A Nuclear Micrococcal-Sensitive, ATP-Dependent Exoribonuclease Degrades Uncapped but Not Capped RNA Substrates

Home , Exoribonuclease, Micrococcal nuclease

\./ 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 10 2685 A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates

K.G.K.Murthy, P.Park and J.L.Manley* Department of Biological Sciences, Columbia University, New York, NY 10027, USA

Received February 13, 1991; Revised and Accepted April 22, 1991

ABSTRACT We have developed an assay for an exoribonuclease likely that a 5'- 3' exoribonuclease that recognizes the uncapped present in HeLa cell nuclear extracts that degrades 5' end of the linearized intron plays an important role in intron capped but not uncapped RNA substrates, and used degradation. Another potential role for such an exonuclease is it to partially purify and characterize such an activity. following the formation of mRNA 3' ends, which involves Capped and uncapped transcripts of different sizes endonucleolytic cleavage of longer precursors (5, 6). While the (37 - 317 nt) were incubated with fractionated nuclear upstream RNA is rapidly polyadenylated, the downstream product extracts, and in all cases the capped RNAs were stable of the cleavage reaction is very unstable both in vivo and in vitro while their uncapped counterparts were completely (7-11), and a likely candidate for the enzyme that degrades this degraded. No changes in activity were detected when downstream uncapped RNA is again a 5'- 3' exonuclease. cap analogs were included in reaction mixtures, Indeed, based on the observations that a functional poly(A) signal suggesting that the stability of capped RNAs was not sequence is required for subsequent transcription termination by due to a cap binding protein. The exoribonuclease was RNA polymerase II (12- 15), and that degradation of the RNA shown to be specific for RNA, and to function downstream of the the cleavage site can apparently occur before processively with either substrates containing transcript release (9), we hypothesized previously that a 5'- 3' 5'-hydroxyl or 5'-phosphorylated ends. The products exonuclease might play an active role in the termination process, were predominantly 5'-mononucleotides, and no perhaps functioning in a manner analogous to E. coli rho factor detectable intermediates were observed at any reaction (14, 16, 17). However, elucidation of the role(s) played by a time points. Sedimentation analysis suggests that the nuclear exoribonuclease specific for uncapped RNAs in native size of the nuclease is 7.4S or -150 kDa. metabolism of pre-mRNAs first requires characterization of the Interestingly, a nucleoside triphosphate was found to enzyme activity. be necessary for specific and complete degradation of The first evidence for the presence of a 5'-3' exoribonuclease the uncapped RNAs. Finally, micrococcal nuclease in eukaryotic cells was provided by Furuichi et al (18) and (MN) pretreatment of the partially purified enzyme Shimotohno et al (19), who showed that reovirus mRNAs with inhibited its activity. As several controls indicated that 5' blocking structures such as m7GpppGm or GpppG are resistant this was not due to non-specific effects of MN, this to degradation relative to mRNAs with unblocked termini, both finding suggests that the exoribonuclease contains an in vivo (in Xenopus laevis oocytes) and in crude extracts of wheat essential RNA component. germ and mouse L cells. Shimotohno and Mura (20) also showed that a 20-fold purified enzyme fraction from the supernatant of INTRODUCTION wheat germ extracts can degrade an RNA substrate in a 5'-3' direction, releasing 5'-mononucleotides. This enzyme apparently An exoribonuclease that specifically degrades uncapped RNAs requires a 5'-hydroxyl group, as both capped and 5'-phosphate may have several roles in the nucleus of a eukaryotic cell. For terminated RNAs were found to be resistant to degradation. Green example, such an activity is very likely involved in degrading et al (21) reported that a capped globin mRNA is more stable introns that are removed during mRNA splicing (1-3). Excised than the same uncapped RNA in Xenopus oocyte nuclei, introns are initially released as lariat structures, in which the 5' suggesting the presence of nucleoplasmic exoribonuclease with phosphate forms a phosphodiester bond with the 2' OH of the specificity for uncapped RNAs. Earlier reports from our 'branch acceptor' nucleotide. Excised introns are usually laboratory also documented the existence of such an activity in extremely unstable in vivo, in most cases virtually undetectable. HeLa cell nuclear extracts active in pre-mRNA splicing and In vitro, and presumably in vivo, lariat introns can be polyadenylation (22, 23), and a similar activity has also been enzymatically 'debranched' to produce linear RNAs (4). It is very observed in yeast extracts (24). Indeed, this activity has proven

