SC35-mediated Reconstitution of Splicing in U2AF-depleted Nuclear Extract

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1997 Jan 7

PMID 8990173

Citations 24

Authors

A M MacMillan

P S McCaw

J D Crispino

P A Sharp

Affiliations

Soon will be listed here.

Abstract

Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate-specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF-dependent and -independent pathways.

Citing Articles

Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Black C, Whelan T, Garside E, MacMillan A, Fast N, Rader S RNA. 2023; 29(5):531-550.

PMID: 36737103 PMC: 10158995. DOI: 10.1261/rna.079273.122.

SAM homeostasis is regulated by CFI-mediated splicing of MAT2A.

Scarborough A, Flaherty J, Hunter O, Liu K, Kumar A, Xing C Elife. 2021; 10.

PMID: 33949310 PMC: 8139829. DOI: 10.7554/eLife.64930.

Nuclear PTEN safeguards pre-mRNA splicing to link Golgi apparatus for its tumor suppressive role.

Shen S, Ji Y, Zhang C, Dong S, Yang S, Xiong Z Nat Commun. 2018; 9(1):2392.

PMID: 29921876 PMC: 6008332. DOI: 10.1038/s41467-018-04760-1.

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

Crisci A, Raleff F, Bagdiul I, Raabe M, Urlaub H, Rain J Nucleic Acids Res. 2015; 43(21):10456-73.

PMID: 26420826 PMC: 4666396. DOI: 10.1093/nar/gkv952.

Genomic functions of U2AF in constitutive and regulated splicing.

Wu T, Fu X RNA Biol. 2015; 12(5):479-85.

PMID: 25901584 PMC: 4615725. DOI: 10.1080/15476286.2015.1020272.

References

Dignam J, Lebovitz R, Roeder R . Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983; 11(5):1475-89. PMC: 325809. DOI: 10.1093/nar/11.5.1475. View

Staknis D, REED R . SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994; 14(11):7670-82. PMC: 359303. DOI: 10.1128/mcb.14.11.7670-7682.1994. View

Konarska M, Sharp P . Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986; 46(6):845-55. DOI: 10.1016/0092-8674(86)90066-8. View

Tarn W, Steitz J . SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 1994; 8(22):2704-17. DOI: 10.1101/gad.8.22.2704. View

Blencowe B, Nickerson J, Issner R, Penman S, Sharp P . Association of nuclear matrix antigens with exon-containing splicing complexes. J Cell Biol. 1994; 127(3):593-607. PMC: 2120221. DOI: 10.1083/jcb.127.3.593. View

MacMillan A, Query C, Allerson C, Chen S, Verdine G, Sharp P . Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 1994; 8(24):3008-20. DOI: 10.1101/gad.8.24.3008. View

Tacke R, Manley J . The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995; 14(14):3540-51. PMC: 394422. DOI: 10.1002/j.1460-2075.1995.tb07360.x. View

Crispino J, Sharp P . A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. Genes Dev. 1995; 9(18):2314-23. DOI: 10.1101/gad.9.18.2314. View

Wang Z, Hoffmann H, Grabowski P . Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity. RNA. 1995; 1(1):21-35. PMC: 1369055. View

10.

Fu X . The superfamily of arginine/serine-rich splicing factors. RNA. 1995; 1(7):663-80. PMC: 1369309. View

11.

Roscigno R, Garcia-Blanco M . SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome. RNA. 1995; 1(7):692-706. PMC: 1369311. View

12.

Crispino J, Mermoud J, Lamond A, Sharp P . Cis-acting elements distinct from the 5' splice site promote U1-independent pre-mRNA splicing. RNA. 1996; 2(7):664-73. PMC: 1369405. View

13.

Valcarcel J, Gaur R, Singh R, Green M . Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected]. Science. 1996; 273(5282):1706-9. DOI: 10.1126/science.273.5282.1706. View

14.

RUSKIN B, Zamore P, Green M . A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988; 52(2):207-19. DOI: 10.1016/0092-8674(88)90509-0. View

15.

Ruby S, Abelson J . An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988; 242(4881):1028-35. DOI: 10.1126/science.2973660. View

16.

Seraphin B, Rosbash M . Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989; 59(2):349-58. DOI: 10.1016/0092-8674(89)90296-1. View

17.

Zamore P, Green M . Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989; 86(23):9243-7. PMC: 298470. DOI: 10.1073/pnas.86.23.9243. View

18.

Barabino S, Blencowe B, Ryder U, Sproat B, Lamond A . Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990; 63(2):293-302. DOI: 10.1016/0092-8674(90)90162-8. View

19.

Zamore P, Green M . Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 1991; 10(1):207-14. PMC: 452631. DOI: 10.1002/j.1460-2075.1991.tb07937.x. View

20.

Guthrie C . Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991; 253(5016):157-63. DOI: 10.1126/science.1853200. View