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U14snoRNAs of the fern, Asplenium nidus, contain large sequence insertions compared with those of higher plants
Biochimica et Biophysica Acta 1397 Ž1998. 325–330
U14snoRNAs of the fern, Asplenium nidus, contain large sequence insertions compared with those of higher plants David J. Leader 1, Gillian P. Clark, John W.S. Brown
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Department of Cell and Molecular Genetics, Scottish Crop Research Institute, InÕergowrie, Dundee DD2 5DA, UK Received 10 December 1997; accepted 30 January 1998
Abstract Northern analyses of U14snoRNAs in different plant species showed the expected hybridising band of approximately 120 nt in monocotyledonous and dicotyledonous angiosperms. In the lower plant, Bird’s nest fern Ž Asplenium nidus ., U14s were larger and three hybridising RNAs of approximately 190, 210 and 250 nt were observed. RT-PCR cloning of all three size variants using primers to the conserved 5X and 3X ends of higher plant U14snoRNAs showed large insertions in one of the plant-specific regions corresponding in position to the yeast U14-specific Y-domain. The insertions are pyrimidine-rich in their 5X halves and purine-rich in their 3X halves and are likely to be sequestered in stem structures consistent with the proposed model of U14snoRNA secondary structure. The 5X flanking regions of one of the fern U14 variants was generated by PCR and lacked classical plant snRNA promoter elements. q 1998 Elsevier Science B.V. Keywords: Small nucleolar RNA; U14; Pre-rRNA processing; ŽPteridiophyte.
1. Introduction Eukaryotic small nuclear RNAs Ž snRNAs. can be divided into two classes: spliceosomal snRNAs involved in pre-mRNA splicing and small nucleolar RNAs ŽsnoRNAs. involved in precursor ribosomal RNA Žpre-rRNA. processing and maturation, and ribosome biogenesis w1–7x. U14 belongs to a major family of snoRNAs called box CrD snoRNAs, is evolutionarily conserved and is required for growth and for 18S rRNA processing in yeast Ž Saccharomyces cereÕisiae. w8–11x. U14 contains se-
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Corresponding author: Fax: q44-1382-562426; E-mail: [email protected] 1 New address: Zeneca Agrochemicals, Jealott’s Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK.
quence elements, required for function and accumulation in yeast w12,13x which are largely conserved in animal and plant U14snoRNAs w14,15x. They include the box C and D sequences, 5X and 3X inverted repeats and two conserved elements Ž18S-A and 18S-B. complementary to regions of 18S rRNA. These elements base-pair with 18S rRNA w11x and the 18S-B element determines a ribose methylation site on 18S rRNA w4,5x. Deletion analyses of Xenopus U14s have demonstrated that a base-paired structure, involving the 5X and 3X terminal inverted repeats flanking boxes C and D, is needed for processing and accumulation of U14s w16,17x. A secondary structure model for yeast U14snoRNA also suggests that the ends of the molecule form a stem while the rest of the molecule is loosely structured w18,19x. Compared with vertebrate U14s, the larger yeast U14 contains a yeastspecific region Ž the Y-domain. which can form a
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stem-loop structure w18,19x. This region is essential for activity in yeast and when introduced into mouse U14, confers activity w11,12,20x. This domain is largely conserved in other yeast species and in all cases is capable of forming a stem-loop structure within which a number of bases in the loop sequence are conserved w20x. More recently, U14 has been shown to cross-link at many positions in the pre-rRNA and to other snoRNAs suggesting that its interactions in pre-rRNA processing are complex w21x. In particular, the Y-domain was essential for viability and cross-linked exclusively to the 35S pre-rRNA but not to mature rRNAs suggesting a role in pre-rRNA processing w21x. In plants, snoRNA genes isolated to date are U3snoRNA, 7-2rMRP snRNA, U14snoRNAs and a number of box CrD and one box HrACA snoRNAs ww15,22–25x; D.J.L. and J.W.S.B., unpublished resultsx. Plant U14snoRNAs showed extensive sequence homology to vertebrate and yeast U14s in containing boxes C and D, terminal inverted repeats and the two regions of complementarity to 18S rRNA sequences. Higher plant U14snoRNAs were intermediate in size Ž 117–120 nt. between animal U14snoRNA Žca. 87 nt. and yeast Ž130 nt. , and contained three plant-specific sequences Ž P1–P3. . These regions are absent in vertebrate U14snoRNAs, and the P2 region corresponds in position to the Y-domain of yeast w15x. In this paper, we have analysed U14snoRNAs from a number of higher plant species and from the Bird’s nest fern, Asplenium nidus. Ferns belong to the Pteridophyta, a lower division of the plant kingdom than the flowering plants ŽSpermatophyta. containing gymnosperms and angiosperms. Surprisingly, the fern U14snoRNAs were found to be much larger than their higher plant counterparts. Three major size variants contained additional sequences in the plant-specific P2 region and could form extensive stem structures.
