Formation of 2′,3′-Cyclic Phosphates at the 3′ End of Human U6 Small Nuclear RNA in Vitro
1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês
10.1074/jbc.272.35.21989
ISSN1083-351X
AutoresJian Gu, Gleb P. Shumyatsky, Nimisha Makan, Ram Reddy,
Tópico(s)RNA modifications and cancer
ResumoApproximately 90% of human U6 small nuclear RNA (snRNA) contains uridine cyclic phosphate (U>p) at its 3′-end (Lund, E., and Dahlberg, J. E. (1992) Science 255, 327–330). We studied the formation of U>p at the 3′ end of human U6 snRNA using an in vitro system where uridylic acid residues are added from UTP precursor and U>p is formed. Analysis of U6 snRNAs with varying number of uridylic acid residues showed that each of these species contains U>p where the phosphate originated from α-phosphate of UTP precursor. The cyclic phosphate formation occurred on U6 snRNA in extracts where essential spliceosomal snRNAs were specifically degraded, thereby indicating that U>p formation is not coupled to pre-mRNA splicing. A subpopulation of human signal recognition particle and mitochondrial RNA processing RNAs isolated from HeLa cells also contained cyclic phosphates at their 3′ ends. These data suggest that U>p in U6 snRNA is unlikely to be related to its participation in splicing of pre-mRNAs. It appears that cyclic phosphate is an intermediate product in the metabolism of these small RNAs. Approximately 90% of human U6 small nuclear RNA (snRNA) contains uridine cyclic phosphate (U>p) at its 3′-end (Lund, E., and Dahlberg, J. E. (1992) Science 255, 327–330). We studied the formation of U>p at the 3′ end of human U6 snRNA using an in vitro system where uridylic acid residues are added from UTP precursor and U>p is formed. Analysis of U6 snRNAs with varying number of uridylic acid residues showed that each of these species contains U>p where the phosphate originated from α-phosphate of UTP precursor. The cyclic phosphate formation occurred on U6 snRNA in extracts where essential spliceosomal snRNAs were specifically degraded, thereby indicating that U>p formation is not coupled to pre-mRNA splicing. A subpopulation of human signal recognition particle and mitochondrial RNA processing RNAs isolated from HeLa cells also contained cyclic phosphates at their 3′ ends. These data suggest that U>p in U6 snRNA is unlikely to be related to its participation in splicing of pre-mRNAs. It appears that cyclic phosphate is an intermediate product in the metabolism of these small RNAs. Many small RNAs are known to participate in important cellular functions. U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) 1The abbreviation used is: snRNA, small nuclear RNA; SRP, signal recognition particle; MRP, mitochondrial RNA processing.1The abbreviation used is: snRNA, small nuclear RNA; SRP, signal recognition particle; MRP, mitochondrial RNA processing. are required co-factors for splicing of nuclear pre-mRNAs (1Steitz J.A. Black D. Gerke V. Parker K.A. Kramer A. Frendeway D. Keller W. Birnstiel M. Structure and Function of Major and Minor snRNPs. Springer-Verlag, Berlin1988: 115-154Google Scholar, 2Steitz J.A. Science. 1992; 257: 888-889Crossref PubMed Scopus (82) Google Scholar, 3Sharp P.A. Cell. 1994; 77: 805-815Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 4Umen J.G. Guthrie C. RNA. 1995; 1: 869-885PubMed Google Scholar). RNaseP, U7 RNA, and many snRNAs are necessary for the site-specific cleavage of pre-tRNA (5Stark B.C. Kole R. Bowman E.J. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3717-3721Crossref PubMed Scopus (203) Google Scholar), histone pre-mRNA (6Schaufele F. Gilmartin G.M. Bannwarth W. Birnstiel M. Nature. 1986; 323: 777-781Crossref PubMed Scopus (101) Google Scholar), and site-specific methylation of pre-ribosomal RNAs (7Kiss-Laszlo Z. Henry Y. Bachellerie J.-P. Caizergues-Ferrer M. Kiss T. Cell. 1996; 85: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 8Cavaille J. Nicoloso M. Bachellerie J.P. Nature. 