Bimolecular exon ligation by the human spliceosome

Science

Volumn 276, Issue 5319, 1997, Pages 1712-1716

  Anderson, Karin ^a   Moore, Melissa J ^a  

^a BRANDEIS UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MESSENGER RNA;

ARTICLE; CONSENSUS SEQUENCE; ENHANCER REGION; EUKARYOTE; EXON; GENE EXPRESSION; HUMAN; INTRON; PRIORITY JOURNAL; RNA SPLICING; SPLICEOSOME;

ADENOVIRIDAE; BASE SEQUENCE; BINDING SITES; EXONS; HUMANS; INTRONS; MOLECULAR SEQUENCE DATA; NUCLEIC ACID CONFORMATION; RNA PRECURSORS; RNA SPLICING; SPLICEOSOMES;

EUKARYOTA;

EID: 0030802754     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5319.1712     Document Type: Article

References (52)

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30. K. Anderson and M. J. Moore, unpublished data.
31. Similar results have been observed for other such truncated splicing substrates [D. Frendewey and W. Keller, Cell 42, 355 (1985); B. Ruskin and M. R. Green, Nature 317, 732 (1985)].
32. Similar results have been observed for other such truncated splicing substrates [D. Frendewey and W. Keller, Cell 42, 355 (1985); B. Ruskin and M. R. Green, Nature 317, 732 (1985)].
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38. 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.
39. 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.
40. The site of exon ligation was confirmed by RT-PCR sequencing as in (22).
41. 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 [A. Parent, S. Zeitlin, A. Esfstrantiadis, J. Biol. Chem. 262, 11284 (1987); A. D. Turnbull-Ross, A. J. Else, I. C. Eperon, Nucleic Acids Res. 16, 395 (1988); P. J. Furdon and R. Kole, Mol. Cell. Biol. 8, 860 (1988)].
42. 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 [A. Parent, S. Zeitlin, A. Esfstrantiadis, J. Biol. Chem. 262, 11284 (1987); A. D. Turnbull-Ross, A. J. Else, I. C. Eperon, Nucleic Acids Res. 16, 395 (1988); P. J. Furdon and R. Kole, Mol. Cell. Biol. 8, 860 (1988)].
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 [A. Parent, S. Zeitlin, A. Esfstrantiadis, J. Biol. Chem. 262, 11284 (1987); A. D. Turnbull-Ross, A. J. Else, I. C. Eperon, Nucleic Acids Res. 16, 395 (1988); P. J. Furdon and R. Kole, Mol. Cell. Biol. 8, 860 (1988)].
44. 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). 27. This sequence corresponds to the last 4 nt of the AdML intron plus a 5′ terminal guanosine for transcription initiation.
45. 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.
46. We are currently investigating this phenomenon (K. Anderson and M. J. Moore, in preparation).
47. 32P]UTP (uridine triphosphate). All AdML/5αSS-BS and 3α substrate RNAs in Figs. 1B, 2A, 2C, and 4 contained G(5α)ppp(5α)G caps. In Figs. 2B, 3A, and 3B, the 3α substrate RNAs were capped with GMP (guanosine monophosphate).
48. 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.
49. A. R. Krainer, T. Maniatis, B. Ruskin, M. R. Green, Cell 36, 993 (1984); J. C. S. Noble, H. Ge, M. Chauduri, J. L. Manley, Mol. Cell. Biol. 9, 2007 (1989).
50. A. R. Krainer, T. Maniatis, B. Ruskin, M. R. Green, Cell 36, 993 (1984); J. C. S. Noble, H. Ge, M. Chauduri, J. L. Manley, Mol. Cell. Biol. 9, 2007 (1989).
51. 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 M. J. Moore and P. A. Sharp, Science 256, 992 (1992) and references therein].
52. 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.