2. Materials and methods 2.1. Materials Restriction enzymes, Taq DNA polymerase and T4 DNA ligase were purchased from Boehringer Ž Mannheim.. Hybond Nq filters were obtained from
Amersham. MMLV reverse transcriptase and RQI RNase-free, DNase were obtained from Gibco. DNA sequencing was carried out with Sequenase II Ž United States Biochemicals. or using the Cycle Sequencing System from BRL Life, on an ABI373 automatic sequencer. The Vectorette II system was purchased from Genosys ŽCambridge, UK. . 2.2. Oligonucleotides U14-2Žy. 5X-CATATGATCAŽGrA.ACATCCAAGGAAGG-3X ; PLU14RT-1 CGGAAGCTTNNAAGGCTTGTTCTC-3X ; F18S 5X-CTCAAGCTTAGG C G G C C A C TG C G A A TG -3 X ; FU 14Ig1 5 X CGGAAGCTTNNGAGAAACAAGCCTT-3X ; Vectorette universal primer 5X-CAACGTGGATCCGAATTCAAGCTTC-3X ; Vectorette nested primer 5X-ACGTGGATCCGAATTCAAGCTTCATG-3X ; ANU145X Ž 5X-CACCGCATGCAGTGTGAAACGATGGCTAG-3X . . The sequences of restriction sites used in cloning are underlined. 2.3. RNA and DNA extraction RNA was isolated from leaf material of maize, barley, potato, tobacco, haricot bean, cucumber, and from fully expanded fronds of asparagus and the Bird’s Nest fern by the guanidinium isothiocyanate method w26x. RNA for use in RT-PCR was DNased twice with RQ1 RNase-free DNase. DNA was extracted from leaf material of the fern by the method of van der Ven et al. w27x. 2.4. Northern blotting and RT-PCR Ten micrograms of total RNA was separated on a denaturing 6% polyacrylamide gel and electroblotted onto Hybond Nq. Blots were hybridised with a probe consisting of the DNA fragment isolated from the plasmid pgMU14.1b which contains the maize U14.1b gene w15x. The probe was labelled with a-w 32 PxdCTP by random priming. RT-PCR reaction conditions were as described w28x. First strand cDNA synthesis was carried out with primer U14-2Žy., complementary to the 18S-B region conserved at the 3X end of all U14s Žw14x; Figs. 2 and 3.. U14 cDNA sequences were amplified by subsequent PCR with U14-2Ž y . and primer
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PLU14RT-1, homologous to a conserved sequence upstream of the 18S-A sequence ŽFigs. 2 and 3.. Amplified PCR products were digested with Bcl I and HindIII, purified by polyacrylamide gel electrophoresis and subcloned into pGEM3ZfŽq. ŽPromega. prior to sequencing. 2.5. Determination of 5X flanking sequences The 5X flanking sequence of a fern U14 gene was generated by PCR using the Vectorette II system according to the manufacturer’s instructions. Briefly, total fern DNA was digested with FspI or Sau3A and appropriate double-stranded adapters ligated to the DNA fragments. The first round of PCR amplification used the vectorette universal primer and the fern-specific primer F18S ŽFig. 3. , complementary to a sequence including the 18S-A region. The products of this reaction were used as the substrate for subsequent doubly nested PCR reactions with the vectorette nested primer and primer FU14Ig1 which is largely complementary to PLU14RT-1 used in the RT-PCR reactions. PCR products were either sequenced directly or digested with HindIII, purified as described above and cloned into pGEM3ZfŽq.. To confirm the identity of the putative 5X flanking sequence obtained by the Vectorette method, the primer ANU145X was designed to a region of the flanking sequence close to the vectorette ligation site Ž Fig. 3.. This primer was used in PCR reactions with A. nidus DNA and the primer U14-2Žy. . PCR products were sequenced directly or subcloned into pGEM3ZfŽq. following digestion with Bcl I and SphI and gel purification.
Fig. 1. Northern analysis of U14snoRNAs from different plant species. Total RNA from the monocotyledonous species: maize Žlane 1., barley Žlane 2. and asparagus Žlane 3., from the dicotyledonous species: potato Žlane 4., tomato Žlane 5., bean Žlane 6. and cucumber Žlane 7., and from the fern, A. nidus Žlane 8. was hybridised with a probe to maize U14.1b w15x. Markers— w 32 Px-end labelled HindIII-digested f X174 DNA.
To investigate the exceptionally large sizes of the fern U14snoRNAs Ž as much as 2–3 times the size of yeast, animal and higher plant U14s. , RT-PCR was carried out on fern RNA using primers designed to conserved regions of higher plant U14snoRNAs Ž Fig. 2.. RT-PCR products of three different sizes were
3. Results and discussion The sizes of U14snoRNAs in a number of plant species were investigated by Northern analysis Ž Fig. 1.. Total RNA of the monocot. species, maize, barley and asparagus ŽFig. 1, lanes 1–3. and the dicot. species potato, tobacco, bean and cucumber Ž Fig. 1, lanes 4–7. all showed a major hybridising band of approximately 120 nt when probed with the maize U14.1b gene. In contrast, total RNA of the fern, A. nidus, showed three bands of approximately 190, 210 and 250 nt ŽFig. 1, lane 8..
Fig. 2. Schematic diagrams of mouse, yeast and higher plant U14s, and fern U14 RT-PCR products. Box CrD sequences are indicated, yeast-specific sequences are shown by open boxes and plant-specific sequences by hatched boxes. Primers to regions conserved in U14s from plants, animals and yeast which were used in RT-PCR analyses on fern leaf total RNA are shown as arrows on the higher plant U14. The three fern U14 RT-PCR products contain additional sequences Žstriped boxes. within the P2 region.
