Nucleotide sequence of U5 RNA from a green alga, Chlamydomonas ...

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Sep 9, 1992 - conserved sequence, G39C40 C41 U42 U43 U44 U45 A46 C47 of loop A, probably involved, by base-pairing with exon sequences,.
k. 1992 Oxford University Press

5224 Nucleic Acids Research, Vol. 20, No. 19

Nucleotide sequence of U5 RNA from Chlamydomonas reinhardtii

alga,

a green

Gabor Jakab, Mihaly Kis and Ferenc Solymosy* Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary EMBL accession no. X67000

Submitted September 9, 1992 From a total RNA extract of haploid vegetative cells of the green alga Chlamydomonas reinhardtii CW 15 strain, we isolated, by the use of anti-m3G antibodies and polyacrylamide gel electrophoresis under denaturing conditions, U5 RNA, one of the major small nuclear RNAs (snRNAs) and an indispensable constituent [cf. (1)] of the spliceosome, the site of pre-mRNA splicing. Consistent with the position of green algae at an ancient branching point of the evolutionary tree (2), the C. reinhardtii US RNA (Figure 1) exhibits an alloy of structural features, some of them reflecting indispensable functional requirements (1) and some others preserved during evolution in certain phyla and lost in certain others (2): (1) (i) An m3G cap structure at the 5' terminus and intramolecular nucleoside modifications in the 5' but not the 3' half of the molecule, (ii) a phylogenetically very highly conserved secondary structure [cf. (4, 5)] encompassing loops A and B as well as internal loops 1 and 2 (ILl and IL2), IL2 being involved in the binding of at least one or a few of the U5-specific proteins in the HeLa 20S U5 RN-P (6), (iii) an Sm antigen-binding site equivalent (A88 to G96) which, in the presence of ILI (I.W.Mattaj, personal communication) is involved in the binding of the common core snRNP proteins in the lOS U5 RNP [cf. (1)], (iv) the phylogenetically absolutely conserved sequence, G39 C40 C41 U42 U43 U44 U45 A46 C47 of loop A, probably involved, by base-pairing with exon sequences, in both transesterification reactions during pre-mRNA splicing (7). (2) (i) A length of 111 nt residues, similar to that of U5 RNA from the lower eukaryotes Tetrahymena (112 nt) and a dinoflagellate (107 nt) and unlike that of U5 RNAs from metazoa ( - 116 nt) and from higher plants (120-126 nt) [cf. (4,5)], (ii) distribution of intramolecular nucleoside modifications according to a pattern found in a lower eukaryotic dinoflagellate U5 RNA [(8) and cf. (5)], (iii) 2'-0-methylation of N1 and N2 (Am1 and Um2) in the molecule, in contrast to higher plant U5 RNAs [cf. (5)], (iv) the plant-specific sequence A82 U83 A84 [cf. (5)] upstream of the Sm antigen-binding site equivalent, and (v) the unique structure of the Sm antigen-binding site: in the C. reinhardtii US RNA, at variance with the consensus sequence RR(U)3-5RR, the stretch of U residues (interrupted by a C residue) is flanked on both sides by only one purine residue, rather than two (one of the 5' flanking purine residues is absent also in US RNAs from the budding yeast, Saccharomyces cerevisiae, the dicot, Arabidopsis thaliana and the monocot, Zea mays [cf.

(4) and (5)]). *

To whom correspondence should be addressed

ACKNOWLEDGEMENTS Thanks are due to Dr C. F.Beck for the C.reinhardtii CW 15 strain and to Professor R. Liihrman for the anti-m3G antibodies. This work was supported by a grant (OTKA 1/3 891/1991) from the Hungarian National Science Foundation.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Liihrann,R. et al. (1990) Biochim. Biophys. Acta 1087, 265-292. Hori,H. and Osawa,S. (1987) Mol. Biol. Evol. 4, 445-472. Peattie,D.A. (1979) Proc. Natl. Acad. Sci. USA 76, 1760-1764. Guthrie,C. and Patterson,B. (1988) Annu. Rev. Genet. 22, 387-419. Solymosy,F. and Polldk,T. (1992) Cnt. Rev. Plant Sci. (in press). Bach,M. and Liihrnann,R. (1991) Biochim. Biophys. Acta 1088, 139-143. Newman,A.J. and Norman,C. (1992) Cell 68, 743-754. Liu,M.-H. et al. (1984) Nucleic Acids Res. 12, 1529-1542.

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reinhardtii. Stems are denoted by roman numerals, terminal loops by capitals. Stem I is interrupted by internal loops 1 and 2 (ILl and IL2) and is divided accordingly into sub-stems marked by lower case letters complementing the roman numerals. The primary structure of the U5 RNA was determined by chemical sequencing (3) and was verified by dideoxy sequencing of its cDNA. Chlm)domonas