THE JOURNAL OF B~LOCICAL CHEMISTRY Vol. 265, No. 16, Issue of June 5, pp. 9491-9495,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, I~c. Printed in U.S.A.

Capping of U6 Small Nuclear RNA in Vitro Can Be Uncoupled from Transcription*

(Received for publication, December 6, 1989)

Shashi Gupta, Ravinder Singh, and Ram Reddy From the Department of Pharmacology, Bay/or College of Medicine, Houston, Texas 77030

U6 small nuclear RNA (snRNA) is a required com- ponent in the splicing of eukaryotic pre-mRNAs. Mam- malian U6 snRNA was synthesized in vitro by T7 RNA polymerase and purified on polyacrylamide gels. This U6 RNA, with pppG on its 5’ end, was accurately capped to CHs-0-pppG, when incubated with HeLa cell extract and this capping was dependent on the capping signal present within the U6 snRNA. When y-32P-la- beled U6 RNA was used as a substrate, the U6 cap formed in vitro retained this labeled y-phosphate, in- dicating that the cap formation involves the methyla- tion of the -y-phosphate incorporated during transcrip- tion. U6 snRNAs with ppG or pG as their 5’ ends, were not capped in this in vitro capping system. Capping of U6 snRNA in vitro requires at least two components, a heat-labile component and S-adenosylmethionine as a methyl group donor. The data presented here show that capping of U6 snRNA can be uncoupled from transcription and that the mechanism of U6 snRNA cap formation differs markedly from the capping mechanism of mRNAs and other U snRNAs where capping is cotranscriptional. While many methyltrans- ferases have been characterized earlier, this is the first report of a methyltransferase that is specific to phos- phate residues. This in vitro capping system will be useful for purification and studies on the U6 snRNA sequence-dependent methyltransferase activity.

U6 small nuclear RNA (snRNA)’ is a component of eukar- yotic spliceosomes (Grabowski and Sharp, 1986; Maniatis and Reed, 1987; Ruby and Abelson, 1988; Steitz et al., 1988) and is required for splicing of nuclear pre-messenger RNAs (Ber- get and Robberson, 1986; Black and Steitz, 1986). In yeasts, U6 snRNA coded by a single copy gene (Brow and Guthrie, 1988; Tani and Ohshima, 1989) is essential for cell viability (Brow and Guthrie, 1988). Although the exact role of U6 snRNA in pre-mRNA splicing is not known, it is suggested that U6 RNA may be directly involved in catalysis during nuclear pre-mRNA splicing (Brow and Guthrie, 1989).

U6 RNA, in addition to being the most conserved of all U snRNAs (Brow and Guthrie, 1988; Roiha et al., 1989), is unique in many respects. First, while other U snRNAs are synthesized by RNA polymerase II (reviewed in Busch et al., 1982; Dahlberg and Lund, 1988), U6 RNA is synthesized by

* This investigation was supported by Grant GM-38320 from The National Institute of Health, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: snRNA, small nuclear RNA; nt(s), nucleotide(s); AdoMet, S-adenosylmethionine; EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid.

polymerase III (Kunkel et al., 1986; Reddy et al., 1987). Second, U6 snRNA does not contain either the trimethyl guanosine cap present on other U snRNAs (Epstein et al., 1980) or the Sm antigen-binding site found in other nucleo- plasmic U snRNAs (Liautard et al., 1982); instead, the 5’ end of U6 snRNA is unique and is blocked by y-monomethyl phosphate (Singh and Reddy, 1989).

Eukaryotic mRNAs and U snRNAs, transcribed by RNA polymerase II, are blocked on their 5’ termini by a guanosine cap; m7GpppN in the case of mRNAs (reviewed in Banerjee, 1980; Shatkin, 1976) and m3’.*s7GpppN in the case of U snRNAs (reviewed in Busch et al., 1982; Reddy and Busch, 1988). In U snRNAs, trimethylation of the monomethyl guan- osine cap occurs post-transcriptionally in cytoplasm and is dependent on the presence of an Sm-binding site (Mattaj, 1986). The cap structure in mRNAs plays important role(s) in mRNA biogenesis including transcription initiation, gen- eration of primers for mRNA synthesis, mRNA splicing, 3’ end formation, and mRNA stability as well as in translation of mRNAs (reviewed in Banerjee, 1980; Shatkin, 1985). How- ever, the function(s) of m32~2~7G cap structure in other U snRNAs or of the methyl phosphate cap structure in U6 snRNA are not known.