1996; 383: 732-735Crossref PubMed Scopus (272) Google Scholar), respectively. SRP particles that contain 7SL RNA recognize the signal peptide of the secretory proteins and participate in the secretion of these proteins (9Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (463) Google Scholar). MRP RNA is found mostly in the nucleolus and is required for the accurate 5′ end formation of the ribosomal 5.8 S RNA (10Clayton D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4615-4617Crossref PubMed Scopus (59) Google Scholar, 11Lygerou Z. Allmang C. Tollervey D. Seraphin B. Science. 1996; 272: 268-270Crossref PubMed Scopus (213) Google Scholar). In addition, there are many other small RNAs that are believed to play important roles in both eukaryotic and prokaryotic cells (1Steitz J.A. Black D. Gerke V. Parker K.A. Kramer A. Frendeway D. Keller W. Birnstiel M. Structure and Function of Major and Minor snRNPs. Springer-Verlag, Berlin1988: 115-154Google Scholar, 12Reddy R. Singh R. Prog. Mol. Subcell. Biol. 1991; 12: 1-36Crossref Google Scholar, 13Balakin A.G. Smith L. Fournier M.J. Cell. 1996; 86: 823-834Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Most RNAs undergo post-transcriptional modifications at their 3′ ends. These modifications include polyadenylation of eukaryotic mRNAs (14Edmonds M. Methods Enzymol. 1990; 181: 161-170Crossref PubMed Scopus (25) Google Scholar) and -CCA addition/turnover on tRNAs (15Deutscher M.P. Prog. Nucleic Acid Res. Mol. Biol. 1990; 39: 209-240Crossref PubMed Scopus (104) Google Scholar). Most eukaryotic small RNAs transcribed by RNA polymerase III, such as ribosomal 5 S, U6, SRP, MRP, RNaseP, 7SK, and plant U3 RNA, terminate with 4–5 uridylic acid residues. Although some of them, like 5 S RNAs, retain their 3′ ends and are found in the functional ribosomes with -UUUU-OH or -UUUUU-OH, several others are processed at their 3′ ends and contain sequences slightly different from the original transcripts. Rinke and Steitz (16Rinke J. Steitz J.A. Nucleic Acids Res. 1985; 13: 2617-2629Crossref PubMed Scopus (93) Google Scholar) identified a small fraction (∼10%) of the total U6 snRNAs that has longer 3′ ends with multiple uridylic acid residues and associates with La protein. Studies in our lab showed that the 3′ end of U6 snRNA in snRNPs specifically get labeled in vitro when [α-32P]UTP is added to the cell extracts (17Reddy R. Henning D. Das G. Harless M. Wright D. J. Biol. Chem. 1987; 262: 75-81Abstract Full Text PDF PubMed Google Scholar). This 3′-uridylation of U6 snRNA has also been studied by Hirai et al. (18Hirai H. Lee D.K. Natori S. Sekimizu S. J. Biochem. ( Tokyo ). 1988; 104: 991-994Crossref PubMed Scopus (16) Google Scholar), Lund and Dahlberg (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar), and Tazi et al. (20Tazi J. Forne T. Jeanteur P. Cathala G. Brunel C. Mol. Cell. Biol. 1993; 13: 1641-1650Crossref PubMed Scopus (37) Google Scholar). Lund and Dahlberg (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar) showed that about 90% of the U6 snRNA in human cells contains U>p at its 3′ end, and Brunel and co-workers concluded that this U>p formation is coupled to its involvement in splicing of pre-mRNAs (20Tazi J. Forne T. Jeanteur P. Cathala G. Brunel C. Mol. Cell. Biol. 1993; 13: 1641-1650Crossref PubMed Scopus (37) Google Scholar, 21Forne T. Labournier E. Rossi F. Gallouzi I. Cathala G. Tazi J. Brunel C. Biochimie ( Paris ). 1996; 78: 436-442Crossref PubMed Scopus (9) Google Scholar). In addition to U6 snRNA, many RNAs are known to contain 2′,3′-cyclic phosphate structures. The autolytic products of the hammerhead cleavage of the satellite RNA of tobacco ringspot virus (22Prody G.A. Bakos J.T. Buzayan J.M. Schneider I.R. Bruening G. Science. 