* To whom correspondence should be addressed 2686 Nucleic Acids Research, Vol. 19, No. 10 useful in studying the interaction of splicing factors (eg, snRNPs) DEAE-Sepharose and heparin agarose chromatography. The with pre-mRNA, as some of these are able to block the protein pellet obtained following precipitation of the 40% AS progression of the exoribonuclease, giving rise to characteristic supernatant with 60% AS was resuspended in 15 ml of buffer RNA products (e.g., ref. 25). D-100 (20 mM Hepes [pH 7.9], 0.08 mM EDTA, 20% glycerol Exoribonuclease activities that degrade synthetic and 100 mM NaCl) and dialyzed against the same buffer. ribohomopolymers in a 5'-3' fashion have been partially Insoluble material was removed by centrifugation and the purified and characterized from yeast (26), Ehrlich ascites tumor supernatant was loaded on a 2.5 x 18 cm DEAE-Sepharose cell nucleoli (27) and human placental nuclei (28). All these column (Pharmacia) equilibrated with buffer D-100, at a flow enzymes appear to be processive in nature and to release rate of 0.4 ml/min. The flow-through fraction was loaded directly 5'-mononucleotides as products. The nucleolar exoribonuclease on a heparin agarose column (2.5 x4.5 cm, Sigma) by coupling has an estimated molecular size of 76,000 and can degrade RNA the DEAE column to the heparin agarose column. After washing substrates from either a 5'-phosphoryl or 5'-hydroxyl terminus both columns with 300 ml of the same buffer, the DEAE column (27). The yeast and placental exonucleases both display a was removed and proteins were eluted from the heparin column preference for 5'-phosphoryl ends and apparent molecular weights with a linear gradient of NaCl (100-1000 mM in 125 ml) at of approximately 160,000 and 113,000, respectively, were a flow rate of 0.4 ml/min. Fractions of 3 ml were collected, and reported (26, 28). None of these enzymes have been purified active fractions, which eluted at approximately 0.3 M NaCl, were to homogeneity, and their ability to distinguish between capped concentrated by using a Centriflo CF025 (Amicon) and then and uncapped substrates is not known. No 5'-3' exoribonuclease dialyzed against buffer D-100. This procedure resulted in 30 fold activity has been detected in bacteria. purification of exonuclease, with only minimal losses of activity In this paper, we describe the partial purification and some during chromatography. Exonuclease activity was stable at of the properties of an exoribonuclease activity present in HeLa -20°C, with no significant loss for at lease four months. nuclear extracts that efficiently degrades uncapped, but not For sedimentation analysis, 0.2 ml of the heparin agarose capped, RNAs. We have used several different pre-mRNA-like fraction was diluted with an equal volume of buffer D containing substrates, and find that in all cases transcripts containing a 5' 400 mM NaCl and lacking glycerol and loaded on the top of a cap structure are stable, while those lacking a cap are completely 4.6 ml glycerol gradient (10-30% v/v) made in buffer D-400. degraded. Characterization of several properties of the enzyme A mixture of protein markers (thyroglobulin, 670 kDa; gamma- suggest that it is processive, that ATP hydrolysis is required for globulin, 158 kDa; ovalbumin, 44 kDa; and myoglobin, 17 kDa) optimal activity, and that the enzyme may contain an essential was loaded on a parallel tube for size estimation. After RNA component. centrifugation at 47000 rpm at 4°C for 14 hr in an SW 50.1 rotor, fractions (ca. 0.36 ml) were collected from the bottom of the tube and dialyzed against buffer D-100. Ribonuclease assay MATERIALS AND METHODS A standard 25 ,dl reaction mixture contained: 10-100 ng of Substrates 32P-labeled capped and uncapped RNAs, 8 mM Hepes-NaOH Several plasmid DNAs were used as templates for in vitro (pH 7.9), 8% (v/v) glycerol, 0.08 mM EDTA, 0.2 mM DTT, transcription with SP6 RNA polymerase, and these are all 40 mM NaCl, 2.0 mM MgCl2, 1.6 mM ATP and 2 ,ul of the diagramed in Figure 1. The plasmid pG3SVL-A contains the heparin agarose fraction of the enzyme. After incubation at 30°C SV40 late poly(A) site and downstream sequences (29). Plasmids for 2.5 hr, reactions were stopped by adding 10 ytg of proteinase pGBG 2a, pGBG 3a and pGBG 4a were constructed by inserting K in 200 td of 100 mM Tris.Cl (pH 7.4), 10 mM EDTA, BgII fragments from the plasmids pB5'SV2-4 (30) into the 150 mM NaCl and 1% SDS, followed by a 30 min. incubation BamHI site of pGEM-3. The resulting plasmids contained at 30°C. RNAs were extracted once with phenol-chloroform fragments from the 3' end of the rabbit I3-globin gene inserted (1:1), precipitated with alcohol and separated on 5% in the orientation such that transcription with SP6RNA polyacrylamide-8.3 M urea sequencing gels. polymerase resulted in