Since U6 RNA has an unusual cap structure, we are study- ing the mechanism of U6 RNA cap formation towards eluci- dating the similarities and differences in the mechanism(s) of cap formation on U6 RNA uersus mRNAs and other U snRNAs. Our earlier mutagenesis studies showed that the capping of U6 snRNA is directed by a conserved stem-loop followed by AUAUAC sequence at the 5’ end of U6 snRNA (Singh et al., 1990). Results obtained in this study show that the transcription and capping of U6 snRNA can be uncoupled. The y-phosphate in the 5’pppG of the U6 RNA incorporated during transcription is retained and methylated; AdoMet acts as a methyl donor in this in vitro capping system. Although many sequence-specific methyltransferases are already known, this is the first report of a methyltransferase specific for the phosphate residues.

MATERIALS AND METHODS

Fine Chemicals-The nuclease Pl, alkaline phosphatase, AdoMet, and S-adenosylhomocysteine were from Sigma; T2 RNase was from Sankyo; T7 polymerase was from Stratagene; polynucleotide kinase was from New England Biolabs; [w~“P]GTP, [r-“‘PIGTP, and [y- ‘“P]ATP were obtained from Amersham Corp.; methyl GTP standard was synthesized from [a-“*P]GTP and methanol (Darzynkiewicz et al., 1985); and pGp was synthesized by labeling Gp with [r-“‘P]ATP and polynucleotide kinase.

Construction of U6 Mutants-The mouse U6 RNA gene (Das et al., 1988) and Syrian hamster 5 S RNA gene (Hart and Folk, 1982) were used as controls. Construction of acered U6 genes was carried out using standard recombinant DNA techniques. The U6 RNA used for in uitro capping studies was constructed in the following manner. The DNA sequence corresponding to the mammalian U6 RNA was ligated to the T7 promoter obtained by annealing two synthetic

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oligonucleotides and was then cloned into HamHI/HindIII sites of the plJC19 vector. When this DNA was truncated with E:coRV and transcribed with T7 polymerase, the resulting RNA was 208 nt long; Nts l-106 corresponded to the mammalian U6 RNA, nts 107-190 corresponded to :l’-nontranscribed spacer of the mouse U6 gene (Das (‘I n/., 1988) and nts 191-208 were from the vector DNA (Fig. 1). The 208 n-long RNA was fractionated on polyacrylamide gels, extracted, precipitated with ethanol and used for studies on capping in uitro.

In Vitro ‘l’mnscription of 5 S, U6, and Mutated U6 Genes-The appropriate plasmid DNAs were transcribed in HeLa cell extract prepared according to Weil et al. (1979). In a typical transcription reaction, a 50-~1 reaction mixture containing 2 fig of supercoiled plasmid DNA was incubated at 30 “C for 3 h. The reaction mixture contained 50% (v/v) HeLa cell extract, 0.6 mM each of unlabeled CTP, UTP, ATP, 0.025 mM GTP, 5 PCi of [cr- “P]GTP, 6 mM creatine phosphate, 20 mM KCI, 1 mM DTT, and 10 mM Tris, pH 8.1. The transcribed RNAs were phenol extracted, precipated with ethanol, and purified by electrophoresing on 5 or 10% polyacrylamide gels. U6 RNA or other RNA transcripts were extracted from the gels and utilized for further studies.

Fractionation of HeLa E.rtract-The HeLa cell extract was loaded on a DEAF,-Sephadex-A25 column in 10 mM Tris buffer, pH 8, containing 100 mM KCI, and was washed with the above buffer. The bound material was eluted step-wise with buffer containing 0.5, 1, 1.5, 2, and 3 M KCI. The eluted material was concentrated and resuspended in buffer containing 100 mM KCI.

In Vitro Copping-Labeled RNA transcripts (usually lO,OOO- 100,000 cpm by Cerenkov counting) were incubated in a 50-~1 reaction mixture at 30 “C for 30-45 min. The reaction mixture contained the suhstrate RNA, 150 mM KCI, 50 mM Tris, pH 8, and 35 ~1 of HeLa cell extract. The reaction was terminated by the addition of sodium dodecyl sulfate/phenol, and the RNA was precipitated from the aqueous phase with ethanol. The RNAs were digested with nuclease Pl, or T2 RNase. In some instances the nuclease Pl digests were also treated with bacterial alkaline phosphatase at pH 8. These digests were fractionated by electrophoresis on DEAE-cellulose paper at pH 3.5 (Epstein (~1 al., 1980). Autoradiography was done for l-7 days at -70 “C using XAR-5 film with Lightning Plus screens.