1986; 231: 1577-1580Crossref PubMed Scopus (445) Google Scholar) and avocado sunblotch viroid RNA (23Hutchins C.J. Rathjen P.D. Forster A.C. Symons R.H. Nucleic Acids Res. 1986; 14: 3627-3639Crossref PubMed Scopus (435) Google Scholar, 24Forster A.G. Symons R.H. Cell. 1987; 50: 9-16Abstract Full Text PDF PubMed Scopus (247) Google Scholar) contain 2′,3′-cyclic phosphates. Several other autolytic motifs, such as hairpin, also cleave to give 2′,3′-cyclic phosphate (25Long D.M. Uhlenbeck O.C. FASEB J. 1993; 7: 25-30Crossref PubMed Scopus (85) Google Scholar). The cleavage of pre-tRNA by an endonuclease results in a 5′ half-molecule containing 2′,3′-cyclic phosphate (26Peebles C.L. Gegenheimer P. Abelson J. Cell. 1983; 32: 525-536Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 27Greer C.L. Peebles C.L. Gegenheimer P. Abelson J. Cell. 1983; 32: 537-546Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 28Filipowicz W. Shatkin A.J. Cell. 1983; 32: 547-557Abstract Full Text PDF PubMed Scopus (111) Google Scholar). The self-excision of a plant pre-tRNA also results in RNAs containing 2′,3′-cyclic phosphate (29Weber U. Beier H. Gross H.J. Nucleic Acids Res. 1996; 24: 2212-2219Crossref PubMed Scopus (15) Google Scholar). Shumyatsky et al. (30Shumyatsky G.P. Tillib S.V. Kramerov D.A. Nucleic Acids Res. 1990; 18: 6347-6351Crossref PubMed Scopus (31) Google Scholar) analyzed Ehrlich ascites carcinoma (mouse) cell RNAs fractionated on sucrose density gradients and analyzed poly(A)+ RNA isolated from light, intermediate, and heavy fractions. Cyclic phosphates (pC>p and pU>p) were found in the poly(A)+ fraction obtained from the intermediate fraction. The synthesis of these cyclic phosphate-containing RNAs was inhibited by low concentrations of α-amanitin, indicating that cyclic phosphate may be present at the 3′ end of mRNAs. Nilsen and his colleagues studied the human pre-rRNA processing in vitro and found 2′,3′-cyclic phosphate cleavage intermediates (31Hannon G.J. Maroney P.A. Branch A.B. Benenfield B.J. Robertson H.D. Nilsen T.W. Mol. Cell. Biol. 1989; 9: 4422-4431Crossref PubMed Scopus (38) Google Scholar). These data show that the formation of cyclic phosphate in RNA is widespread in nature. In this study, we further characterized the formation of cyclic phosphate at the 3′ end of U6 snRNA. We also report that cyclic phosphate is present at the 3′ end of other small RNAs. In addition, the U>p appears to be an intermediate after the removal of a nucleoside from RNAs containing 3′-OH by an exonuclease. Fine chemicals, including nucleoside 2′,3′-cyclic phosphates, were obtained from Sigma. [α-32P]ATP, [α-32P]CTP, and [α-32P]UTP were obtained from Amersham Corp., and [32P]orthophosphate was obtained from ICN. For preparation of uniformly labeled RNAs, HeLa cells were incubated at 37 °C with [32P]orthophosphate for 16 h in phosphate-free medium (32Reddy R. Tan E.M. Henning D. Nohga K. Busch H. J. Biol. Chem. 1983; 258: 1383-1386Abstract Full Text PDF PubMed Google Scholar). The 4–8 S RNA was prepared by centrifugation of the whole cell RNA on a sucrose density gradient and pooling the fractions corresponding to 4–8 S RNAs (33Reddy R. Li W.-Y. Henning D. Choi Y.C. Nohga K. Busch H. J. Biol. Chem. 1981; 256: 8452-8457Abstract Full Text PDF PubMed Google Scholar). The RNAs were fractionated and purified on a 10% denaturing polyacrylamide gel containing 7m urea. DNA dot hybridizations were carried out as described by Kafatos et al. (34Kafatos F.C. Jones C.W. Etstratiadis A. Nucleic Acids Res. 1979; 7: 1541-1552Crossref PubMed Scopus (904) Google Scholar). 5 μg each of cloned DNA was immobilized on nitrocellulose discs and hybridized at 42 °C for 16 h with uniformly 32P-labeled HeLa 4–8 S RNAs. Hybridization solution contained 50% formamide, 80 mmTris, pH 7.5, 600 mm NaCl, 4 mm EDTA, 1.5 × Denhardt's reagent, 0.01% SDS, and 5 μg/ml yeast tRNA. The filters were washed at 42 °C with 3 × SSC, 1.5 × SSC, and 0.5 × SSC containing 0.1% SDS. The hybridized RNAs were eluted with sterile water at 100 °C for 5 min., precipitated in presence of carrier yeast tRNA, and electrophoresed on denaturing polyacrylamide gels. The RNAs were digested with nuclease P1 at a 1:1500 (w/w) enzyme to substrate ratio at 37 °C for 30 min. Usually, 30 μg of RNA was digested with 20 ng of nuclease P1 in 25 μl