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. 1997 Apr;17(4):2291–2300. doi: 10.1128/mcb.17.4.2291

The Drosophila suppressor of sable protein binds to RNA and associates with a subset of polytene chromosome bands.

M V Murray 1, M A Turnage 1, K J Williamson 1, W R Steinhauer 1, L L Searles 1
PMCID: PMC232078  PMID: 9121479

Abstract

Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5' splice site and a sequence that is found near the 5' end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands. Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.

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Selected References

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  1. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  4. Cherbas L., Cherbas P. The arthropod initiator: the capsite consensus plays an important role in transcription. Insect Biochem Mol Biol. 1993 Jan;23(1):81–90. doi: 10.1016/0965-1748(93)90085-7. [DOI] [PubMed] [Google Scholar]
  5. Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
  6. Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
  7. Fischer U., Meyer S., Teufel M., Heckel C., Lührmann R., Rautmann G. Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA. EMBO J. 1994 Sep 1;13(17):4105–4112. doi: 10.1002/j.1460-2075.1994.tb06728.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fridell R. A., Pret A. M., Searles L. L. A retrotransposon 412 insertion within an exon of the Drosophila melanogaster vermilion gene is spliced from the precursor RNA. Genes Dev. 1990 Apr;4(4):559–566. doi: 10.1101/gad.4.4.559. [DOI] [PubMed] [Google Scholar]
  9. Fridell R. A., Searles L. L. Evidence for a role of the Drosophila melanogaster suppressor of sable gene in the pre-mRNA splicing pathway. Mol Cell Biol. 1994 Jan;14(1):859–867. doi: 10.1128/mcb.14.1.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  11. Geyer P. K., Chien A. J., Corces V. G., Green M. M. Mutations in the su(s) gene affect RNA processing in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7116–7120. doi: 10.1073/pnas.88.16.7116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guo L. H., Stepień P. P., Tso J. Y., Brousseau R., Narang S., Thomas D. Y., Wu R. Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli. Gene. 1984 Jul-Aug;29(1-2):251–254. doi: 10.1016/0378-1119(84)90186-0. [DOI] [PubMed] [Google Scholar]
  13. Horowitz D. S., Krainer A. R. Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet. 1994 Mar;10(3):100–106. doi: 10.1016/0168-9525(94)90233-x. [DOI] [PubMed] [Google Scholar]
  14. Izaurralde E., Lewis J., Gamberi C., Jarmolowski A., McGuigan C., Mattaj I. W. A cap-binding protein complex mediating U snRNA export. Nature. 1995 Aug 24;376(6542):709–712. doi: 10.1038/376709a0. [DOI] [PubMed] [Google Scholar]
  15. Izaurralde E., Lewis J., McGuigan C., Jankowska M., Darzynkiewicz E., Mattaj I. W. A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell. 1994 Aug 26;78(4):657–668. doi: 10.1016/0092-8674(94)90530-4. [DOI] [PubMed] [Google Scholar]
  16. Izaurralde E., Mattaj I. W. RNA export. Cell. 1995 Apr 21;81(2):153–159. doi: 10.1016/0092-8674(95)90323-2. [DOI] [PubMed] [Google Scholar]
  17. Jarmolowski A., Boelens W. C., Izaurralde E., Mattaj I. W. Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol. 1994 Mar;124(5):627–635. doi: 10.1083/jcb.124.5.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim N., Kim J., Park D., Rosen C., Dorsett D., Yim J. Structure and expression of wild-type and suppressible alleles of the Drosophila purple gene. Genetics. 1996 Apr;142(4):1157–1168. doi: 10.1093/genetics/142.4.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lynch K. W., Maniatis T. Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 1995 Feb 1;9(3):284–293. doi: 10.1101/gad.9.3.284. [DOI] [PubMed] [Google Scholar]
  20. Malim M. H., Cullen B. R. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol Cell Biol. 1993 Oct;13(10):6180–6189. doi: 10.1128/mcb.13.10.6180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mancebo R., Lo P. C., Mount S. M. Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein. Mol Cell Biol. 1990 Jun;10(6):2492–2502. doi: 10.1128/mcb.10.6.2492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mattaj I. W. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. doi: 10.1016/0092-8674(93)90265-r. [DOI] [PubMed] [Google Scholar]