Capping E//iicienc>f-The autoradiograms were scanned with a LKB laser beam densitometer. The capping efficiency was determined from the relative amounts of pppG and CH,-0-pppG at the end of the capping reaction.

RESULTS

Transcription and Capping of U6 RNA Can Be Uncoupled- When the mammalian U6 gene was transcribed in uitro using HeLa cell extract, a fraction of the synthesized U6 RNA was accurately capped (Fig. 2, lane 2 and Singh et al., 1990). To see whether U6 RNA can be capped post-transcriptionally, we prepared U6 RNA using T7 polymerase and purified it on polyacrylamide gels; this RNA is referred to as T7-U6 RNA. The T7-U6 RNA contained pppG as its 5’ end (Fig. 2, lane 3) and when incubated with HeLa cell extract for 30 min, a fraction of the T7-U6 RNA was capped accurately (Fig. 2, lane 4). These data show that transcription and capping of U6 snRNA can be uncoupled. The T7-U6 transcript used in this study contained 102 extra nucleotides on its 3’ end. These additional sequences are unlikely to have any significant effect

U61-106 v +I02

FIN:. 1. Diagramatic representation of the plasmid DNA construct used for generation of T7-U6 snRNA. pUC19 vector, digested with HanHI/HindIII, was used to clone T7 promoter/U6 DNA as shown above. The DNA was linearized with EcoRV and transcribed with T7 RNA polymerase. The 208-nt longT7-U6 snRNA contained l-106 nucleotides of mammalian U6 snRNA, 84 nucleo- tides of the 3’ spacer from the mouse U6 gene (Das et al., 1988), and 18 nts from the vector.

FIG. 2. Analysis of cap structure formed in vitro during transcription and post-transcriptionally with different RNA templates. Lane 1, synthetic methyl GTP; lane 2, plasmid DNA containing mouse U6 gene transcribed in vitro; lane 3, T7-U6 RNA; lane 4, T7-U6 RNA incubated with HeLa cell extract; lane 5, 5 S RNA obtained by transcribing 5 S gene in uitro; lane 6, 5 S RNA incubated with HeLa cell extract; lane 7, U6 RNA lacking 2-25 nucleotides prepared by transcribing dl(k)26 DNA (Singh et al., 1990); lane 8, U6 RNA lacking 2-25 nts incubated with HeLa cell extract. Lane 9 is a longer exposure of lane 8. All RNAs were labeled with [TV-““PIGTP; the labeled RNAs were digested with nuclease Pl, electrophoresed on DEAE-cellulose paper, and subjected to autora- diography. Syn. cap, synthetic methyl GTP; 0’, RNAs before incu- bation with HeLa extract; 30’, period of incubation with HeLa extract.

on the efficiency of capping, since our earlier studies showed that the 5’ 25 nucleotides of U6 snRNA are required and sufficient for capping; additional sequences of up to 350 nucleotides had no detectable effect on capping (Singh et al., 1990).

The optimum conditions for capping U6 snRNA in uitro were determined and are given under “Materials and Meth- ods.” The efficiency of conversion ranged from 30 to 80% in a typical 30 min reaction; there was detectable cap structure formed after 2 min incubation, the shortest time period tested (results not shown). The capping efficiency varied depending on the conditions used for this reaction. In some experiments, the reaction mixture also contained nucleotides, creatine phosphate, and low salt concentration and under these con- ditions the capping efficiency was less. Both KC1 and Mg”+ were required as there was no detectable capping in their absence. The capping reaction was not dependent on ATP and was not affected by the addition of EGTA, indicating that Ca”+ ions are not required for the capping reaction. Addition of EDTA resulted in extensive degradation of added labeled RNA, and there was no detectable pppG at the end of incubation (results not shown).

Post-transcriptional Capping in Vitro Was Dependent on a Capping Signal in U6 RNA-We tested whether the cap structure formed post-transcriptionally requires the capping determinant characterized previously (Singh et al., 1990). While the T7-U6 RNA was capped after incubation with HeLa cell extract (Fig. 2, lane 4), the 5 S RNA (Fig. 2, lane 6) or U6 dl(k)26 RNA in which the capping signal (nucleo- tides between l-26 of U6 snRNA) was deleted (Fig. 2, lanes 8 and 9) were not capped. These data show that the capping observed in this in uitro system is not random but is specific to U6 snRNA containing the capping signal.

