Bimolecular Exon Ligation by the Human Spliceosome
10.1126/science.276.5319.1712
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Intron excision is an essential step in eukaryotic gene expression, but the molecular mechanisms by which the spliceosome accurately identifies splice sites in nuclear precursors to messenger RNAs (pre-mRNAs) are not well understood. A bimolecular assay for the second step of splicing has now revealed that exon ligation by the human spliceosome does not require covalent attachment of a 3′ splice site to the branch site. Furthermore, accurate definition of the 3′ splice site in this system is independent of either a covalently attached polypyrimidine tract or specific 3′ exon sequences. Rather, in this system 3′ splice site selection apparently occurs with a 5′ → 3′ directionality.

Bibliography

Anderson, K., & Moore, M. J. (1997). Bimolecular Exon Ligation by the Human Spliceosome. Science, 276(5319), 1712–1716.

Authors 2
  1. Karin Anderson (first)
  2. Melissa J. Moore (additional)
References 60 Referenced 52
  1. M. J. Moore C. C. Query P. A. Sharp in The RNA World R. F. Gestland and J. F. Atkins Eds. (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1993) pp. 303–357;
  2. Nilsen T. W., Cell 78, 1 (1994); (10.1016/0092-8674(94)90563-0) / Cell by Nilsen T. W. (1994)
  3. ; A. Krämer in Pre-mRNA Processing A. I. Lamond Ed. (Landes Austin TX 1995) pp. 35–64. (10.1007/978-3-662-22325-3_3)
  4. 10.1126/science.6206566
  5. Moore M. J., Sharp P. A., Nature 365, 364 (1993); (10.1038/365364a0) / Nature by Moore M. J. (1993)
  6. Maschhoff K. L., Padgett R. A., Nucleic Acids Res. 21, 5456 (1993). (10.1093/nar/21.23.5456) / Nucleic Acids Res. by Maschhoff K. L. (1993)
  7. Ruskin B., Krainer A. R., Maniatis T., Green M. R., Cell 38, 317 (1984). (10.1016/0092-8674(84)90553-1) / Cell by Ruskin B. (1984)
  8. In this report the term “3′ splice site” refers to the site of exon ligation only in contrast to many other papers in which this same term is used to refer collectively to all of the sequences near the 3′ end of an intron (that is the branch site PPT and site of exon ligation).
  9. 10.1016/0076-6879(90)83018-5
  10. Roscigno R. F., Weiner M., Garcia-Blanco M. A., J. Biol. Chem. 268, 11222 (1993). (10.1016/S0021-9258(18)82114-7) / J. Biol. Chem. by Roscigno R. F. (1993)
  11. and references therein.
  12. 10.1016/S0959-437X(96)80053-0
  13. Umen J., Guthrie C., RNA 1, 869 (1995). / RNA by Umen J. (1995)
  14. All examples of alternative 3′ splice site use in metazoans that have been investigated are actually attributable to alternative branch site use [
  15. Smith C. W. J., Patton J. G., Nadal-Ginard B., Annu. Rev. Genet. 23, 527 (1989); (10.1146/annurev.ge.23.120189.002523) / Annu. Rev. Genet. by Smith C. W. J. (1989)
  16. McKeown M., Annu. Rev. Cell Biol. 8, 133 (1992)]. (10.1146/annurev.cb.08.110192.001025) / Annu. Rev. Cell Biol. by McKeown M. (1992)
  17. Patterson B., Guthrie C., Cell 64, 181 (1991); (10.1016/0092-8674(91)90219-O) / Cell by Patterson B. (1991)
  18. 10.1101/gad.9.7.855
  19. 10.1101/gad.3.12b.2113
  20. Smith C. W. J., Porro E. B., Patton J. G., Nadal-Ginard B., Nature 342, 243 (1989); (10.1038/342243a0) / Nature by Smith C. W. J. (1989)
  21. Smith C. W. J., Chu T. T., Nadal-Ginard B., Mol. Cell. Biol. 13, 4939 (1993). / Mol. Cell. Biol. by Smith C. W. J. (1993)
  22. Solnick D., Cell 42, 157 (1985); (10.1016/S0092-8674(85)80111-2) / Cell by Solnick D. (1985)
  23. ; M. M. Konarska R. A. Padgett P. A. Sharp ibid. p. 165; J. P. Bruzik and T. Maniatis Nature 360 692 (1992); (10.1038/360692a0)