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obtained ŽFig. 2. and subcloned into pGEM3ZfŽ q. prior to sequencing. Control reactions with no reverse transcriptase gave no PCR products Žresults not shown. demonstrating that the RT-PCR products were RNA-derived. A sequence alignment of the three expressed fern U14snoRNA size variants with the higher plant U14snoRNA consensus is shown in Fig. 3. Extensive sequence homology clearly exists between the 5X and 3X halves of the fern U14snoRNAs and higher plant U14snoRNAs Ž Fig. 3b and d.. Additional sequences of 71, 91 and 135 nt were present in the fern U14.2, U14.3 and U14.4 RT-PCR clones respectively Ž Fig. 3c.. These sequences were in the plant-specific region P2. To demonstrate that the size
variants were the products of three different genes, PCR was carried out on genomic fern DNA using the same primers as above, and all three size variants were obtained Žresults not shown. . The presence of three U14 variants in fern is of interest since higher plants and animals contain multiple genes of similar size and which differ by at most a few nucleotides in sequence. The additional sequences contained within the three fern U14 gene variants are related to one another in regions of primary sequence Ž see alignment in Fig. 3c.. Of particular note is that the 5X halves of these regions are pyrimidine-rich while the 3X halves are purine-rich. Secondary structure predictions using
Fig. 3. Alignment of the three fern U14 sequences with those of yeast Ž S. cereÕisiae ., mouse and maize. The sequences have been separated into different sections Ža–d. to better illustrate the conservation of the 5X and 3X portions of all of the U14s. The three plant-specific regions, P1, P2 and P3 w15x are marked below the maize sequence and the conserved 18S-A and 18S-B, and yeast-specific Y-domain is shown above the yeast sequence. Inverted repeats ŽIR., boxes C and D and the positions of certain oligonucleotide primers are indicated. The 5X and 3X regions of the fern U14s which show conservation with other U14s are aligned in Žb. and Žd.. The additional fern sequences in the P2 region are shown separately Žc. from the conserved 5X and 3X regions. The 5X flanking sequence is that generated by the Vectorette PCR system Ža..
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MFOLD ŽUWGCG. fold each of the regions into elongated stem structures of different sizes Ž Fig. 4. . MFOLD predictions of higher plant U14s Ž e.g., potato and maize. suggest formation of a short stem-loop structure where the P2 region forms the upper half of
Fig. 4. Secondary structure predictions ŽUWGCG-MFOLD. for a region of potato, maize and fern U14s. The region consisting of P2 and immediately adjacent sequences can potentially form stem-loops. The P2 region is indicated by the thin line in the maize and potato structures and forms the upper part of the stem and loop. Sequence variation in maize U14 variants are labelled. The heavy line across all five structures indicates a sequence, including part of P2, conserved at the base of each stem.
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the stem and the loop sequence Ž Fig. 4.. Although the higher plant P2 regions can also be modelled to form loops of 10–12 bases w20x, alternatively they may form more extensive stems with only small loops ŽFig. 4.. This stem structure is almost completely conserved in the three fern U14 sequences including the side bulge in the 3X half of the stem, consisting of two uridines ŽFig. 4.. However, in place of the loop sequence found in the short stem-loops in higher plant U14s, the additional sequences in the fern U14s form long stem structures. The predicted maximum free energies of the fern U14s were DG s y44.6 kcalrmole Ž U14.2 ., DG s y50.5 kcalrmole Ž U14.3 . and DG s y66.2 kcalrmole Ž U14.4 ., compared to DG s y8.8–9.2 kcalrmole for the potato or maize U14s. Assuming that all three variants are functional in fern pre-rRNA processing, the size and sequences of the stem structures may not be important to function as large deletions or additions are tolerated. This may suggest that the secondary structures act to position and hold the important regions of the U14s Ž e.g., 18S-A and 18S-B. in the correct conformation for activity, as suggested for the yeast Y-domain stemloop w19x. However, in yeast nucleotides within the Y-domain loop are highly conserved, are required for function in S. cereÕisiae and may be cross-linked to 18S rRNA suggesting that they may directly contact the rRNA or form the binding site for proteins mediating RNA:RNA contacts or processing w21x. The loop sequences in the secondary structure models of the three fern U14 variants are not conserved in the models but it is possible that other secondary structures form to allow base-pairing interactions to occur as suggested for the Y-domain loop w20,21x. Such interactions would presumably involve the regions of primary sequence conservation seen among the three genes. In the absence of a system for analysing plant snoRNA function in plants, it will be of interest to analyse the ability of plant, and particularly fern, U14 P2 sequences in yeast–plant U14 hybrids. Due to the positions of the primers used in RTPCR, 5X and 3X terminal inverted repeats and box C would not be amplified in the fern clones, and box D and adjoining sequences are primer-derived. To examine the sequences at the 5X end of the fern U14s and to investigate their genomic organisation, the 5X half of the fern U14s and 5X flanking sequences were
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generated by PCR ŽFig. 3a. using the Genosys Vectorette II system Žsee Section 2. . The authenticity of this sequence was confirmed by PCR on total fern DNA using the internal U14 primer and a primer ŽANU145X . to the Vectorette-generated 5X flanking sequence. The flanking sequence contained the conserved box C sequence ŽUGAUGA. and a short region, P1, found only in plant U14s w23x ŽFig. 3b.. The estimated sizes of the fern U14s based on the position of the box C sequence and assumed position of box D are 190, 210 and 255 nt which correspond well to the sizes observed from Northern analyses ŽFig. 1.. On the basis of small sequence differences among the fern U14 variants, the 5X flanking sequence would have been derived from either the U14.2 or U14.3 gene. None of the established plant snRNA promoter elements involved in UsnRNA, U3snoRNA and MRP transcription Ž TATA-box; Upstream Sequence Element—USE wTCCCACATCGx and Monocot-Specific Promoter— MSP-element wRGCCCRx w29,30x. were present in the 5X flanking region. Computer simulated translation identified two potential protein coding sequences but neither contained homology to known polypeptides in databases. Finally, the flanking sequence did not appear to contain further U14 coding regions or those of other box CrD or box HrACA snoRNAs. Thus, either lower plant snRNA genes contain different promoter elements from higher plants or the fern U14s may be clustered as found for other plant U14s, or the presence of potential exon sequences may suggest that they are intronic. Acknowledgements This research was supported by the Scottish Office of Agriculture, Environment and Fisheries Department and Gene Shears. References w1x J.E. Dahlberg, E. Lund, in: M. Birnstiel ŽEd.., Structure and Function of Major and Minor Small Ribonucleoprotein, Springer-Verlag, Berlin, 1988, pp. 38–70.