The y-Phosphate of T7-U6 RNA Was Retained in the Cap Structure-During the mTG cap formation in mRNAs and other U RNAs, the y-phosphate from the pppN is removed from the pre-mRNA transcript and the a-phosphate of the capping G is retained (reviewed in Banerjee, 1980; Shatkin, 1976). To understand the origin of the three phosphate resi- dues in the U6 snRNA cap, the RNA was labeled with [r-‘“PI GTP and the fate of this labeled y-phosphate was followed during the in vitro capping reaction. As shown earlier (Singh

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Post-transcriptional Capping of U6 snRNA

et al., 1990), the cap was labeled when U6 RNA with label in the a-phosphate was used as substrate (Fig. 3, lane 3). The cap structure formed with the -y-phosphate-labeled U6 RNA, was also labeled (Fig. 3, lane 6). This cap structure had the same electrophoretic mobility as the cap formed with other U6 RNA substrates (Fig. 3, lanes 1 and 3), and it was resistant to digestion with alkaline phosphatase (Fig. 3, lane 7). These data show that during U6 RNA cap formation, the r-phos- phate is not removed from the substrate; instead, the y- phosphate gets methylated.

U6 snRNAs Containing pG or ppG on Their 5’ Ends Are Not Recognized as Substrates for Methylation--When the U6 RNA gene or 5 S RNA gene was transcribed in vitro using the HeLa cell extract, the RNAs initially made with pppG were found to be converted partly to ppG and pG (Fig. 4, lanes 1, 2, 7, and 9). This is presumably due to phosphatase activity present in the extract. If the capping machinery is able to cap U6 RNA at a or fi positions, these U6 RNA substrates with ppG or pG termini present during transcrip- tion would have been converted to CH,-0-ppG and CH:$-O- pG, respectively. However, there were no detectable spots corresponding to CH:,-0-ppG or CHs-0-pG (Fig. 4, lane 4), indicating that ppG and pG-containing U6 RNAs are not substrates for capping and the capping machinery appears to be specific to the y-phosphate of U6 snRNA.

To directly test whether mono- or diphosphate-containing U6 RNA can be capped in vitro, Lu-‘“P-labeled T7-U6 RNA and U6 RNA transcribed from U6 gene were incubated with HeLa cell extract. Again only CHa-0-pppG was detectable and there was no detectable CH,%-0-ppG or CH,<-0-pG (Fig. 4, lanes 11 and 12), suggesting that the capping machinery is rather stringent in requiring y-phosphate for capping.

Effect of Distance between pppG and the Capping Signal- We carried out studies with three different T7 polymerase- transcribed RNAs, where the pppG was shifted 32,51, or 361 nts away with reference to the capping determinant. In all these cases, there was no readily detectable cap structure formed (results not shown). These data confirm our earlier data and show that the y-phosphate has to be in close prox- imity to the capping determinant for optimal capping.

Factors Involved in the Capping of U6 RNA-Since the y- phosphate is retained and gets methylated during the capping of U6 RNA, we carried out studies to identify the methyl group donor and the factor(s) involved in this methylation

ORIGIN 0 l l

1 2 3 4 5 6 7

FIG. 3. Analysis of cap structures formed from a- and y- labeled T7-U6 RNAs. Lane I, RNA obtained by transcribing mouse U6 DNA in uitro; lane 2, T7-U6 RNA labeled with [(u-‘“P]GTP; lane 9, tu-““P-labeled T7-U6 RNA incubated with HeLa cell extract; lane 4, same as in lane 3 hut also digested with alkaline phosphatase; lane 5, T7-U6 RNA labeled with [r-“‘P]GTP; lane 6, y-“‘P-labeled T7-U6 RNA incubated with HeLa cell extract; lane 7, same as in lane 6 hut also digested with alkaline phosphatase. The RNAs were digested with nuclease Pl (and also with alkaline phosphatase in lanes 4 and 7), electrophoresed on DEAE-cellulose paper, and subjected to auto- radiography. 0’, RNAs before incubation with HeLa extract; 30’, period of incubation with HeLa extract.