of 10 mm acetate buffer, pH 5.0. The digests were either spotted on DEAE-cellulose paper and subjected to electrophoresis or spotted on cellulose plate and subjected to chromatography. Digestion with T1 RNase was done in 10 mmTris-HCl, pH 7.4, at a 1:10 (w/w) enzyme to substrate ratio at 37 °C for 60 min. Labeled standard pU>p was prepared by labeling U>p with polynucleotide kinase and [γ-32P]ATP. The RNase H digestion of U1 or U2 snRNAs was done according to Black et al. (35Black D.L. Chabot B. Steitz J.A. Cell. 1985; 42: 737-750Abstract Full Text PDF PubMed Scopus (317) Google Scholar). The oligonucleotides used in this study were 5′-TGCCAGGTAAGTAT-3′ (complementary to U1 snRNA nucleotide 1–14) and 5′-GAACAGATACTACACTTGA-3′ (complementary to U2 snRNA nucleotide 27–45). Electrophoresis on 57–80-cm-long DEAE-cellulose paper was carried out at 500 V for 4–5 h in Savant high voltage electrophoresis unit using 5% acetic acid/ammonium hydroxide buffer, pH 3.5. Chromatography on cellulose plates was performed as described by Silberklang et al.(36Silberklang M. Gillum A.M. Raj Bhandary U.L. Methods Enzymol. 1979; 59B: 58-109Crossref Scopus (352) Google Scholar). The first dimension solvent was isobutyric acid/water/ammonium hydroxide (66:33:1, v/v/v), and the second dimension solvent was 0.1m sodium phosphate buffer, pH 6.8/ammonium sulfate/n-propanol (100:60:2, v/w/v). In some cases, only the second dimension was performed for the separation of pU and pU>p. As shown earlier by Lund and Dahlberg (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar), U6 snRNA from HeLa cells labeled in vivowith [32P]orthophosphate contained pU>p (Fig. 1 D). Incubation of HeLa cell extract with [α-32P]UTP resulted in rapid labeling of U6 snRNA, which was the major labeled RNA (Fig. 1 A,lane 3). The labeled U6 snRNA was then digested with different enzymes and subjected to chromatography. Digestion with nuclease P1 yielded pU and pU>p (Fig. 1 B, lane 3), and digestion with nuclease P1 followed by alkaline phosphatase resulted in Pi and U>p (Fig. 1 B,lane 4). When the nuclease P1 digest was fractionated by two-dimensional chromatography, radioactivity was observed in pU>p, pU, and pUp (Fig. 1 C). In several independent experiments, the amount of pU>p and pUp were 10–30 and 1–2% of the total radioactivity, respectively. The isolation and subsequent digestion of pU>p with alkaline phosphatase yielded two labeled spots corresponding to Pi and U>p (data not shown). These data show that some of the U6 snRNA 3′ ends formed in vitro contain U>p; in addition, these data show that both the 5′- and 3′-phosphate residues in pU>p originated from the labeled α-phosphate of the precursor UTP. Addition of [α-32P]CTP or [α-32P]ATP to HeLa cell extract resulted in labeling of tRNA 3′ ends (Fig. 1 A, lanes 1 and 2). Digestion of labeled tRNA with nuclease P1 yielded only pC or pA (Fig. 1 B, lanes 1 and 2), and no detectable cyclic phosphate was found. These data show that formation of cyclic phosphate is specific to U6 snRNA and does not occur in all cases where nucleotides are added to the 3′ end of RNAs. Previous data showed that there are multiple U6 snRNAs differing in the number of uridylic acid residues at the 3′ end (16Rinke J. Steitz J.A. Nucleic Acids Res. 1985; 13: 2617-2629Crossref PubMed Scopus (93) Google Scholar, 37Das G. Henning D. Wright D. Reddy R. EMBO J. 1988; 7: 503-512Crossref PubMed Scopus (154) Google Scholar). When labeled U6 snRNA was fractionated on a short polyacrylamide gel (Fig. 1 A, lane 3), multiple U6 snRNA species did not separate. To facilitate separation of U6 snRNA differing in number of uridylic acid residues at the 3′ end, the U6 snRNA labeled in vitro (Fig. 1 A, lane 3) was digested with T1 RNase to completion and then fractionated by electrophoresis on a long 20% polyacrylamide gel (Fig. 2 A, lane 2). A U6 snRNA T1 RNase digestion fragment corresponding to the 3′ end with four U residues was electrophoresed to serve as a standard (Fig. 2 A, lane 1). The position