synthesis of anti-sense transcripts. Two small RNAs (37nt and 58nt) were transcribed from the polylinker End product analysis region of pGEM-3. Capped and uncapped RNAs were Capped or uncapped precursors were incubated with synthesized with SP6 RNA polymerase essentially as described exoribonuclease under standard reaction conditions. At different previously (31, 32), using ac-32P GTP to label the RNA time points, 5 ILI samples were removed from the reaction transcripts. Uniformly labeled single-stranded (ss) DNAs were mixture, diluted to 100 ,ul with water, extracted with phenol- prepared from an M13 mpl8 template. The universal primer chloroform, dried under vacuum and suspended in 5 il of water. (17 mer) was annealed to the single-stranded template and Two iul aliquots either with or without treatment with calf extended with the Klenow fragment of DNA polymerase. DNA intestinal alkaline phosphatase, were spotted on Whatman #1 was digested with Pvul, products were separated on a denaturing paper along with unlabelled guanosine 3'- and 5'-mononucleotide polyacrylamide sequencing gel, and DNAs of 93 and 272 bases markers and a 32p inorganic phosphate marker. Before use the were eluted and purified. paper was saturated with 0.4 M ammonium sulfate solution and dried. Chromatograms were developed in 76% (v/v) ethanol (34) Protein Fractionation for 16 hrs. The positions of the UV markers were determined Nuclear extracts were prepared from 45 1 of HeLa cell culture and the paper was exposed to X-ray film. To quantitate the by the method of Dignam et al. (33), with minor modifications amount of 5' GMP released, reaction products were separated (23, 29). Exoribonuclease was purified from a 40-60% on a poly(ethylenimine)-impregnated cellulose thin-layer plate ammonium sulfate (AS) fraction of nuclear extract by (35). The plate was developed with 2.0 N HCOOH-0.5 M LiCl Nucleic Acids Research, Vol. 19, No. 10 2687 (1:1, V/V). Regions of the PEI-plate corresponding to 5' GMP sequences. Typical assays (see Materials and Methods) contained and the origin were cut and radioactivity counted in a scintillation 2-6 RNA substrates (of which 1-2 were capped), Mg++, counter. ATP and fractionated nuclear extract. Nuclear extracts were prepared from HeLa cells essentially Micrococcal nuclease digestion as described by Dignam et al (33) with minor modifications (29), Micrococcal nuclease pretreatment of the exonuclease was and assayed for their ability to degrade uncapped but not capped performed by mixing 1 td of 5 mM CaCl2 and 1 Al of RNA substrates. Extracts were initially fractionated by 5- 15U/4l MN (Boehringer-Mannheim) with 5 11 of the heparin precipitation with different concentrations of (NH4)2SO4 (data agarose-purified enzyme. After incubation at 30°C for 30 min, not shown). Most of the activity was recovered in the 40-60% 2 Al of 10 mM EGTA was added to the reaction mixture to (NH4)2SO4 fraction, although a significant amount of inactivate the MN. To heat inactivate the MN-treated or exoribonuclease was also detected in the 20-40% fraction. -untreated enzyme, samples were heated at 65°C for 5 min. However, because the latter fraction also contained considerable non-specific nuclease activity, the 40-60 fraction was used in further purification. The 40-60 fraction was applied to a RESULTS DEAE-Sepharose column and nearly 100% of the activity was recovered in the flow-through. This fraction was loaded directly We have used as an assay for exonuclease activity preferential onto a heparin agarose column and bound proteins were eluted degradation of uncapped mRNA-like transcripts relative to similar with a linear gradient of NaCl. Details ofthe fractionation scheme capped RNAs. RNAs ranged in size from 37 to 317 nt (see are contained in Materials and Methods. Figure 1), and were prepared by in vitro transcription of template DNAs as described in Materials and Methods. Different substrates used in single reactions had similar nucleotide An exonuclease activity specifically degrades uncapped RNAs sequences. For example, RNAs of 317 and 233 nt were made Several of the RNA substrates described above were incubated from a plasmid pG3SVL-A containing the SV40 late 3' end with heparin agarose-purified enzyme and the products were processing signals, digested with either PvuIl or DraI restriction analyzed by denaturing polyacrylamide gel electrophoresis enzymes. Another set of substrates (260, 185, 148 nt) were made (Figure 2). In all cases, regardless of size, capped RNAs were from a segment of the rabbit-,B globin gene containing (on the stable while similar uncapped RNAs were extensively degraded. opposite strand) the conserved AATAAA sequence, poly(A) (In this and subsequent figures, capped RNAs are indicated by addition site and different amounts of 3' downstream sequences arrows). Similar results (some not shown) were