  23. Matunis M. J., Matunis E. L., Dreyfuss G. Isolation of hnRNP complexes from Drosophila melanogaster. J Cell Biol. 1992 Jan;116(2):245–255. doi: 10.1083/jcb.116.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Meyer B. E., Malim M. H. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 1994 Jul 1;8(13):1538–1547. doi: 10.1101/gad.8.13.1538. [DOI] [PubMed] [Google Scholar]
  25. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ohno M., Sakamoto H., Shimura Y. Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5187–5191. doi: 10.1073/pnas.84.15.5187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pret A. M., Searles L. L. Splicing of retrotransposon insertions from transcripts of the Drosophila melanogaster vermilion gene in a revertant. Genetics. 1991 Dec;129(4):1137–1145. doi: 10.1093/genetics/129.4.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rio D. C. Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts. Proc Natl Acad Sci U S A. 1988 May;85(9):2904–2908. doi: 10.1073/pnas.85.9.2904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rio D. C., Laski F. A., Rubin G. M. Identification and immunochemical analysis of biologically active Drosophila P element transposase. Cell. 1986 Jan 17;44(1):21–32. doi: 10.1016/0092-8674(86)90481-2. [DOI] [PubMed] [Google Scholar]
  30. Robbins A., Dynan W. S., Greenleaf A., Tjian R. Affinity-purified antibody as a probe of RNA polymerase II subunit structure. J Mol Appl Genet. 1984;2(4):343–353. [PubMed] [Google Scholar]
  31. Robbins S. G., Frana M. F., McGowan J. J., Boyle J. F., Holmes K. V. RNA-binding proteins of coronavirus MHV: detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay. Virology. 1986 Apr 30;150(2):402–410. doi: 10.1016/0042-6822(86)90305-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sachs A. B., Kornberg R. D. Purification and characterization of polyadenylate-binding protein. Methods Enzymol. 1990;181:332–352. doi: 10.1016/0076-6879(90)81134-g. [DOI] [PubMed] [Google Scholar]
  33. Searles L. L., Ruth R. S., Pret A. M., Fridell R. A., Ali A. J. Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles. Mol Cell Biol. 1990 Apr;10(4):1423–1431. doi: 10.1128/mcb.10.4.1423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Siebel C. W., Kanaar R., Rio D. C. Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI. Genes Dev. 1994 Jul 15;8(14):1713–1725. doi: 10.1101/gad.8.14.1713. [DOI] [PubMed] [Google Scholar]
  35. Smith P. A., Corces V. G. Drosophila transposable elements: mechanisms of mutagenesis and interactions with the host genome. Adv Genet. 1991;29:229–300. doi: 10.1016/s0065-2660(08)60109-1. [DOI] [PubMed] [Google Scholar]
  36. Takagaki Y., Manley J. L. A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein. Nature. 1994 Dec 1;372(6505):471–474. doi: 10.1038/372471a0. [DOI] [PubMed] [Google Scholar]
  37. Tuerk C., Eddy S., Parma D., Gold L. Autogenous translational operator recognized by bacteriophage T4 DNA polymerase. J Mol Biol. 1990 Jun 20;213(4):749–761. doi: 10.1016/S0022-2836(05)80261-X. [DOI] [PubMed] [Google Scholar]
  38. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  39. Voelker R. A., Gibson W., Graves J. P., Sterling J. F., Eisenberg M. T. The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins. Mol Cell Biol. 1991 Feb;11(2):894–905. doi: 10.1128/mcb.11.2.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Weeks J. R., Hardin S. E., Shen J., Lee J. M., Greenleaf A. L. Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev. 1993 Dec;7(12A):2329–2344. doi: 10.1101/gad.7.12a.2329. [DOI] [PubMed] [Google Scholar]
  41. Williams D. C., Van Frank R. M., Muth W. L., Burnett J. P. Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science. 1982 Feb 5;215(4533):687–689. doi: 10.1126/science.7036343. [DOI] [PubMed] [Google Scholar]
  42. Witherell G. W., Wu H. N., Uhlenbeck O. C. Cooperative binding of R17 coat protein to RNA. Biochemistry. 1990 Dec 18;29(50):11051–11057. doi: 10.1021/bi00502a006. [DOI] [PubMed] [Google Scholar]
  43. Zachar Z., Chou T. B., Bingham P. M. Evidence that a regulatory gene autoregulates splicing of its transcript. EMBO J. 1987 Dec 20;6(13):4105–4111. doi: 10.1002/j.1460-2075.1987.tb02756.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zachar Z., Kramer J., Mims I. P., Bingham P. M. Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans. J Cell Biol. 1993 May;121(4):729–742. doi: 10.1083/jcb.121.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]

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