w2x E.S. Maxwell, M.J. Fournier, Annu. Rev. Biochem. 35 Ž1995. 897–934. w3x B. Sollner-Webb, K.T. Tycowski, J.A. Steitz, in: R.A. Zimmerman, A.E. Dahlberg ŽEds.., Ribosomal RNA, CRC Press, Boca Raton, FL, 1995, pp. 469–490. w4x Z. Kiss-Laszlo, ´ ´ Y. Henry, J.-P. Bachellerie, M. CaizerguesFerrer, T. Kiss, Cell 85 Ž1996. 1077–1088. w5x M. Nicoloso, L.-H. Qu, B. Michot, J.-P. Bachellerie, J. Mol. Biol. 260 Ž1996. 178–195. w6x J. Ni, A.L. Tien, M.J. Fournier, Cell 89 Ž1997. 563–573. w7x P. Ganot, M.-L. Bortolin, T. Kiss, Cell 89 Ž1997. 799–809. w8x D. Tollervey, EMBO J. 6 Ž1987. 4169–4175. w9x J. Zagorski, D. Tollervey, M.J. Fournier, Mol. Cell Biol. 8 Ž1988. 3282–3290. w10x H.V. Li, J. Zagorski, M.J. Fournier, Mol. Cell Biol. 10 Ž1990. 1145–1152. w11x W.-Q. Liang, M.J. Fournier, Genes Dev. 9 Ž1995. 2433– 2443. w12x A. Jarmolowski, J. Zagorski, H.V. Li, M.J. Fournier, EMBO J. 9 Ž1990. 4503–4509. w13x G.M. Huang, A. Jarmolowski, J.C. Struck, M.J. Fournier, Mol. Cell Biol. 12 Ž1992. 4456–4463. w14x J. Liu, E.S. Maxwell, Nucleic Acids Res. 18 Ž1990. 6565– 6571. w15x D.J. Leader, J.F. Sanders, R. Waugh, P.J. Shaw, J.W.S. Brown, Nucleic Acids Res. 22 Ž1994. 5196–5200. w16x N.J. Watkins, R.D. Leverette, L. Xia, M.T. Andrews, E.S. Maxwell, RNA 2 Ž1996. 118–133. w17x L. Xia, N.J. Watkins, E.S. Maxwell, RNA 3 Ž1997. 17–26. w18x J. Li, M.J. Fournier, EMBO J. 11 Ž1992. 683–689. w19x A.G. Balakin, R.A. Lempicki, G.M. Huang, M.J. Fournier, J. Cell Biol. 269 Ž1994. 739–746. w20x D.A. Samarsky, G.S. Schneider, M.J. Fournier, Nucleic Acids Res. 24 Ž1996. 2059–2066. w21x J.P. Morrisey, D. Tollervey, Chromosoma 105 Ž1997. 515– 522. w22x T. Kiss, F. Solymosy, Nucleic Acids Res. 18 Ž1990. 1941– 1949. w23x D.J. Leader, S. Connelly, W. Filipowicz, J.W.S. Brown, Biochim. Biophys. Acta 1219 Ž1994. 145–147. w24x T. Kiss, C. Marshallsay, W. Filipowicz, Cell 65 Ž1992. 517–526. w25x D.J. Leader, G.P. Clark, J. Watters, A.F. Beven, P.J. Shaw, J.W.S. Brown, EMBO J. 16 Ž1997. 5742–5751. w26x P. Chomczynski, N. Sacchi, Anal. Biochem. 162 Ž1987. 156–159. w27x W.T.G. van der Ven, W. Powell, G. Ramsay, R. Waugh, Heredity 65 Ž1990. 329–342. w28x C.G. Simpson, R. Sinibaldi, J.W.S. Brown, Plant J. 2 Ž1992. 835–836. w29x P. Vankan, W. Filipowicz, EMBO J. 8 Ž1989. 3875–3882. w30x S. Connelly, C. Marshallsay, D. Leader, J.W.S. Brown, W. Filipowicz, Mol. Cell Biol. 14 Ž1994. 5910–5919.
