results (data not shown). We also tested whether another phosphoguanidine compound, arginine phosphate (AP), which exists only in invertebrates (reviewed in Ref. 46), could substitute for CP in the cleavage assay. Strikingly, AP also activated cleavage, and it did so as efficiently as CP (Fig. 2C). This implies that the phosphoguanido group may be an important structural motif of the cofactor. Importantly, the fact that AP can substitute for CP further argues against the possibility that the CP requirement in fact reflects an ATP requirement, through the activity of undetectable contaminating CPK in the purified factors, because AP can not be used as a substrate by CPK (47).

In the above experiments, we utilized highly purified preparations of CPSF and CstF, whereas the CFI and CFII fractions were less pure (although, as mentioned above, they were free of detectable CPK and ATPase). To provide additional evidence that contaminants in the protein fractions were not in some way responsible for the CP effect, both CFI and CFII were subjected to additional purification steps (see "Experimental Procedures"), and two different preparations of each were used in cleavage assays, as above. Under standard reaction conditions (a solution containing 2 mM EDTA and no ATP), CP was again found to be essential for cleavage, activating the reaction in a concentration-dependent manner, nearly identical to the results observed with the less pure cleavage factors (Fig. 3).

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The above results suggest that CP, not ATP, can be a necessary and sufficient cofactor for efficient 3 cleavage in vitro. However, given the unexpected nature of this finding, we investigated more extensively the possible role of ATP in 3 cleavage of the SV40 pre-mRNA. We first tested the effect of increasing concentrations of exogenous ATP on cleavage. Fig. 4A shows that ATP concentrations as high as 10 mM did not allow detectable cleavage in the presence of either 0.5 mM MgCl₂ (lanes 2-4) or 2 mM EDTA (lanes 7-9), whereas CP alone allowed efficient cleavage under both reaction conditions (lanes 5 and 10). As shown above (Fig. 1A), cleavage in the presence of both ATP and CP appeared less efficient than cleavage in the presence of CP alone. We therefore examined the effect of increasing ATP concentrations on cleavage activated by 40 mM CP. Fig. 4B shows that ATP indeed inhibited cleavage effected by CP in a concentration-dependent manner in the presence of either 0.5 mM MgCl₂ (lanes 2-4) or 2 mM EDTA (lanes 6-8). In contrast, high concentrations of EDTA (up to 8 mM) had no significant effect on cleavage efficiency (Fig. 4C, lanes 1-4). These findings strengthen the conclusions that divalent cations are not required for cleavage and that ATP is not only not required but can in fact be inhibitory.

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We next examined the possibility that a bound form of ATP, perhaps tightly associated with a purified factor(s), might participate in cleavage. To this end, cleavage was assayed using factors treated with hexokinase and glucose to remove any endogenous ATP. Depletion of endogenous ATP by conversion to glucose 6 phosphate was monitored via addition of trace amounts of [-³²P]ATP and was analyzed by TLC. After incubation of purified factors with hexokinase and glucose for 10 min in the presence of MgCl₂, the exogenous ATP was entirely depleted (Fig. 5A, lanes 1 and 5). EDTA and substrate pre-mRNA were then added, and reaction mixtures were incubated for the times indicated in Fig. 5, after which RNA products and ATP were both analyzed. Although no labeled ATP was generated at any time during the incubation (Fig. 5A, lanes 2-4 and lanes 6-8), efficient cleavage was detected when CP was added (Fig. 5B, lanes 2-4), but not when ATP was added (Fig. 5B, lanes 6-8). Identical results (not shown) were obtained in the absence of added EDTA, which would allow the hexokinase to remain active throughout the incubation. Taken together, the above results indicate that 3 cleavage occurs efficiently in the absence of ATP.

CP has a large free energy of hydrolysis (10.3 kcal/mol; reviewed in Ref. 46) and, as suggested above, is a necessary cofactor for efficient 3 cleavage. These points raise the possibility that CP could function as a direct energy donor during the cleavage reaction. To examine this, we tested whether hydrolysis of CP occurs during the cleavage reaction. ³²P-Labeled CP was prepared by enzymatic reaction from [-³²P]ATP and creatine and purified as described under "Experimental Procedures." Hydrolysis of CP during the cleavage reaction was monitored by removing small aliquots from reaction mixtures containing trace amounts of ³²P-labeled CP and 40 mM unlabeled CP at the times indicated in Fig. 6 and then analyzing the fate of the CP by TLC. Whereas efficient cleavage proceeded during the incubation (Fig. 6A), neither hydrolyzed inorganic phosphate (Fig. 6B) nor ATP, conceivably generated by transferring the phosphate group from CP to possible endogenous ADP (Fig. 6C), was detected. These results suggest that CP is neither an energy donor for cleavage nor a phosphate source to produce ATP. This latter finding further excludes the possibility that the observed CP requirement reflects ATP regeneration by CPK contamination in the purified factors. Also, SDS gel electrophoresis of reaction mixtures following incubation with labeled CP failed to provide any evidence of protein phosphorylation (results not shown).