of this fragment was designated as 0, because this fragment with four uridylic acid residues represents the U6 snRNA transcript as transcribed by RNA polymerase III (37Das G. Henning D. Wright D. Reddy R. EMBO J. 1988; 7: 503-512Crossref PubMed Scopus (154) Google Scholar, 38Sakonju S. Bogenhagen D. Brown D.D. Cell. 1980; 19: 13-25Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 39Kunkel G.R. Maser R. Calvet J. Pederson T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8575-8579Crossref PubMed Scopus (158) Google Scholar). Thirteen labeled bands were visualized with varying intensities; these fragments correspond to seven additional U residues to five deleted nucleotides when compared with U6 snRNA transcribed by RNA polymerase III. These labeled RNA fragments were purified and analyzed for the presence of cyclic phosphate. Both longer and shorter U6 snRNA forms contained U>p (Fig. 2 B). These data show that the presence of U>p is not restricted to mature U6 snRNA with 4–5 uridylic acid residues. Taziet al. (20Tazi J. Forne T. Jeanteur P. Cathala G. Brunel C. Mol. Cell. Biol. 1993; 13: 1641-1650Crossref PubMed Scopus (37) Google Scholar) studied the formation of U>p at the 3′ end of U6 snRNA and concluded that U>p formation is coupled to an event arising during splicing. Complementary oligonucleotide-directed RNase H cleavage was employed to degrade U1 or U2 snRNAs, which are essential for splicing of pre-mRNAs (35Black D.L. Chabot B. Steitz J.A. Cell. 1985; 42: 737-750Abstract Full Text PDF PubMed Scopus (317) Google Scholar). Fig. 3 A shows the analysis of snRNAs after targeted degradation. There was no detectable intact U1 snRNA when oligonucleotide complementary to U1 snRNA was added (Fig. 3 A, lane 2), and a similar result was obtained when U2 snRNA was targeted (Fig. 3 A, lane 3). These data show that essential snRNAs were effectively degraded by this procedure. Next, the U1 and U2 snRNA depleted extracts were used for 3′ end labeling of U6 snRNA in vitro (Fig. 3 B). The labeled U6 snRNAs from the control (Fig. 3 B, lane 1) and snRNA-depleted extracts (Fig. 3 B, lanes 2 and 3) were purified and analyzed for the presence of U>p. There was U>p formation in each case, and the amounts of pU>p in the control, U1, and U2 oligonucleotide-treated extracts were 29, 23, and 22% of total radioactivity, respectively (Fig. 3 C,lanes 1–3). These data show that the formation of U>p at the 3′ end of U6 snRNA does not require its participation in the splicing of pre-mRNAs. Because the cyclic phosphate formation is occurring in RNP particles, the marginal reduction in pU>p formation upon U1 and U2 oligonucleotide treatment may be due to nonobligate linkage of cyclic phosphate formation to pre-mRNA splicing (see “Discussion”). HeLa cell 4–8 S RNA uniformly labeled with [32P]orthophosphate was fractionated on a 50-cm-long polyacrylamide gel and subjected to autoradiography (Fig. 4 A). Two distinct bands of roughly equal intensity were observed for SRP RNA (also known as 7SL RNA), the RNA component of the SRP. The faster migrating band was designated SRP-1 RNA, and the slower band was designated SRP-2 RNA. Similar results were obtained with MRP RNA (also known as 7SM or 7–2 RNA). There were two bands of similar intensity where one was designated MRP-1 RNA and the other was designated MRP-2 RNA. Hybrid selection was carried out to confirm that these four distinct bands correspond to SRP or MRP RNAs. Human SRP DNA immobilized on nitrocellulose paper hybrid-selected both SRP-1 and SRP-2 RNAs (Fig. 4 B, lane 2), and human MRP DNA hybrid-selected both MRP-1 and MRP-2 RNAs (Fig. 4 B, lane 3). These data provide evidence for the identity of these RNAs. Under similar experimental conditions, U6 snRNA also fractionated into two major bands and several slower migrating bands of less intensity (Fig. 4 B, lane 4), whereas 7SK RNA electrophoresed as one band (Fig. 4 B, lane 1). The SRP and MRP RNAs were purified, digested with nuclease P1, and fractionated on a DEAE-cellulose paper. The SRP RNA is not known to contain any modified nucleotides (33Reddy R. Li W.