U14snoRNAs of the fern, Asplenium nidus, contain large sequence insertions compared with those of higher plants David J. Leader 1, Gillian P. Clark, John W.S. Brown
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Department of Cell and Molecular Genetics, Scottish Crop Research Institute, InÕergowrie, Dundee DD2 5DA, UK Received 10 December 1997; accepted 30 January 1998
Abstract Northern analyses of U14snoRNAs in different plant species showed the expected hybridising band of approximately 120 nt in monocotyledonous and dicotyledonous angiosperms. In the lower plant, Bird’s nest fern Ž Asplenium nidus ., U14s were larger and three hybridising RNAs of approximately 190, 210 and 250 nt were observed. RT-PCR cloning of all three size variants using primers to the conserved 5X and 3X ends of higher plant U14snoRNAs showed large insertions in one of the plant-specific regions corresponding in position to the yeast U14-specific Y-domain. The insertions are pyrimidine-rich in their 5X halves and purine-rich in their 3X halves and are likely to be sequestered in stem structures consistent with the proposed model of U14snoRNA secondary structure. The 5X flanking regions of one of the fern U14 variants was generated by PCR and lacked classical plant snRNA promoter elements. q 1998 Elsevier Science B.V. Keywords: Small nucleolar RNA; U14; Pre-rRNA processing; ŽPteridiophyte.
1. Introduction Eukaryotic small nuclear RNAs Ž snRNAs. can be divided into two classes: spliceosomal snRNAs involved in pre-mRNA splicing and small nucleolar RNAs ŽsnoRNAs. involved in precursor ribosomal RNA Žpre-rRNA. processing and maturation, and ribosome biogenesis w1–7x. U14 belongs to a major family of snoRNAs called box CrD snoRNAs, is evolutionarily conserved and is required for growth and for 18S rRNA processing in yeast Ž Saccharomyces cereÕisiae. w8–11x. U14 contains se-
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Corresponding author: Fax: q44-1382-562426; E-mail: [email protected] 1 New address: Zeneca Agrochemicals, Jealott’s Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK.
quence elements, required for function and accumulation in yeast w12,13x which are largely conserved in animal and plant U14snoRNAs w14,15x. They include the box C and D sequences, 5X and 3X inverted repeats and two conserved elements Ž18S-A and 18S-B. complementary to regions of 18S rRNA. These elements base-pair with 18S rRNA w11x and the 18S-B element determines a ribose methylation site on 18S rRNA w4,5x. Deletion analyses of Xenopus U14s have demonstrated that a base-paired structure, involving the 5X and 3X terminal inverted repeats flanking boxes C and D, is needed for processing and accumulation of U14s w16,17x. A secondary structure model for yeast U14snoRNA also suggests that the ends of the molecule form a stem while the rest of the molecule is loosely structured w18,19x. Compared with vertebrate U14s, the larger yeast U14 contains a yeastspecific region Ž the Y-domain. which can form a
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stem-loop structure w18,19x. This region is essential for activity in yeast and when introduced into mouse U14, confers activity w11,12,20x. This domain is largely conserved in other yeast species and in all cases is capable of forming a stem-loop structure within which a number of bases in the loop sequence are conserved w20x. More recently, U14 has been shown to cross-link at many positions in the pre-rRNA and to other snoRNAs suggesting that its interactions in pre-rRNA processing are complex w21x. In particular, the Y-domain was essential for viability and cross-linked exclusively to the 35S pre-rRNA but not to mature rRNAs suggesting a role in pre-rRNA processing w21x. In plants, snoRNA genes isolated to date are U3snoRNA, 7-2rMRP snRNA, U14snoRNAs and a number of box CrD and one box HrACA snoRNAs ww15,22–25x; D.J.L. and J.W.S.B., unpublished resultsx. Plant U14snoRNAs showed extensive sequence homology to vertebrate and yeast U14s in containing boxes C and D, terminal inverted repeats and the two regions of complementarity to 18S rRNA sequences. Higher plant U14snoRNAs were intermediate in size Ž 117–120 nt. between animal U14snoRNA Žca. 87 nt. and yeast Ž130 nt. , and contained three plant-specific sequences Ž P1–P3. . These regions are absent in vertebrate U14snoRNAs, and the P2 region corresponds in position to the Y-domain of yeast w15x. In this paper, we have analysed U14snoRNAs from a number of higher plant species and from the Bird’s nest fern, Asplenium nidus. Ferns belong to the Pteridophyta, a lower division of the plant kingdom than the flowering plants ŽSpermatophyta. containing gymnosperms and angiosperms. Surprisingly, the fern U14snoRNAs were found to be much larger than their higher plant counterparts. Three major size variants contained additional sequences in the plant-specific P2 region and could form extensive stem structures.
