Abstract
Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions.
Full Text
The Full Text of this article is available as a PDF (5.9 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adema G. J., Bovenberg R. A., Jansz H. S., Baas P. D. Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA. Nucleic Acids Res. 1988 Oct 25;16(20):9513–9526. doi: 10.1093/nar/16.20.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Arning S., Grüter P., Bilbe G., Krämer A. Mammalian splicing factor SF1 is encoded by variant cDNAs and binds to RNA. RNA. 1996 Aug;2(8):794–810. [PMC free article] [PubMed] [Google Scholar]
- Arnott S., Hukins D. W., Dover S. D., Fuller W., Hodgson A. R. Structures of synthetic polynucleotides in the A-RNA and A'-RNA conformations: x-ray diffraction analyses of the molecular conformations of polyadenylic acid--polyuridylic acid and polyinosinic acid--polycytidylic acid. J Mol Biol. 1973 Dec 5;81(2):107–122. doi: 10.1016/0022-2836(73)90183-6. [DOI] [PubMed] [Google Scholar]
- Berglund J. A., Chua K., Abovich N., Reed R., Rosbash M. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell. 1997 May 30;89(5):781–787. doi: 10.1016/s0092-8674(00)80261-5. [DOI] [PubMed] [Google Scholar]
- Borer P. N., Lin Y., Wang S., Roggenbuck M. W., Gott J. M., Uhlenbeck O. C., Pelczer I. Proton NMR and structural features of a 24-nucleotide RNA hairpin. Biochemistry. 1995 May 16;34(19):6488–6503. doi: 10.1021/bi00019a030. [DOI] [PubMed] [Google Scholar]
- Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
- Cate J. H., Gooding A. R., Podell E., Zhou K., Golden B. L., Kundrot C. E., Cech T. R., Doudna J. A. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science. 1996 Sep 20;273(5282):1678–1685. doi: 10.1126/science.273.5282.1678. [DOI] [PubMed] [Google Scholar]
- Cate J. H., Hanna R. L., Doudna J. A. A magnesium ion core at the heart of a ribozyme domain. Nat Struct Biol. 1997 Jul;4(7):553–558. doi: 10.1038/nsb0797-553. [DOI] [PubMed] [Google Scholar]
- Chiara M. D., Gozani O., Bennett M., Champion-Arnaud P., Palandjian L., Reed R. Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly. Mol Cell Biol. 1996 Jul;16(7):3317–3326. doi: 10.1128/mcb.16.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Costa M., Michel F. Frequent use of the same tertiary motif by self-folding RNAs. EMBO J. 1995 Mar 15;14(6):1276–1285. doi: 10.1002/j.1460-2075.1995.tb07111.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cowtan K., Main P. Miscellaneous algorithms for density modification. Acta Crystallogr D Biol Crystallogr. 1998 Jul 1;54(Pt 4):487–493. doi: 10.1107/s0907444997011980. [DOI] [PubMed] [Google Scholar]
- Dickerson R. E. DNA bending: the prevalence of kinkiness and the virtues of normality. Nucleic Acids Res. 1998 Apr 15;26(8):1906–1926. doi: 10.1093/nar/26.8.1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dyhr-Mikkelsen H., Kjems J. Inefficient spliceosome assembly and abnormal branch site selection in splicing of an HIV-1 transcript in vitro. J Biol Chem. 1995 Oct 13;270(41):24060–24066. doi: 10.1074/jbc.270.41.24060. [DOI] [PubMed] [Google Scholar]
- Ennifar E., Yusupov M., Walter P., Marquet R., Ehresmann B., Ehresmann C., Dumas P. The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges. Structure. 1999 Nov 15;7(11):1439–1449. doi: 10.1016/s0969-2126(00)80033-7. [DOI] [PubMed] [Google Scholar]
- Ferré-D'Amaré A. R., Doudna J. A. RNA folds: insights from recent crystal structures. Annu Rev Biophys Biomol Struct. 1999;28:57–73. doi: 10.1146/annurev.biophys.28.1.57. [DOI] [PubMed] [Google Scholar]
- Greenbaum N. L., Radhakrishnan I., Patel D. J., Hirsh D. Solution structure of the donor site of a trans-splicing RNA. Structure. 1996 Jun 15;4(6):725–733. doi: 10.1016/s0969-2126(96)00078-0. [DOI] [PubMed] [Google Scholar]
- Gutell R. R., Weiser B., Woese C. R., Noller H. F. Comparative anatomy of 16-S-like ribosomal RNA. Prog Nucleic Acid Res Mol Biol. 1985;32:155–216. doi: 10.1016/s0079-6603(08)60348-7. [DOI] [PubMed] [Google Scholar]
- Hartmuth K., Barta A. Unusual branch point selection in processing of human growth hormone pre-mRNA. Mol Cell Biol. 1988 May;8(5):2011–2020. doi: 10.1128/mcb.8.5.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
- Laing L. G., Gluick T. C., Draper D. E. Stabilization of RNA structure by Mg ions. Specific and non-specific effects. J Mol Biol. 1994 Apr 15;237(5):577–587. doi: 10.1006/jmbi.1994.1256. [DOI] [PubMed] [Google Scholar]