obtained with (30). These three RNAs lack any known RNA processing signals, all of the substrates shown in Figure 1. The results presented and also contain the same 5' and 3' sequences, as the size in Figure 2 also show that the same RNAs that were stable when differences result from internal deletions. Two small RNAs, capped were susceptible to degradation when synthesized without 37 and 58 nt, were made from transcription ofpGEM polylinker a cap. These findings indicate that the stability of a capped RNA was indeed due to its cap structure rather than to its size or nucleotide sequence, and therefore strongly suggest that An Dr I FPvu nt degradation proceeds in a 5'-3' direction. SP6 AATAAAt------1 pG3SVL-A 10 MM4. It was conceivable from the above data that the stabilizing effect of the cap was not direct, but resulted instead from the interaction 317 nt of a cofractionating cap binding protein with the capped RNAs. Dr. EcoR SP6 l Such an interaction could conceivably block the action of an pGBG-4 _0 AAATAA An " nt a b a b a b a b a b 260 nt EcoR SP6 pGBG-3l --- a. a ~ AAATAA An ...... 185 nt Ave EcoR SP6 317 - 0 pGBG-2 - t AAATAA 260 - de"4- - 233 - l-.l. _ 4- 132 nt 148 nt 1 85 -

SP6 Bam Hi Eco RI pGEM-3 + 1 48- S~~~~~~AL4 37 nt +t 58 nt 1. SchematicpGBG-3 diagram~...... of the plasmid DNAs used to synthesize RNA Figure 2. A nuclear activity extensively degrades uncapped but not capped RNAs. Figure substrates. DNAs were cleaved with one of the indicated restriction enzymes and SP6 transcripts were made by using several of the plasmid DNAs indicated in transcribed by SP6 polymerase. The structure and size of each RNA are Figure 1 digested with the appropriate restriction enzymes as templates. Each diagrammed. Shaded rectangles indicate inserted DNA sequences (see text), and assay contained both capped and uncapped RNAs of different sizes with (b) or dashed lines indicate the position of deleted sequences in the DNA and RNA. without (a) the heparin agarose fraction of the exoribonuclease. Reaction conditions AATAAA and An indicate polyadenylation signals. These signals are present were as described in Materials and Methods. Numbers indicate the sizes of the in the transcript pG3SVL-A, but are on the opposite strand of the pGBG plasmids. RNAs (in nucleotides). Arrows indicate the positions of capped RNAs. 2688 Nucleic Acids Research, Vol. 19, No. 10 exonuclease that would otherwise not be affected by a cap. To a 5' triphosphate, suggesting that the only requirement of the test this, we took advantage of the fact that characterized cap RNA 5' end is that it lack a cap structure. binding proteins recognize cap analogues, and that reactions dependent upon cap recognition can be inhibited by such Mode of attack and nature of products structures (e.g., ref. 36). In this case, addition of cap analogues An exonuclease may act by either of two distinct mechanisms, to reaction mixtures would be predicted to destabilize the capped distributive or processive (37, 38). The processive mode of attack transcripts if a cap binding protein was in fact responsible for results in the complete degradation of one RNA molecule before their stabilization. However, the results in Figure 3A show that enzyme release. In the distributive mode, a general shortening addition of high concentrations of m7GpppG (0.5 or 1.0 mM) of the substrate occurs as a function of the extent ofthe reaction, had no effect on exoribonuclease activity, providing strong indicating that the enzyme dissociates from the substrate before evidence that the stability of the capped RNA was not due to digestion is complete. To determine the mode of action of the cap binding proteins. Figure 3A also shows that human placental HeLa nuclear exoribonuclease, timed aliquots of an RNA ribonuclease inhibitor (RNasin) did not affect exonuclease digestion were analyzed (Figure 4). The amount of enzyme used activity, ruling out the involvement of a number of non-specific in this (and all other) assays was sub-saturating, as doubling the RNases. enzyme concentration resulted in significantly more rapid We also tested whether single-stranded DNA (ssDNA) could degradation under standard reaction conditions (results not serve as a substrate for the exonuclease. For this, two ssDNAs, shown). Exonuclease activity was assayed at both 37°C and 272 and 93 nt, were prepared as described in Materials and 30°C. At 37°C, the reaction was nearly complete by 60 min, Methods and incubated with partially purified exonuclease, while at 30°C it took 150 min. At both temperatures a significant together with a 148 nt uncapped RNA and a 132 nt capped lag time ( - 30 min) was observed before degradation transcript. Gel electrophoresis (Figure 3B) revealed that the two commenced. As the time of incubation increased, the amount ssDNAs were completely stable, while the uncapped, but not the