2. Materials and methods 2.1. Materials Restriction enzymes, Taq DNA polymerase and T4 DNA ligase were purchased from Boehringer Ž Mannheim.. Hybond Nq filters were obtained from
Amersham. MMLV reverse transcriptase and RQI RNase-free, DNase were obtained from Gibco. DNA sequencing was carried out with Sequenase II Ž United States Biochemicals. or using the Cycle Sequencing System from BRL Life, on an ABI373 automatic sequencer. The Vectorette II system was purchased from Genosys ŽCambridge, UK. . 2.2. Oligonucleotides U14-2Žy. 5X-CATATGATCAŽGrA.ACATCCAAGGAAGG-3X ; PLU14RT-1 CGGAAGCTTNNAAGGCTTGTTCTC-3X ; F18S 5X-CTCAAGCTTAGG C G G C C A C TG C G A A TG -3 X ; FU 14Ig1 5 X CGGAAGCTTNNGAGAAACAAGCCTT-3X ; Vectorette universal primer 5X-CAACGTGGATCCGAATTCAAGCTTC-3X ; Vectorette nested primer 5X-ACGTGGATCCGAATTCAAGCTTCATG-3X ; ANU145X Ž 5X-CACCGCATGCAGTGTGAAACGATGGCTAG-3X . . The sequences of restriction sites used in cloning are underlined. 2.3. RNA and DNA extraction RNA was isolated from leaf material of maize, barley, potato, tobacco, haricot bean, cucumber, and from fully expanded fronds of asparagus and the Bird’s Nest fern by the guanidinium isothiocyanate method w26x. RNA for use in RT-PCR was DNased twice with RQ1 RNase-free DNase. DNA was extracted from leaf material of the fern by the method of van der Ven et al. w27x. 2.4. Northern blotting and RT-PCR Ten micrograms of total RNA was separated on a denaturing 6% polyacrylamide gel and electroblotted onto Hybond Nq. Blots were hybridised with a probe consisting of the DNA fragment isolated from the plasmid pgMU14.1b which contains the maize U14.1b gene w15x. The probe was labelled with a-w 32 PxdCTP by random priming. RT-PCR reaction conditions were as described w28x. First strand cDNA synthesis was carried out with primer U14-2Žy., complementary to the 18S-B region conserved at the 3X end of all U14s Žw14x; Figs. 2 and 3.. U14 cDNA sequences were amplified by subsequent PCR with U14-2Ž y . and primer
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PLU14RT-1, homologous to a conserved sequence upstream of the 18S-A sequence ŽFigs. 2 and 3.. Amplified PCR products were digested with Bcl I and HindIII, purified by polyacrylamide gel electrophoresis and subcloned into pGEM3ZfŽq. ŽPromega. prior to sequencing. 2.5. Determination of 5X flanking sequences The 5X flanking sequence of a fern U14 gene was generated by PCR using the Vectorette II system according to the manufacturer’s instructions. Briefly, total fern DNA was digested with FspI or Sau3A and appropriate double-stranded adapters ligated to the DNA fragments. The first round of PCR amplification used the vectorette universal primer and the fern-specific primer F18S ŽFig. 3. , complementary to a sequence including the 18S-A region. The products of this reaction were used as the substrate for subsequent doubly nested PCR reactions with the vectorette nested primer and primer FU14Ig1 which is largely complementary to PLU14RT-1 used in the RT-PCR reactions. PCR products were either sequenced directly or digested with HindIII, purified as described above and cloned into pGEM3ZfŽq.. To confirm the identity of the putative 5X flanking sequence obtained by the Vectorette method, the primer ANU145X was designed to a region of the flanking sequence close to the vectorette ligation site Ž Fig. 3.. This primer was used in PCR reactions with A. nidus DNA and the primer U14-2Žy. . PCR products were sequenced directly or subcloned into pGEM3ZfŽq. following digestion with Bcl I and SphI and gel purification.
Fig. 1. Northern analysis of U14snoRNAs from different plant species. Total RNA from the monocotyledonous species: maize Žlane 1., barley Žlane 2. and asparagus Žlane 3., from the dicotyledonous species: potato Žlane 4., tomato Žlane 5., bean Žlane 6. and cucumber Žlane 7., and from the fern, A. nidus Žlane 8. was hybridised with a probe to maize U14.1b w15x. Markers— w 32 Px-end labelled HindIII-digested f X174 DNA.
To investigate the exceptionally large sizes of the fern U14snoRNAs Ž as much as 2–3 times the size of yeast, animal and higher plant U14s. , RT-PCR was carried out on fern RNA using primers designed to conserved regions of higher plant U14snoRNAs Ž Fig. 2.. RT-PCR products of three different sizes were
3. Results and discussion The sizes of U14snoRNAs in a number of plant species were investigated by Northern analysis Ž Fig. 1.. Total RNA of the monocot. species, maize, barley and asparagus ŽFig. 1, lanes 1–3. and the dicot. species potato, tobacco, bean and cucumber Ž Fig. 1, lanes 4–7. all showed a major hybridising band of approximately 120 nt when probed with the maize U14.1b gene. In contrast, total RNA of the fern, A. nidus, showed three bands of approximately 190, 210 and 250 nt ŽFig. 1, lane 8..
Fig. 2. Schematic diagrams of mouse, yeast and higher plant U14s, and fern U14 RT-PCR products. Box CrD sequences are indicated, yeast-specific sequences are shown by open boxes and plant-specific sequences by hatched boxes. Primers to regions conserved in U14s from plants, animals and yeast which were used in RT-PCR analyses on fern leaf total RNA are shown as arrows on the higher plant U14. The three fern U14 RT-PCR products contain additional sequences Žstriped boxes. within the P2 region.