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FIG. 4. Analysis of cap structure from U6 snRNA contain- ing mono-, di-, or triphosphates on their 5’ termini. Lane I, U6 DNA transcribed in uitro and digested with nuclease Pl; lane 2, 5 S RNA transcribed from Syrian hamster 5 S DNA transcribed in uitro and digested with nuclease Pl; lanes 3 and IO, synthetic methyl GTP as standard; lane 4, same as lane I hut also digested with alkaline phosphatase; lane 5, same as in lane 2 hut digested also with alkaline phosphatase; lane 6, standard pGp; lane 7, same as lane 1 hut digested with T2 RNase: lane 8. T7-U6 RNA divested with T2 RNas< lane 9, same as lane i hut digested with T2 RNase; lane I I, U6 RNA transcribed from mouse U6 DNA in oitro, U6 RNA purified and incubated with HeLa cell extract and digested with nuclease Pl and alkaline phosphatase. Lane 12, T7-U6 RNA, labeled with [n-‘“PI GTP, was incubated with HeLa cell extract and digested with nu- clease Pl and alkaline phosphatase. The digested RNAs were electro- phoresed on DEAE-cellulose paper and subjected to autoradiography. The positions of CHzl-0-ppG and CH:,-0-pG are indicated from the observed mobility of radioactive standards run in earlier experiments. In lanes 7,8, and 9, only mononucleotides Gp, Ap and Cp are included; Up, which migrates faster, is not included in this figure.

reaction. The HeLa extract was heated for 5 min at 65 “C and tested for its ability to cap U6 snRNA. While the normal HeLa cell extract capped T7-U6 snRNA in vitro (Fig. 5, lane I), the heat-treated extract was not capable of capping the T7-U6 snRNA (Fig. 5, lane 3), indicating that one or more heat-labile factor(s) are required for the capping reaction.

When a U6 capping-permissive extract was fractionated on a DEAE-Sephadex column, several fractions were obtained upon elution with different concentrations of KCl. These fractions were tested for their ability to cap T7-U6 snRNA in vitro. The fraction eluted with 0.5 M KC1 was not capable of capping the T7-U6 RNA (Fig. 5, lane 5); however, this fraction when supplemented with AdoMet supported the capping re- action (Fig. 5, lane 6). The cap structure formed in the presence of AdoMet was resistant to digestion by alkaline phosphatase (Fig. 5, lane 7). In addition, the U6 snRNA capping was inhibited by the addition of S-adenosylhomocy- steine, a known inhibitor of methylation reactions mediated by AdoMet (Fig. 5, lane 8). These data show that AdoMet serves as a methyl group donor during in vitro capping of U6 snRNA.

DISCUSSION

Data presented in this study show that presynthesized U6 snRNA can be accurately capped in vitro by HeLa cell extract. During this capping reaction the y-phosphate is retained; AdoMet serves as the methyl group donor for the U6 snRNA sequence-dependent methylation of the y-phosphate.

There are several lines of evidence to suggest that transcrip- tion and capping of mRNAs and U snRNAs are concurrent.

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FIN:. 5. Requirement of a heat-labile component and S-adenosyl methionine (SAM) for capping in uitro. I,anes I and IO, T7-LJ6 RNA incubated with HeLa cell extract; lanes 2, 4, and 9, T7-U6 RNA at zero time; Intjc 3, T7-UG RNA incubated with HeLa cell extract heat-treated f’or 5’ at 65 “C; lane 5, T7-U6 RNA incubated with DEAE-Sephadex column (0.6 M KC1 eluted) f’raction f’or 30 min; lane 6, same as lane S in the presence of added 2 mM AdoMet. Lane 7, same as lane 6 but also digested with alkaline phosphatase. Lane 8, T7-U6 RNA incubated with HeLa cell extract and 2 mM S-adenosyl homocysteine (SAH). The RNA samples were digested with nuclease PI and were electrophoresed on DEAE-cellulose paper and subjected to autoradiography. O’, SO’, -/.i’, periods of incubation. a, heat-treated extract.

In RNA polymerase II transcripts, capping is cotranscrip- tional as studied in viral and cellular mRNAs (Furuichi, 1978 and references therein; also reviewed in Banerjee, 1980), as well as in U snRNAs (Skuzeski et al., 1984). Furthermore, only 5-10% of the T7 mRNA transcripts in mammalian cells contained terminal cap structure presumably because of the lack of coupling between capping enzyme and heterologous T7 polymerase (Fuerst and Moss, 1989). These data further confirm that capping is tightly linked to transcription by polymerase II. The results obtained in this study show that accurate capping of U6 snRNA can be carried out in HeLa cell extracts post-transcriptionally. In this respect, the U6 snRNA-capping mechanism differs from the capping mecha- nism of pre-mRNAs and other characterized U snRNAs.