of uncapped RNA decreased, but there was no change in the size capped, RNA was extensively degraded. From these results we of the remaining substrate. As above, the size and amount of conclude that the exoribonuclease is specific for RNA substrates the capped RNAs were essentially unchanged at all time points. and is free of detectable endo- or exo-deoxyribonuclease activity. These results strongly suggest that the exonuclease is processive. Previous studies (26, 28) indicated that 5'- 3' exoribonucleases To determine the identity of the end products of exonuclease isolated from yeast and human placental nuclei preferred digestion, reactions containing single RNA substrates were substrates containing 5' phosphates. The possible requirement performed. Reactions were stopped at different time points and of a 5'-phosphate group on the HeLa nuclear exoribonuclease products were loaded directly on a 20% polyacrylamide-urea gel was tested by treating uncapped RNAs with calf intestinal alkaline (Figure 5A). The digested RNA moved exclusively as a single phosphatase prior to incubation with the exonuclease (Figure 3C). band at the position of mononucleotides, within the resolution Such substrates were degraded as efficiently as RNAs containing of the gel system. Longer exposures of this gel did not reveal any intermediate bands, consistent with the conclusion that degradation is processive. When a capped RNA was used as a substrate (arrow), a small amount of label was released. This was likely due to the presence of uncapped RNAs, as the efficiency of capping during in vitro transcription was estimated to be only - 80% (results not shown). To determine whether the products were indeed mononucleotides, and, if so, whether they were 3' or 5' monophosphates, aliquots of the same reaction

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Figure 3. Properties of the exoribonuclease. (A) Cap structure analogue and ribonuclease inhibitor (RNasin) do not affect exoribonuclease activity. Exonuclease activity was assayed under standard reaction conditions in the absence of any additions (EXO) or in the presence of cap structure analogue or RNasin. Concentrations of the cap structure analogue (in mM) and RNasin (in units) are indicated. Arrows indicate the positions of capped RNAs. (B) Exoribonuclease degrades uncapped RNA but not capped RNA or single-standed DNA. Uniformly labeled ss DNAs were prepared from M13 mpl8 DNA as described in Materials and Methods. Reactions containing the indicated substrates were incubated in the presence (EXO) or absence (PRECURSORS) of exonuclease under standard Figure 4. Time course of exoribonuclease activity. RNAs were incubated with conditions. M indicates DNA size markers. The sizes (in nucleotides) and nature exonuclease at 30° or 37°. At each of the indicated times, a small aliquot of ofthe substrates used in the assay are indicated on the right. (C) RNA substrates each sample was removed and the reaction was stopped by addition of EDTA were preincubated in the presence (CIP/EXO) or absence (EXO) of alkaline to 5.0 mM. Samples were processed and analyzed as in Figure 2. Arrows indicate phosphatase prior to treatment with exonuclease. the position of capped RNAs. Nucleic Acids Research, Vol. 19, No. 10 2689 mixture were chromatographed on Whatman # 1 paper under exclusively 5' GMP is consistent with the fact that a-32P GTP conditions that would distinguish the various mononucleotides was used to label the RNA substrate, and indicates that the as well as 5' and 3' mononucleotides (34). Reaction products nuclease cleaves on the 3' side of the phosphodiester bond, were observed to migrate exclusively with a guanosine releasing 5'-mononucleotides. This conclusion is strengthened 5'-mononucleotide marker (Figure SB). The generation of by the observation that treatment of the reaction products with alkaline phosphatase completely converts the label to a species that comigrates with inorganic phosphate (Figure 5B). To estimate A O 6 -o 6 0 0 Co r: Ew_M _ 0 cn 40 el . the efficiency of the reaction, spots corresponding to undigested dl-. 260 -- ---L substrate (which remained at the origin) and to 5' GMP were 1 32 I -o' 'm- 4- quantitated by liquid scintillation counting as described in Materials and Methods. At the 150 min time point, more than 85% of the substrate was converted to 5'-GMP. Properties of the partially purified exonuclease Several requirements for exonuclease activity were analyzed, and the results are shown in Figures 6 and 7. The enzyme displayed an absolute requirement for a divalent cation (Figure 6A). Mg++ and Mn++ functioned equivalently, each giving rise to maximal enzyme activity between 0.5 to 2.0 mM. Ca++ and Zn+ + could not be substituted, and EDTA (2.0 mM) completely inhibited the reaction. The temperature sensitivity of the exoribonuclease was examined by