-Y. Henning D. Choi Y.C. Nohga K. Busch H. J. Biol. Chem. 1981; 256: 8452-8457Abstract Full Text PDF PubMed Google Scholar); therefore, only pppG and mononucleotides were expected. However, in addition to pppG and mononucleotides, another labeled compound comigrating with pU>p was observed. This spot was more prominent in the SRP-1 RNA species than SRP-2 RNA (Fig. 4 C, lanes 3 and 4). Similar results were obtained when MRP-1 and MRP-2 RNAs were analyzed after nuclease P1 treatment. The faster migrating MRP-1 RNA species contained significantly greater quantities of pU>p than MRP-2 RNA (Fig. 4 C, lanes 1 and 2). As controls, human U6 snRNA and ribosomal 5 S RNA were also analyzed after nuclease P1 digestion. As expected, a spot consistent with pU>p was observed in U6 snRNA (Fig. 4 C, lane 6), whereas there was no detectable pU>p in 5 S RNA (Fig. 4 C, lane 5). The nuclease P1 digests of SRP-1 RNA and MRP-1 RNA were also subjected to two-dimensional chromatography, and in both cases pU>p was observed (Fig. 5, A and B). These data show that in addition to U6 snRNA, a subset of SRP and MRP RNAs also contain U>p. The main findings of this study are: 1) U>p is not restricted to mature U6 snRNA with 4–5 uridylic acid residues because longer and shorter U6 snRNA species contain U>p; 2) Splicing is not required for the formation of U>p at the 3′ end of U6 snRNA. These data and data from other investigators are consistent with a mechanism shown in Fig. 6 where trimming and addition of uridylic acid residues to the 3′ end of U6 snRNA is constantly occurring in a dynamic equilibrium. In addition, this mechanism also implies that U>p is an intermediate in the trimming process. The evidence for this mechanism is as follows. This mechanism (Fig. 6) predicts three populations of U6 snRNA: 3′-OH, 3′-p, and 2′,3′>p. These products are formed in vitro and have been identified by us in this study and also by other investigators (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar, 20Tazi J. Forne T. Jeanteur P. Cathala G. Brunel C. Mol. Cell. Biol. 1993; 13: 1641-1650Crossref PubMed Scopus (37) Google Scholar). Because [α-32P]UTP is the source of the radioactivity and is added to the 3′-OH of U6 snRNA, the necessary RNA substrates with 3′-OH should be present in vitro. These substrates varied from 1 to 10 U residues (see Fig. 2 A, lane 2). We found that the 3′-p in pU>p was also labeled (Fig. 1 B, lane 4); and this is possible only by the addition of UMP and the subsequent removal of a nucleoside. Therefore, these data provide evidence for a trimming reaction in vitro involving deletion of a nucleoside. Though the yield was rather low (1–2%), we also found pUp (Fig. 1 C), which is a result of decyclization of U>p. Removal of the 2′,3′-cyclization is required to make the 3′-OH group available for the elongation reaction. The fact that U6 snRNAs-1 to -5 species were found to be labeled in vitro (Fig. 2 A, lane 2) shows that the U6 snRNAs where nucleotides are deleted are also used for elongation in vitro. These data are also consistent with the observation of Lund and Dahlberg (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar), who showed that the terminal nucleotides of U6 snRNA are turning over in a nontemplate-dependent manner. The predicted intermediates with 3′-OH, 3′-p, and 2′,3′>p are also found in vivo. The identification of U6 snRNA with up to about 20 U residues (16Rinke J. Steitz J.A. Nucleic Acids Res. 1985; 13: 2617-2629Crossref PubMed Scopus (93) Google Scholar) shows that necessary U6 snRNA substrates for the addition of uridylic acid residues are available in vivo. Because only RNAs with 3′-OH are substrates for the addition of uridylic acids, different sizes of these U6 snRNAs must be present in vivo. We also prepared cDNAs of U6 snRNA obtained from immunoprecipitates of La antibodies and sequenced them. Some of the resulting cloned cDNAs contained more as well as less than 4–5 U residues as predicted from mature and predominant form of human U6 snRNA. 