  24. Ghetti A., Abelson J. N., Proc. Natl. Acad. Sci. U.S.A. 92, 11461 (1995); (10.1073/pnas.92.25.11461) / Proc. Natl. Acad. Sci. U.S.A. by Ghetti A. (1995)
  25. Pasman Z., Garcia-Blanco M. A., Nucleic Acids Res. 24, 1638 (1996). (10.1093/nar/24.9.1638) / Nucleic Acids Res. by Pasman Z. (1996)
  26. Chiara M. D., Reed R., Nature 375, 510 (1995). (10.1038/375510a0) / Nature by Chiara M. D. (1995)
  27. Konforti B. B., Konarska M. A., RNA 1, 815 (1995). / RNA by Konforti B. B. (1995)
  28. Bruzik J. P., Maniatis T., Proc. Natl. Acad. Sci. U.S.A. 92, 7056 (1995). (10.1073/pnas.92.15.7056) / Proc. Natl. Acad. Sci. U.S.A. by Bruzik J. P. (1995)
  29. Nilsen T. W., Infect. Agents Dis. 1, 212 (1992); / Infect. Agents Dis. by Nilsen T. W. (1992)
  30. ; Mol. Biochem. Parasitol. 73 1 (1995). (10.1016/0166-6851(95)00111-D)
  31. K. Anderson and M. J. Moore unpublished data.
  32. Similar results have been observed for other such truncated splicing substrates [
  33. 10.1016/S0092-8674(85)80131-8
  34. Ruskin B., Green M. R., Nature 317, 732 (1985)]. (10.1038/317732a0) / Nature by Ruskin B. (1985)
  35. Ramchatesingh J., Zahler A. M., Neugebauer K. M., Roth M. B., Cooper T. A., Mol. Cell. Biol. 15, 4898 (1995). (10.1128/MCB.15.9.4898) / Mol. Cell. Biol. by Ramchatesingh J. (1995)
  36. Fu X. D., RNA 1, 663 (1995); / RNA by Fu X. D. (1995)
  37. 10.1146/annurev.bi.65.070196.002055
  38. Manley J. L., Tacke R., Genes Dev. 10, 1569 (1996); (10.1101/gad.10.13.1569) / Genes Dev. by Manley J. L. (1996)
  39. Valcarcel J., Green M. R., Trends Biochem. Sci. 21, 296 (1996). (10.1016/S0968-0004(96)10039-6) / Trends Biochem. Sci. by Valcarcel J. (1996)
  40. The ligated exon products from both 3′ substrates were purified by electrophoresis and subjected to RT-PCR. The RT-PCR products were then cloned and sequenced. Correct splice junction usage was also observed on direct sequencing of the AdML/TNT RT-PCR product.
  41. All splicing gels were quantitated with a Molecular Dynamics PhosphorImager. Even on the darkest exposures no product was detected from exon ligation at either downstream CAG of PPT-CAG/AdML with the limit of detection being 1/17 that of the 5′-most CAG.
  42. The site of exon ligation was confirmed by RT-PCR sequencing as in (22).
  43. Splicing the 3′-most CAG has not been observed. Although the reason for this is unknown the use of this site would result in a 3′ exon of only 12 nt. Several studies on 3′ exon length with full-length pre-mRNA constructs have shown that terminal 3′ exons containing <20 nt are inefficient second-step substrates [
  44. Parent A., Zeitlin S., Esfstrantiadis A., J. Biol. Chem. 262, 11284 (1987); (10.1016/S0021-9258(18)60957-3) / J. Biol. Chem. by Parent A. (1987)
  45. Turnbull-Ross A. D., Else A. J., Eperon I. C., Nucleic Acids Res. 16, 395 (1988); (10.1093/nar/16.2.395) / Nucleic Acids Res. by Turnbull-Ross A. D. (1988)
  46. Furdon P. J., Kole R., Mol. Cell. Biol. 8, 860 (1988)]. / Mol. Cell. Biol. by Furdon P. J. (1988)
  47. To date there is no indication that any of the 3′ substrates described here has any effect on the efficiency by which the 5′ substrate undergoes the first step (18).
  48. This sequence corresponds to the last 4 nt of the AdML intron plus a 5′ terminal guanosine for transcription initiation.
  49. Most of the AdML(as) sequence is part of the coding region for AdML DNA polymerase and so is normally part of an exon whereas the AdML(i) sequence is not exonic. Therefore splicing efficiency in this system may correlate with the presence of exonic sequences.