We next wished to determine whether our findings could be extended to other pre-mRNAs, which, unlike SV40 late, require PAP for cleavage. We therefore examined the substrate specificity of the CP effect on cleavage by using a pre-mRNA containing the adenovirus L3 poly(A) signal (48), purified factors as above, and recombinant bovine PAP (see "Experimental Procedures"). Fig. 7 shows that no cleavage was detected in the absence of CP (lanes 1 and 5), but processing was observed as the concentration of CP was increased (lanes 2-4 and 6-8). However, in this case, cleavage was inefficient, and addition of 1 mM ATP with CP greatly enhanced cleavage efficiency (compare lanes 2-4 and lanes 6-8). Note, however, that reaction mixtures contained no divalent cation, suggesting that the role of ATP is likely unusual. It is also noteworthy that assays with ATP yielded two different size upstream cleavage products (Fig. 7, lanes 6-8), whereas assays without ATP yielded only the longer one (Fig. 7, lanes 2-4). These results suggest that ATP can influence the precise site of endonucleolytic cleavage. Such heterogeneity in cleavage site specification with L3 pre-mRNA has also been observed by other groups in the presence of 3 dATP (49, 50). We also observed similar heterogeneity in the presence of 3 dATP, but in this case, the shorter upstream cleavage product was less prominent than in the presence of ATP (data not shown). Taken together, our data suggest that CP also functions as a necessary cofactor for cleavage of L3 pre-mRNA, but ATP enhances cleavage efficiency and affects the local choice of cleavage sites.

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DISCUSSION

ATP has until now been thought to be an essential cofactor for endonucleolytic cleavage of pre-mRNAs containing 3 end processing signals. In contrast, CP has been assumed to act as a reservoir for ATP regeneration during the cleavage reaction. CP is used for this purpose in many ATP-requiring reactions in which contaminating ATPases might otherwise deplete the ATP pools, including, for example, such complicated processes as transcription, translation, and splicing. Using highly purified 3 processing factors, we have shown that CP does not function in 3 end formation to regenerate ATP. Instead, it is able to function by itself as a necessary and sufficient cofactor for the reaction. Importantly, the ability of CP to act as a high energy donor is not required, and indeed, our data have shown that no energy source is required for RNA 3 cleavage. Although our experiments have established that ATP is not necessary, they have also shown that it can affect cleavage either positively or negatively, dependent on the substrate.

ATP appears to be required for 3 cleavage in yeast extracts (51). Whether or not this reflects a significant difference between the yeast and mammalian catalytic machinery is not clear. However, recent studies have revealed unexpected similarities between several mammalian and yeast polyadenylation factors (reviewed in Ref. 52), and it could be that the ATP requirement reflects the absence of CP and related compounds in yeast.

CP is a member of the phosphagen family, which consists of high energy phosphorylated guanidine compounds that are believed to provide temporary energy buffers in tissues with high energy demands (reviewed in Ref. 53). CPK catalyzes the reversible transfer of phosphate groups between CP and ADP (46). Several lines of evidence exclude the possibility that the observed CP requirement in the 3 cleavage reaction in fact reflects an ATP requirement through generation of ATP by undetected CPK activity in the purified factors. First, we were not able to detect any CPK activity in any of the purified factors. Second, AP could substitute for CP, even though AP exists only in invertebrates and cannot be a substrate of CPK (47). Third, cleavage proceeds normally even in the presence of 8 mM EDTA, although CPK requires a divalent cation for catalytic activity. Fourth, during cleavage, phosphate transfer from ³²P-labeled CP to possible endogenous ADP was not detected. Finally, ATP depletion (by glucose and hexokinase) was without effect on cleavage. Since four different preparations of commercially available CP and one preparation of AP gave identical results, we believe that CP or a related guanidinophosphate compound is required for efficient cleavage. However, our findings that high concentrations of CP (20-60 mM) are necessary for optimal cleavage and that similar concentrations of sodium phosphate allow a lower level of cleavage leave some question as to the identity of the physiologically relevant cofactor. Our data have shown conclusively, however, that the factor is not ATP.

Is it even reasonable to consider the possibility that CP, at concentrations of ~40 mM, is the physiologically relevant cofactor? Although the answer to this question is probably no (the only tissue where this high a level of CP has been documented is skeletal muscle (54, 55)), many other tissues (e.g. brain, smooth muscle, and kidney), have been shown to have CP concentrations of 5-10 mM (56). Thus, if CP can perform its essential function in polyadenylation (see below) at this level in vivo, then it would appear that the concentration of CP could be adequate. But another requirement is that CP be in the nucleus. Although the bulk of CPK is found in the cytoplasm, a number of studies have found significant levels of the enzyme in the nucleus (57-59). In addition, although to our knowledge, the intracellular localization of CP has not been measured, diffusion through the nuclear pores would likely allow accumulation in the nucleus (60).