heating the enzyme to different temperatures prior to incubation with substrates (Figure 6B). The BPB enzyme displayed a sharp heat inactivation profile. Exoribonuclease activity was resistant to 50°C, but heating to 55°C for 5 min completely inactivated the enzyme. Activity was also sensitive to pronase and to N-ethylmaleimide (results not shown). Figure 7A presents the results of experiments examining the requirement of a nucleoside triphosphate for exonuclease activity. When ATP was omitted from reaction mixtures, degradation of B uncapped RNAs was significantly reduced. However, all RNAs, capped as well as uncapped, were partly destabilized. This 'non- specific' degradation was observed with all RNAs tested, required Mg++, and was not inhibited by RNAsin (results not shown). Furthermore, the limited shortening of the substrates is most 3.GMP consistent with an exonucleolytic activity. For these reasons, and

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_ 260- __0m1014 so ______* 260 - 0 e 18 5- 0 It O0 X 1 48- 4 W -_ C-)6O UJ u n_O 0 CLLC: 185 - **@_ oQ 4-

148 - * Figure 5. Exoribonuclease activity is processive and releases 5'-mononucleotides. A (A) Either a capped (132 nt) or uncapped (260 nt) RNA was incubated with 132 - * .4- exonuclease under standard reaction conditions. At the indicated times, a small aliquot of each sample was removed. Samples were extracted with phenol, with chloroform, dried, suspended in loading buffer and separated on a 20% polyacrylamide-8.3M urea sequencing gel. The positions of the capped RNA (-) Figure 6. Exonuclease requires a divalent cation and is heat sensitive. (A) Divalent and bromophenol blue (BPB) are indicated on the right. (B) Uncapped RNA (260 cation requirement. Reaction mixtures were as described in Materials and Methods, nt) was incubated with (EXO) or without (PRECURSORS) exonuclease at 30°C except that MgCl2 was omitted, and substituted with 1.0 mM of the indicated for 150 min. Samples were extracted, dried, dissolved in water and separated divalent cation or with EDTA. RNAs were analyzed as in Figure 2. (B) Heat on Whatman # 1 paper either with (EXO/CIP) or without (EXO) treatment with inactivation. Aliquots of the exonuclease were heated at the indicated temperatures alkaline phosphatase as described in Materials and Methods. The positions of for 5 min., cooled on ice and assayed for exoribonuclease activity. Arrows indicate cochomatographed standards are indicated. the positions of the capped RNAs. 2690 Nucleic Acids Research, Vol. 19, No. 10 others (see Discussion), we believe that the ATP-dependent and during the preincubation with Ca++ (results not shown), no -independent activities are both properties of the same enzyme. effect on exonuclease activity was observed. Similar results were Figure 7A also shows that any of the four nucleoside triphosphates obtained with several smaller RNAs, as shown in lanes 9-12. could bring about 'specific' degradation of uncapped RNAs, While the above results are consistent with the hypothesis that although GTP and ATP were somewhat more effective than CTP the exonuclease contains an MN-sensitive component, other and UTP. In addition, relatively high concentrations of ATP explanations were possible. For example, MN could have resulted (> 1.5 mM) were required for optimal activity. Neither a-: nor in partial, but not complete, degradation of RNA contaminating f-'y methylene derivatives of ATP could substitute for ATP the exonuclease, and these small RNA fragments might have (Figure 7B). However, both analogues, in a concentration- functioned as competitive inhibitors of the exonuclease. To test dependent manner, prevented the partial degradation observed whether MN inhibition was due to the generation of inhibitors in the absence of ATP. As discussed below, these findings suggest such as this, we performed an experiment in which equal amounts that binding of ATP to the exonuclease may be sufficient to block of MN-treated and -untreated enzyme were mixed prior to its non-specific activity, while ATP hydrolysis is necessary for addition to reaction mixtures. The results obtained revealed that the efficient and processive degradation of uncapped substrates. the activity of the untreated exonuclease was not only not We have also estimated the native size of the heparin agarose- inhibited, but actually slightly increased by addition of the purified enzyme by glycerol gradient centrifugation (see Materials MN-treated sample (compare lanes 2 and 5 in Figure 8). These and Methods). Fractions were assayed with the same substrates results strongly suggest that MN pretreatment does not generate used in Figure 7, and the results (not shown) revealed a single an inhibitor. However, we decided to test an additional possibility, peak of activity, with a size of 7.4S, or, assuming a globular which was that the hypothetical