  50. We are currently investigating this phenomenon (K. Anderson and M. J. Moore in preparation).
  51. Plasmids pAdMLpar and pAdMLDAG were as described [
  52. 10.1002/j.1460-2075.1994.tb06638.x
  53. ]. The sequences of these plasmids have been extensively modified compared to the original adenovirus L1-IVS1-L2 sequences from which they were derived. The beginning of the second exon of pAdMLpar and therefore the beginning of the downstream exons of all of the AdML 3′ substrates described here differs from wild-type AdML exon L2 in that /CUCGCG was replaced by /CUGCAGGUCGAC thereby creating Pst I and Sal I restriction sites in the exon. For both the full-length AdML and AdML/5′SS-BS substrates the 5′ exon 5′ splice site ( l ) and intron sequences up to the branch point ( A ) are: In Figs. 2B 2C 3 and 4 a truncated version of the 5′ substrate was used in which nucleotides 5 to 46 of the 5′ exon had been deleted. The TNT exon sequence is identical to the optimized exon 5 mutation N of chicken cardiac troponin T (20). Full-length AdML RNA was transcribed with T7 polymerase from pAdMLpar that had been linearized with Bam HI. All other RNAs were transcribed from T7 templates generated by PCR with either self-complementary primers (TNT 3′ substrates) or primers complementary to either pAdMLpar (AdML 3′ substrates) or pAdMLDAG (AdML/5′SS-BS RNA). RNAs were labeled internally with either [α- 32 P]ATP (adenosine triphosphate) or [α- 32 P]UTP (uridine triphosphate). All AdML/5′SS-BS and 3′ substrate RNAs in Figs. 1B 2A 2C and 4 contained G(5′)ppp(5′)G caps. In Figs. 2B 3A and 3B the 3′ substrate RNAs were capped with GMP (guanosine monophosphate).
  54. Splicing reactions were performed at 30°C in volumes of 5 or 10 μl containing 40% (w/v) HeLa nuclear extract (HeLa cells were obtained from Cellex Biosciences Minneapolis MN) 70 to 85 mM KCl 2 mM MgCl 2 1 mM ATP and 5 mM creatine phosphate. When present the 5′ substrate was 35 nM and the 3′ substrates were 175 nM. RNAs were extracted and separated by denaturing polyacrylamide gel electrophoresis. All gels were subjected to autoradiography and quantitated with a Molecular Dynamics PhosphorImager.
  55. Krainer A. R., Maniatis T., Ruskin B., Green M. R., Cell 36, 993 (1984); (10.1016/0092-8674(84)90049-7) / Cell by Krainer A. R. (1984)
  56. Noble J. C. S., Ge H., Chauduri M., Manley J. L., Mol. Cell. Biol. 9, 2007 (1989). / Mol. Cell. Biol. by Noble J. C. S. (1989)
  57. The slight fuzziness of the bands in Figs. 2B and 3B was likely due to heterogeneity at the 3′ end of the 3′ substrate. Blurred bands were observed only in experiments with the truncated version of the 5′ substrate and AdML 3′ substrates separated on low-percentage gels (8 to 10% polyacrylamide). The AdML 3′ substrate terminates at a Bam HI restriction site (Fig. 1A) which is known to cause extensive 3′ end heterogeneity in run-off transcripts [for example see figure 2B in
  58. 10.1126/science.1589782
  59. and references therein].
  60. We thank R. Reed for plasmids pAdMLpar and pAdMLDAG; T. Cooper and A. Zahler for cTNT sequences; and L. Davis J. Gelles C. Miller C. Query M. Rosbash and P. Sharp for critical reading of the manuscript. This work was supported by NIH grant GM53007 a Packard Fellowship and a Searle Scholarship.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:35 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:04 p.m.)
Indexed 1 year, 7 months ago (Jan. 13, 2024, 12:01 a.m.)
Issued 28 years, 2 months ago (June 13, 1997)
Published 28 years, 2 months ago (June 13, 1997)
Published Print 28 years, 2 months ago (June 13, 1997)
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@article{Anderson_1997, title={Bimolecular Exon Ligation by the Human Spliceosome}, volume={276}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.276.5319.1712}, DOI={10.1126/science.276.5319.1712}, number={5319}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Anderson, Karin and Moore, Melissa J.}, year={1997}, month=jun, pages={1712–1716} }