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obtained ŽFig. 2. and subcloned into pGEM3ZfŽ q. prior to sequencing. Control reactions with no reverse transcriptase gave no PCR products Žresults not shown. demonstrating that the RT-PCR products were RNA-derived. A sequence alignment of the three expressed fern U14snoRNA size variants with the higher plant U14snoRNA consensus is shown in Fig. 3. Extensive sequence homology clearly exists between the 5X and 3X halves of the fern U14snoRNAs and higher plant U14snoRNAs Ž Fig. 3b and d.. Additional sequences of 71, 91 and 135 nt were present in the fern U14.2, U14.3 and U14.4 RT-PCR clones respectively Ž Fig. 3c.. These sequences were in the plant-specific region P2. To demonstrate that the size
variants were the products of three different genes, PCR was carried out on genomic fern DNA using the same primers as above, and all three size variants were obtained Žresults not shown. . The presence of three U14 variants in fern is of interest since higher plants and animals contain multiple genes of similar size and which differ by at most a few nucleotides in sequence. The additional sequences contained within the three fern U14 gene variants are related to one another in regions of primary sequence Ž see alignment in Fig. 3c.. Of particular note is that the 5X halves of these regions are pyrimidine-rich while the 3X halves are purine-rich. Secondary structure predictions using
Fig. 3. Alignment of the three fern U14 sequences with those of yeast Ž S. cereÕisiae ., mouse and maize. The sequences have been separated into different sections Ža–d. to better illustrate the conservation of the 5X and 3X portions of all of the U14s. The three plant-specific regions, P1, P2 and P3 w15x are marked below the maize sequence and the conserved 18S-A and 18S-B, and yeast-specific Y-domain is shown above the yeast sequence. Inverted repeats ŽIR., boxes C and D and the positions of certain oligonucleotide primers are indicated. The 5X and 3X regions of the fern U14s which show conservation with other U14s are aligned in Žb. and Žd.. The additional fern sequences in the P2 region are shown separately Žc. from the conserved 5X and 3X regions. The 5X flanking sequence is that generated by the Vectorette PCR system Ža..
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MFOLD ŽUWGCG. fold each of the regions into elongated stem structures of different sizes Ž Fig. 4. . MFOLD predictions of higher plant U14s Ž e.g., potato and maize. suggest formation of a short stem-loop structure where the P2 region forms the upper half of
Fig. 4. Secondary structure predictions ŽUWGCG-MFOLD. for a region of potato, maize and fern U14s. The region consisting of P2 and immediately adjacent sequences can potentially form stem-loops. The P2 region is indicated by the thin line in the maize and potato structures and forms the upper part of the stem and loop. Sequence variation in maize U14 variants are labelled. The heavy line across all five structures indicates a sequence, including part of P2, conserved at the base of each stem.
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the stem and the loop sequence Ž Fig. 4.. Although the higher plant P2 regions can also be modelled to form loops of 10–12 bases w20x, alternatively they may form more extensive stems with only small loops ŽFig. 4.. This stem structure is almost completely conserved in the three fern U14 sequences including the side bulge in the 3X half of the stem, consisting of two uridines ŽFig. 4.. However, in place of the loop sequence found in the short stem-loops in higher plant U14s, the additional sequences in the fern U14s form long stem structures. The predicted maximum free energies of the fern U14s were DG s y44.6 kcalrmole Ž U14.2 ., DG s y50.5 kcalrmole Ž U14.3 . and DG s y66.2 kcalrmole Ž U14.4 ., compared to DG s y8.8–9.2 kcalrmole for the potato or maize U14s. Assuming that all three variants are functional in fern pre-rRNA processing, the size and sequences of the stem structures may not be important to function as large deletions or additions are tolerated. This may suggest that the secondary structures act to position and hold the important regions of the U14s Ž e.g., 18S-A and 18S-B. in the correct conformation for activity, as suggested for the yeast Y-domain stemloop w19x. However, in yeast nucleotides within the Y-domain loop are highly conserved, are required for function in S. cereÕisiae and may be cross-linked to 18S rRNA suggesting that they may directly contact the rRNA or form the binding site for proteins mediating RNA:RNA contacts or processing w21x. The loop sequences in the secondary structure models of the three fern U14 variants are not conserved in the models but it is possible that other secondary structures form to allow base-pairing interactions to occur as suggested for the Y-domain loop w20,21x. Such interactions would presumably involve the regions of primary sequence conservation seen among the three genes. In the absence of a system for analysing plant snoRNA function in plants, it will be of interest to analyse the ability of plant, and particularly fern, U14 P2 sequences in yeast–plant U14 hybrids. Due to the positions of the primers used in RTPCR, 5X and 3X terminal inverted repeats and box C would not be amplified in the fern clones, and box D and adjoining sequences are primer-derived. To examine the sequences at the 5X end of the fern U14s and to investigate their genomic organisation, the 5X half of the fern U14s and 5X flanking sequences were
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generated by PCR ŽFig. 3a. using the Genosys Vectorette II system Žsee Section 2. . The authenticity of this sequence was confirmed by PCR on total fern DNA using the internal U14 primer and a primer ŽANU145X . to the Vectorette-generated 5X flanking sequence. The flanking sequence contained the conserved box C sequence ŽUGAUGA. and a short region, P1, found only in plant U14s w23x ŽFig. 3b.. The estimated sizes of the fern U14s based on the position of the box C sequence and assumed position of box D are 190, 210 and 255 nt which correspond well to the sizes observed from Northern analyses ŽFig. 1.. On the basis of small sequence differences among the fern U14 variants, the 5X flanking sequence would have been derived from either the U14.2 or U14.3 gene. None of the established plant snRNA promoter elements involved in UsnRNA, U3snoRNA and MRP transcription Ž TATA-box; Upstream Sequence Element—USE wTCCCACATCGx and Monocot-Specific Promoter— MSP-element wRGCCCRx w29,30x. were present in the 5X flanking region. Computer simulated translation identified two potential protein coding sequences but neither contained homology to known polypeptides in databases. Finally, the flanking sequence did not appear to contain further U14 coding regions or those of other box CrD or box HrACA snoRNAs. Thus, either lower plant snRNA genes contain different promoter elements from higher plants or the fern U14s may be clustered as found for other plant U14s, or the presence of potential exon sequences may suggest that they are intronic. Acknowledgements This research was supported by the Scottish Office of Agriculture, Environment and Fisheries Department and Gene Shears. References w1x J.E. Dahlberg, E. Lund, in: M. Birnstiel ŽEd.., Structure and Function of Major and Minor Small Ribonucleoprotein, Springer-Verlag, Berlin, 1988, pp. 38–70.