inhibitor could not function 'in structure, approximately 150 kD. trans'. For example, perhaps small RNA fragments were completely bound by the enzyme (or contaminating proteins) in Micrococcal nuclease pretreatment destroys exonuclease the initial sample, and thus, while inhibiting the endogenous activity exonuclease, could not inhibit the added enzyme. To address this possibility, MN-pretreated, as well as untreated, samples were Previous studies from our laboratory showed that the stability heated to 65°C for 10 min to denature the enzyme, cooled, and of uncapped pre-mRNAs in micrococcal nuclease (MN)- mixed with untreated enzyme. As above, addition of the pretreated nuclear extracts active in pre-mRNA splicing and inactivated samples had no inhibitory effect on untreated polyadenylation was significantly greater than in untreated exonuclease (lanes 7 and 8). Note that mixing MN- and heat- extracts (23). Although there are several possible explanations inactivated enzymes together did not restore activity (Figure 8, for this result, one is that the exonuclease characterized here is lane 6). This argues against a final, albeit unlikely, possibile sensitive to MN. To test this, we carried out a series of experiments to examine the effect of MN pretreatment on the activity of the partially purified exonuclease. The data in Figure 8 provides evidence that the exonuclease is indeed sensitive to MN. Lane 4 shows that the activity of the exonuclease was substantially reduced when the enzyme was preincubated with MN in the presence of Ca+ + (followed by inactivation of MN by addition of EGTA). In contrast, when EGTA, Ca++ and MN were added simultaneously (lane 3), or when MN was not included

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.4 Figure 8. Exoribonuclease activity is micrococcal nuclease sensitive. MN pretreatment, heat inactivation and reaction conditions were as described in Materials and Methods. Lane 1, no enzyme; lane 2, standard exonuclease assay; lane 3, exonuclease pretreated with MN in the presence of EGTA; lane 4, Figure 7. Effects of nucleoside triphosphates on exonuclease activity. (A) MN-pretreated enzyme (EXO MN); lane 5, equal amounts of EXO MN and Nucleoside triphosphates. Reaction mixtures were as described in Materials and untreated exonuclease; lane 6, equal amounts of EXO MN and heat inactivated Methods, except that ATP was omitted and the indicated nucleotides were added exonuclease; lane 7, equal amounts of EXO MN heated to 650 and untreated to 1.6 mM, or ATP to the concentrations shown. (B) ATP analogues. Reaction enzyme; lane 8, equal amount of heat inactivated exonuclease and untreated conditions were the same as in panel A, except that 1.5 or 3.0 mM ATP, enzyme; lanes 9- 12, identical to lanes 1 -4, except that the RNAs indicated a-f-methylene ATP (AMP-CPP), or f--y-methylene ATP (AMP-PCP) were added were used as substrates. Arrows indicate the position of capped RNA. The mixing to reaction mixtures as indicated. experiments are grouped by the bracket. Nucleic Acids Research, Vol. 19, No. 10 2691 artifact, which was that MN treatment inhibited exonuclease processive and complete degradation of uncapped RNAs. We activity by destroying contaminating RNA, which would, by this therefore believe that ATP is indeed required directly for scenario, be required to provide a minimal mass of RNA (e.g., exonuclease function. see ref. 23). Taken together, these results provide strong evidence We propose the following model, which is consistent with the that the exonuclease is itself sensitive to micrococcal nuclease, available data, to explain the role of ATP in exonuclease function. suggesting that a nucleic acid component (presumably RNA) is In the absence of ATP, we suggest that the conformation of the required for activity. enzyme is such that it is unable to differentiate between capped and uncapped RNAs, and is able to bind to both types of substrates and carry out very limited, non-processive 5'-3' DISCUSSION degradation. ATP binding would then bring about a conformational change in the enzyme that prevents it from The results described here provide an initial characterization of recognizing capped RNAs, perhaps increasing its affinity for a HeLa cell nuclear exoribonuclease that may play an important uncapped substrates. The increased stability of uncapped as well role in the metabolism of pre-mRNAs. The enzyme, which was as capped RNAs in the presence of non-hydrolyzable ATP detected in nuclear but not cytoplasmic extracts (unpublished analogues suggests further that the enzyme may be inactive in data), totally degraded a variety of uncapped transcripts. In this conformation. ATP hydrolysis might then bring about another contrast, the identical RNAs, when capped, were stable, as were conformational change, resulting in cleavage of the first similarly sized