2Y. Chen and R. Reddy, unpublished data. U6 snRNA with 3′-p was also found in several species in vivo (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar). In yeast U6 snRNA, the 3′-p containing population was predominant. In fruit fly, the 3′-OH, 3′-p, and 2′,3′>p populations were present at a ratio of 1:1:1. Therefore, the U6 snRNA in vivo has three populations with 3′-OH, 3′-p, and 2′,3′>p. These data are consistent with the proposed mechanism shown in Fig. 6. The enzymes involved in the trimming and elongation of the 3′ end of U6 snRNA are not known. An enzymatic activity capable of forming cyclic phosphate at the 3′ end of RNAs has been characterized from HeLa cells (40Filipowicz W. Vincente O. Methods Enzymol. 1990; 181: 499-510Crossref PubMed Scopus (16) Google Scholar). However, the RNA substrate for this enzyme requires a 3′-phosphate, which is then converted into a cyclic phosphate. In the case of U6 snRNA, the 3′-cyclic phosphate originates from the 5′-α-phosphate of the pppU used as the precursor. Therefore, enzymes involved in the cyclic phosphate formation in U6 snRNA should be different from this enzyme characterized by Filipowicz and Vincente (40Filipowicz W. Vincente O. Methods Enzymol. 1990; 181: 499-510Crossref PubMed Scopus (16) Google Scholar). There are many instances where an endonuclease or self-cleavage of RNA results in RNA fragments with cyclic phosphate at the 3′ ends (see Introduction). In fact, any time the ester bond between 3′-phosphate and 5′-hydroxyl group in an RNA molecule is cleaved, the 2′,3′-cyclic phosphate is the initial product. However, the cyclic phosphate formation in U6 snRNA 3′ end could not be due to the action of an endonuclease. Because our data identified a series of cyclic phosphate-containing U6 snRNAs that differ by one nucleotide in length, it appears that an exonuclease deletes a terminal nucleoside with a cleavage of the ester bond between 3′-phosphate and 5′-hydroxyl resulting in the formation of cyclic phosphate. Data from our lab (17Reddy R. Henning D. Das G. Harless M. Wright D. J. Biol. Chem. 1987; 262: 75-81Abstract Full Text PDF PubMed Google Scholar) and other groups (20Tazi J. Forne T. Jeanteur P. Cathala G. Brunel C. Mol. Cell. Biol. 1993; 13: 1641-1650Crossref PubMed Scopus (37) Google Scholar) have shown that this 3′ end modification is occurring in RNP particles, and it is likely that the enzymes responsible for 3′ end modification in U6 snRNA recognize one or more proteins associated with U6 snRNA. It would be necessary to reconstitute U6 snRNPs in vitro for characterization and eventual purification of the various enzymes involved in this modification. Tazi et al. (20Tazi J. Forne T. Jeanteur P. Cathala G. Brunel C. Mol. Cell. Biol. 1993; 13: 1641-1650Crossref PubMed Scopus (37) Google Scholar) studied the U>p formation at the 3′ end of U6 snRNA and concluded that this formation is coupled to its involvement in splicing of pre-mRNA. Our data show that the formation of spliceosome and U6 snRNA participation in the splicing are not required for the U>p formation at its 3′ end. The 3′ end turnover and U>p formation of U6 snRNA may be occurring within the spliceosome; however, our data indicate that it may be incidental to U6 snRNA being present, and U>p formation is not coupled to its participation in splicing. The observation of Lund and Dahlberg (19Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar) that U6 snRNA in some species has very little U>p at its 3′ end is also indicative that U>p is not likely to be functionally relevant or coupled to a step in splicing of pre-mRNAs. In addition, the presence of U>p at the 3′ ends of SRP and MRP RNAs, whose function is unrelated to splicing of pre-mRNAs, also suggests that U>p formation is more likely to be related to the metabolism of these small RNAs. We thank M. Finley for providing HeLa cells and Y. Chen for valuable discussions.
Referência(s)