w2x E.S. Maxwell, M.J. Fournier, Annu. Rev. Biochem. 35 Ž1995. 897–934. w3x B. Sollner-Webb, K.T. Tycowski, J.A. Steitz, in: R.A. Zimmerman, A.E. Dahlberg ŽEds.., Ribosomal RNA, CRC Press, Boca Raton, FL, 1995, pp. 469–490. w4x Z. Kiss-Laszlo, ´ ´ Y. Henry, J.-P. Bachellerie, M. CaizerguesFerrer, T. Kiss, Cell 85 Ž1996. 1077–1088. w5x M. Nicoloso, L.-H. Qu, B. Michot, J.-P. Bachellerie, J. Mol. Biol. 260 Ž1996. 178–195. w6x J. Ni, A.L. Tien, M.J. Fournier, Cell 89 Ž1997. 563–573. w7x P. Ganot, M.-L. Bortolin, T. Kiss, Cell 89 Ž1997. 799–809. w8x D. Tollervey, EMBO J. 6 Ž1987. 4169–4175. w9x J. Zagorski, D. Tollervey, M.J. Fournier, Mol. Cell Biol. 8 Ž1988. 3282–3290. w10x H.V. Li, J. Zagorski, M.J. Fournier, Mol. Cell Biol. 10 Ž1990. 1145–1152. w11x W.-Q. Liang, M.J. Fournier, Genes Dev. 9 Ž1995. 2433– 2443. w12x A. Jarmolowski, J. Zagorski, H.V. Li, M.J. Fournier, EMBO J. 9 Ž1990. 4503–4509. w13x G.M. Huang, A. Jarmolowski, J.C. Struck, M.J. Fournier, Mol. Cell Biol. 12 Ž1992. 4456–4463. w14x J. Liu, E.S. Maxwell, Nucleic Acids Res. 18 Ž1990. 6565– 6571. w15x D.J. Leader, J.F. Sanders, R. Waugh, P.J. Shaw, J.W.S. Brown, Nucleic Acids Res. 22 Ž1994. 5196–5200. w16x N.J. Watkins, R.D. Leverette, L. Xia, M.T. Andrews, E.S. Maxwell, RNA 2 Ž1996. 118–133. w17x L. Xia, N.J. Watkins, E.S. Maxwell, RNA 3 Ž1997. 17–26. w18x J. Li, M.J. Fournier, EMBO J. 11 Ž1992. 683–689. w19x A.G. Balakin, R.A. Lempicki, G.M. Huang, M.J. Fournier, J. Cell Biol. 269 Ž1994. 739–746. w20x D.A. Samarsky, G.S. Schneider, M.J. Fournier, Nucleic Acids Res. 24 Ž1996. 2059–2066. w21x J.P. Morrisey, D. Tollervey, Chromosoma 105 Ž1997. 515– 522. w22x T. Kiss, F. Solymosy, Nucleic Acids Res. 18 Ž1990. 1941– 1949. w23x D.J. Leader, S. Connelly, W. Filipowicz, J.W.S. Brown, Biochim. Biophys. Acta 1219 Ž1994. 145–147. w24x T. Kiss, C. Marshallsay, W. Filipowicz, Cell 65 Ž1992. 517–526. w25x D.J. Leader, G.P. Clark, J. Watters, A.F. Beven, P.J. Shaw, J.W.S. Brown, EMBO J. 16 Ž1997. 5742–5751. w26x P. Chomczynski, N. Sacchi, Anal. Biochem. 162 Ž1987. 156–159. w27x W.T.G. van der Ven, W. Powell, G. Ramsay, R. Waugh, Heredity 65 Ž1990. 329–342. w28x C.G. Simpson, R. Sinibaldi, J.W.S. Brown, Plant J. 2 Ž1992. 835–836. w29x P. Vankan, W. Filipowicz, EMBO J. 8 Ž1989. 3875–3882. w30x S. Connelly, C. Marshallsay, D. Leader, J.W.S. Brown, W. Filipowicz, Mol. Cell Biol. 14 Ž1994. 5910–5919.