Given that our data have established that the role of CP in polyadenylation is not to function as an energy source, what could it be doing to facilitate 3 cleavage? The earliest steps in the reaction have been well studied; they involve specification of the poly(A) site by the cooperative interaction of CstF and CPSF with the pre-mRNA (20, 27, 28). A variety of experiments suggest that this interaction, which is frequently measured by the UV-cross-linking of the CstF-64 polypeptide to the pre-mRNA, is independent of CP (or ATP) (20, 23, 61-64). Therefore, it is likely that an interaction involving CFI and/or CFII requires CP. These factors are not well characterized, although CFI appears to be a heterotrimer, and all three subunits can be UV-cross-linked to RNA (31). We propose that CP is able to facilitate a conformational change important for the function of one or both of these factors. Interestingly, our early studies are consistent with the idea that CPSF, CstF, CFI, and CFII all coexist in a single large complex (15), and it is thus possible that a conformational change within this complex is facilitated by CP or a related compound. Also supporting the involvement of CFI and/or CFII, when a cruder fraction containing both these factors (MonoQ; see Ref. 16) was employed with CstF and CPSF, a low but significant level of cleavage could be detected in the absence of CP (and ATP).³ Although we do not have a clear explanation for this phenomenon, it was not observed when cruder fractions of CstF or CPSF were used, and thus it again implicates CFI and/or II as the target of CP.

Another possibility is that CP (or AP) does not in fact play any direct role in 3 end formation in vivo and functions in vitro by mimicking another component. An intriguing possibility is that this might be an unknown phosphoprotein, perhaps containing phosphoarginine. Although rare, phosphoarginine is known to exist in eukaryotic proteins (65). By this model, the high concentrations of CP required allow it to be recognized by a component of the polyadenylation machinery that naturally recognizes a phosphoprotein. Alternatively, CP (or AP) might bind directly to the RNA, influencing its structure and thereby facilitating cleavage. However, attempts to detect an interaction between [³²P]CP and unlabeled substrate RNA (SV40 late and AdL3) were unsuccessful.³ Furthermore, neither Mg²⁺ nor spermidine, which are known to influence RNA structure, had any affect on cleavage of the SV40 late pre-mRNA.

Our data have ruled out a requirement for ATP in 3 cleavage. However, they also show that ATP can influence the reaction, either positively or negatively, dependent on the pre-mRNA substrate. How does this occur? We suggest the possibility that the inhibitory effect is due to competition between CP and ATP. Given our lack of understanding of how CP functions, it is not possible to comment on the details of this putative competition, or on the possible physiological significance of the inhibition. Indeed, given that the SV40 late pre-mRNA is unusual in its lack of a requirement of PAP for cleavage, it could be that the enhancement of cleavage, observed with the AdL3 pre-mRNA, is most relevant. The sequences within the SV40 RNA that confer PAP independence are limited to nine bases surrounding the cleavage site (48); it may be that CP facilitates interaction of cleavage factors with this region, whereas for most pre-mRNAs, PAP and ATP are also required for optimal cleavage. It is also notable that ATP, in addition, affects the precise site of cleavage in the AdL3 RNA. Interestingly, a possibly related phenomenon was observed over a decade ago by Sperry and Berget (66). These authors observed that in nuclear extract, an SV40 early pre-mRNA (which requires PAP) was cleaved 18-20 bases downstream of the authentic cleavage site when ATP was omitted from reaction mixtures. This apparently aberrant cleavage appeared to be catalyzed by the normal polyadenylation machinery, as judged by its poly(A) signal dependence, and was suppressed by ATP in a concentration-dependent manner. We believe that both the enhancement of cleavage and the switch in site selection resulting from ATP are mediated by PAP. Perhaps the processing complex that assembles when PAP contains bound ATP is different from the complex that assembles in the absence of ATP. We do not know whether ATP hydrolysis is required for these effects, but we suspect that it is not, as our experiments were performed in the absence of divalent cation. It is intriguing to suggest that other treatments that affect PAP activity (e.g. phosphorylation; Ref. 67) could also influence cleavage efficiency or site selection. Future work may elucidate this type of regulation.

To summarize, our studies have shown that contrary to expectation, endonucleolytic 3 cleavage of mammalian pre-mRNAs does not require energy. Creatine phosphate can function as a necessary and sufficient cofactor, but CP hydrolysis does not occur. Finally, ATP can influence the reaction quantitatively and qualitatively but is not essential.