- MacMillan A. M., Query C. C., Allerson C. R., Chen S., Verdine G. L., Sharp P. A. Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 1994 Dec 15;8(24):3008–3020. doi: 10.1101/gad.8.24.3008. [DOI] [PubMed] [Google Scholar]
- Parker R., Siliciano P. G., Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell. 1987 Apr 24;49(2):229–239. doi: 10.1016/0092-8674(87)90564-2. [DOI] [PubMed] [Google Scholar]
- Perrotta A. T., Nikiforova O., Been M. D. A conserved bulged adenosine in a peripheral duplex of the antigenomic HDV self-cleaving RNA reduceskinetic trapping of inactive conformations. Nucleic Acids Res. 1999 Feb 1;27(3):795–802. doi: 10.1093/nar/27.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pley H. W., Flaherty K. M., McKay D. B. Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. Nature. 1994 Nov 3;372(6501):111–113. doi: 10.1038/372111a0. [DOI] [PubMed] [Google Scholar]
- Podar M., Perlman P. S. Photocrosslinking of 4-thio uracil-containing RNAs supports a side-by-side arrangement of domains 5 and 6 of a group II intron. RNA. 1999 Feb;5(2):318–329. doi: 10.1017/s1355838299981724. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Portmann S., Grimm S., Workman C., Usman N., Egli M. Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage. Chem Biol. 1996 Mar;3(3):173–184. doi: 10.1016/s1074-5521(96)90260-4. [DOI] [PubMed] [Google Scholar]
- Qin P. Z., Pyle A. M. The architectural organization and mechanistic function of group II intron structural elements. Curr Opin Struct Biol. 1998 Jun;8(3):301–308. doi: 10.1016/s0959-440x(98)80062-6. [DOI] [PubMed] [Google Scholar]
- Query C. C., Moore M. J., Sharp P. A. Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model. Genes Dev. 1994 Mar 1;8(5):587–597. doi: 10.1101/gad.8.5.587. [DOI] [PubMed] [Google Scholar]
- Query C. C., Strobel S. A., Sharp P. A. Three recognition events at the branch-site adenine. EMBO J. 1996 Mar 15;15(6):1392–1402. [PMC free article] [PubMed] [Google Scholar]
- Ruskin B., Pikielny C. W., Rosbash M., Green M. R. Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2022–2026. doi: 10.1073/pnas.83.7.2022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
- Rutz B., Séraphin B. Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly. RNA. 1999 Jun;5(6):819–831. doi: 10.1017/s1355838299982286. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith J. S., Nikonowicz E. P. NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein. Biochemistry. 1998 Sep 29;37(39):13486–13498. doi: 10.1021/bi981558a. [DOI] [PubMed] [Google Scholar]
- Spingola M., Grate L., Haussler D., Ares M., Jr Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae. RNA. 1999 Feb;5(2):221–234. doi: 10.1017/s1355838299981682. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Staley J. P., Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998 Feb 6;92(3):315–326. doi: 10.1016/s0092-8674(00)80925-3. [DOI] [PubMed] [Google Scholar]
- Su L., Chen L., Egli M., Berger J. M., Rich A. Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot. Nat Struct Biol. 1999 Mar;6(3):285–292. doi: 10.1038/6722. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thiviyanathan V., Guliaev A. B., Leontis N. B., Gorenstein D. G. Solution conformation of a bulged adenosine base in an RNA duplex by relaxation matrix refinement. J Mol Biol. 2000 Jul 28;300(5):1143–1154. doi: 10.1006/jmbi.2000.3931. [DOI] [PubMed] [Google Scholar]
- Valcárcel J., Gaur R. K., Singh R., Green M. R. Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected]. Science. 1996 Sep 20;273(5282):1706–1709. doi: 10.1126/science.273.5282.1706. [DOI] [PubMed] [Google Scholar]
- Valegård K., Murray J. B., Stockley P. G., Stonehouse N. J., Liljas L. Crystal structure of an RNA bacteriophage coat protein-operator complex. Nature. 1994 Oct 13;371(6498):623–626. doi: 10.1038/371623a0. [DOI] [PubMed] [Google Scholar]
- Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
- Zabolotny J. M., Krummenacher C., Fraser N. W. The herpes simplex virus type 1 2.0-kilobase latency-associated transcript is a stable intron which branches at a guanosine. J Virol. 1997 Jun;71(6):4199–4208. doi: 10.1128/jvi.71.6.4199-4208.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhuang Y., Weiner A. M. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 1989 Oct;3(10):1545–1552. doi: 10.1101/gad.3.10.1545. [DOI] [PubMed] [Google Scholar]