Recently, one U4/U6 snRNP protein has been character- ized (Banroques and Abelson, 1989), and SPG-transcribed U6 RNA was shown to reconstitute into snRNP particles and incorporate into spliceosomes (Pikielny et al., 1989). It is not known whether the capping enzyme is part of the U6 snRNP particle or whether cap structure is required for U6 snRNP function in the spliceosome. Since the capping of T7-U6 snRNA is inhibited by S-adenosylhomocysteine (Fig. 5), it is possible to study the requirement of cap structure for U6 snRNP function. If the cap structure in U6 snRNA is required for spliceosome assembly and splicing of pre-mRNAs, addi- tion of S-adenosylhomocysteine should result in inhibiting these reactions.

Where in the cell does U6 snRNA get capped? The capping of other U snRNAs takes place in two stages: the addition m’G is coupled to transcription in the nucleus (Skuzeski et al., 1984) and the trimethylation of the m5G cap is cytoplasmic (Mattaj, 1986). The capping of U6 snRNA appears to be cytoplasmic. When T7-U6 snRNA was injected into enu- cleated frog oocytes, accurate capping of U6 RNA was ob- served.” These data suggest that capping of U6 snRNA occurs post-transcriptionally in the cytoplasm.

The DNA and RNA methylation reactions involving AdoMet characterized earlier do not require magnesium ions (Martin and Moss, 1975; Yuan and Hamilton, 1984); however, some methylation reactions are stimulated in the presence of Mg?’ ions (reviewed in Yuan and Hamilton, 1984). The meth- ylation reaction characterized here requires magnesium ions, and there was no detectable capping with HeLa extract de- pleted of Mg’+ ions. Since the stem-loop structure in U6

’ S. Clupta, R. Singh, D. Wright, and R. Reddy, unpublished results.

snRNA is required for the U6 snRNA cap formation (Singh et al., 1990), it is likely that the Mg’+ ions are necessary to facilitate U6 RNA in acquiring the appropriate secondary structure required to direct the cap formation. It is also possible that the U6 RNA sequence-dependent methyltrans- ferase is different from other methyltransferases in requiring magnesium ions.

During the mRNA cap formation from mRNA and pppG, the y-phosphate of the mRNA is removed and does not appear in the mature mRNA. In contrast, data obtained in this study show that the y-phosphate of U6 snRNA is not removed during cap formation. Therefore, the mechanism of this U6 cap formation is very different from the mechanism that has been well characterized for mRNAs (reviewed in Banerjee, 1980).

In addition to the requirement for the capping signal pres- ent within the U6 snRNA (Singh et al., 1990, and Fig. 2), the capping machinery methylated only the y-phosphate although U6 snRNAs with pG and ppG on their 5’ ends were also present. This suggests that the y-phosphate has to be in the right context with reference to the capping signal. Whenever the ppG was moved away from the capping signal, the effi- ciency of capping was ~1%. These data suggest that the only requirement in the U6 snRNA substrate for the capping is the capping signal and the y-phosphate in the right position.

Many DNA sequence-dependent methyltransferases have been purified and characterized (Holliday, 1989; Syzf et al., 1989; Yuan and Hamilton, 1984, and references therein). Since most cellular RNAs, including ribosomal, transfer, and messenger RNAs, are methylated post-transcriptionally (Perry, 1981), many RNA sequence-dependent methyltrans- ferases must exist in the cells. Recently, RNA sequence- dependent accurate methylation of internal adenosine resi- dues in messenger RNA was reported (Narayan and Rottman, 1988); however, all the known methyltransferases modify either the base or the sugar moieties in nucleic acids. This is the first instance of RNA sequence-dependent methyltrans- ferase that modifies the phosphate residue. The ability to reconstitute the capping with a DEAE-Sephadex fraction and AdoMet provides evidence that there are at least two com- ponents necessary for capping the T7-U6 snRNA. This assay system will be useful in purifying and studying the mechanism of action of the capping enzyme.

Acknou,le~~ment.s-We would like to thank the following: Dr. Harris Busch f’or encouragement, Dr. Wei Wei Zhaing for generously

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Post-transcriptional Capping of U6 snRNA 9495

providing DEAE-Sephadex-fractionated HeLa cell extract, Dr. Mi- chael Barattain for the use of LKB densitometer, Dr. Ohshima and Dr. Folk for mouse U6 and Syrian hamster 5 S genes, respectively, Minyone Finley for HeLa cells, and Dr. Lawrence Rothblum and Dr. Robert Larson for synthesizing deoxyoligonucleotides.

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S Gupta, R Singh and R ReddyCapping of U6 small nuclear RNA in vitro can be uncoupled from transcription.

1990, 265:9491-9495.J. Biol. Chem. 

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