single-stranded DNA molecules. As seen in phosphodiester bond, and initiating efficient and processive several previous studies that characterized 5'-3' exonuclease degradation of the substrate. activities (26-28), intermediates were not detected at any reaction Other than glycerol gradient sedimentation, our attempts to time tested, and the end products of the reaction were exclusively purify the exonuclease beyond the heparin agarose fraction used 5' mononucleotides. The nuclease thus appears to function here have been largely unsuccessful. Frequently, we detect an processively, and not to contain any endonucleolytic activities. activity that partially degrades capped as well as uncapped Together, these findings strongly suggest that the nuclease activity substrates, even in the presence of ATP (unpublished data). While we have characterized is a 5'- 3' exoribonuclease that cannot it may be that this is due to a contaminating non-specific RNAse act on capped RNAs. We note that we have not directly that is activated by fractionation away from an inhibitor, the demonstrated that degradation actually proceeds in a 5'-3' behavior of this nuclease is similar in a number of ways to that direction, but this is certaintly the simplest explanation for the ofthe exonuclease. The 'non-specific' activity absolutely requires data presented. Mg++, is resistant to RNAsin, and produces 5' mononucleo- The properties of this enzyme are consistent with the roles tides as the only detectable degradation products (unpublished postulated for a nuclear exoribonuclease that would degrade data). Perhaps the exonuclease consists of separable subunits, uncapped but not capped RNAs. RNA polymerase II transcripts one of which, a 'specificity subunit', is responsible for selection are capped very shortly after, or perhaps simultaneously with, of the appropriate (ie, uncapped) 5' end, while the other, a initiation (8, 39, 40), and would thus be completely protected 'catalytic subunit', is responsible for degradation of the RNA. from degradation. However, linearized introns and the By this model, the separated catalytic subunit could degrade downstream products of polyadenylation / 3' cleavage lack cap capped as well as uncapped RNA. The partial instability ofcapped structures (both are probably terminated with 5' Pi), providing RNAs in the absence of ATP, discussed above, is compatible a clear explanation for their extreme instability. The processive with this model, which additionally suggests that proper substrate nature of the nuclease would also ensure the rapid and complete recognition by the putative specificity subunit requires ATP. turnover of these RNA processing by-products, and might also These properties of the exonuclease, together with the observed be important if the exonuclease does indeed play a role in micrococcal nuclease sensitivity of the enzyme, suggest that transcription termination (see Introduction). RNA polymerase I exonuclease function may involve a complex, multi-step and m transcripts are of course uncapped, but nonetheless mechanism. The observed lag of approximately 30 min before resistant to degradation. This could be because oftheir localization significant activity is detected, perhaps reflecting the formation in the nucleus, the binding of specific proteins and/or the of a 'degradation complex' on the RNA, is consistent with this formation of stable secondary or tertiary structures. proposal. The requirement of a nucleoside triphosphate for exonuclease We believe the micrococcal nuclease sensitivity of the activity is intriguing. To our knowledge, the only previously exonuclease reflects the requirement of an RNA component for described exonuclease that requires ATP is the E. coli recBC activity. While studies with MN can be subject to experimental nuclease (41, 42). This DNAse has multiple activities that require artifact (e.g., 23, 43), the controls we have performed argue ATP, including 5'- 3 and 3'-5 exonuclease, endonuclease, and strongly that the MN sensitivity reflects an intrinsic property of DNA helicase. We do not yet know with certainty that the ATP the enzyme; ie, the presence of an RNA component. To date, requirement is an intrinsic property of the exoribonuclease we have been unable to identify a clear candidate for this putative described here. It is conceivable that ATP hydrolysis is required RNA. Analysis (by pCp labelling) of the RNAs present in the in some manner for 'preparation' of the substrate prior to active fraction obtained by glycerol gradient centrifugation degradation; for example, perhaps regions of secondary structure revealed a number of distinct RNAs, one of which ( 85 nt) must be melted by a cofractionating RNA helicase before the cofractionated with activity (unpublished data). However, this nuclease can function. 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