A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro

SV40 early pre-mRNA is alternatively spliced by utilization of two different 5' splice sites and a shared 3' splice site to produce large T and small t mRNAs. The ratio of small t to large T mRNAs produced in human embryonic kidney 293 cells is 10- to 20-fold greater than in other mammalian cells, suggesting the existence of a 293 cell-specific factor that modulates alternative splicing. Here we show that nuclear extracts from 293 cells give rise to significantly more small t splicing than do extracts from HeLa cells. Using an in vitro complementation assay, we have characterized and extensively purified a factor from 293 extracts that brings about striking increases in small t splicing with concomitant decreases in large T splicing. The factor is heat sensitive and micrococcal nuclease resistant, suggesting that it is a protein lacking an accessible RNA component. Purification of the alternative splicing factor indicates that the activity is contained in one of several possibly related polypeptides of 30-35 kd.

[1]  C. Prives,et al.  Alternative splicing of SV40 early pre-mRNA is determined by branch site selection. , 1988, Genes & development.

[2]  R. Lührmann,et al.  Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. , 1986, The EMBO journal.

[3]  J. Manley,et al.  The role of the polypyrimidine stretch at the SV40 early pre‐mRNA 3′ splice site in alternative splicing. , 1988, The EMBO journal.

[4]  A. Weiner,et al.  A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation , 1986, Cell.

[5]  J. Manley,et al.  Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA , 1988, Molecular and cellular biology.

[6]  J. Manley,et al.  Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. , 1989, Genes & development.

[7]  Tom Maniatis,et al.  A role for exon sequences and splice-site proximity in splice-site selection , 1986, Cell.

[8]  P. Sharp,et al.  Structure of the adenovirus 2 early mRNAs , 1978, Cell.

[9]  M. Streuli,et al.  Regulation of tissue‐specific alternative splicing: exon‐specific cis‐elements govern the splicing of leukocyte common antigen pre‐mRNA. , 1989, The EMBO journal.

[10]  G. Rubin,et al.  Analysis of the cis-acting requirements for germ-line-specific splicing of the P-element ORF2-ORF3 intron. , 1989, Genes & development.

[11]  I. Mattaj,et al.  Regulated splicing in early development and stage-specific U snRNPs. , 1989, Development.

[12]  B. Nadal-Ginard,et al.  Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. , 1987, Annual review of biochemistry.

[13]  G. Dreyfuss,et al.  RNA-binding proteins as developmental regulators. , 1989, Genes & development.

[14]  B. S. Baker,et al.  The sex determination locus transformer-2 of Drosophila encodes a polypeptide with similarity to RNA binding proteins , 1989, Cell.

[15]  D. Helfman,et al.  Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation , 1986, Molecular and cellular biology.

[16]  Christine Guthrie,et al.  Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA , 1987, Cell.

[17]  R. Spritz,et al.  Alternative splicing of SV40 early pre-mRNA in vitro. , 1986, Nucleic acids research.

[18]  G. Rubin,et al.  Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing , 1986, Cell.

[19]  D. Solnick.  Trans splicing of mRNA precursors , 1985, Cell.

[20]  Stephen M. Mount,et al.  A catalogue of splice junction sequences. , 1982, Nucleic acids research.

[21]  F. Graham,et al.  Characteristics of a human cell line transformed by DNA from human adenovirus type 5. , 1977, The Journal of general virology.

[22]  A. Berk,et al.  Spliced early mRNAs of simian virus 40. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Steitz,et al.  Isolation of small nuclear ribonucleoproteins containing U1, U2, U4, U5, and U6 RNAs. , 1983, The Journal of biological chemistry.

[24]  G. Dreyfuss,et al.  RNA binding specificity of hnRNP proteins: a subset bind to the 3′ end of introns. , 1988, The EMBO journal.

[25]  F. Baralle,et al.  A role for exon sequences in alternative splicing of the human fibronectin gene. , 1987, Nucleic acids research.

[26]  J. Tazi,et al.  A protein that specifically recognizes the 3′ splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein , 1986, Cell.

[27]  P. Bingham,et al.  Developmental expression of a regulatory gene is programmed at the level of splicing. , 1987, The EMBO journal.

[28]  B. S. Baker,et al.  Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns. , 1990, Genes & development.

[29]  T. Maniatis,et al.  Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus , 1990, Nature.

[30]  Adrian R. Krainer,et al.  The essential pre-mRNA splicing factor SF2 influences 5′ splice site selection by activating proximal sites , 1990, Cell.

[31]  P. Sharp,et al.  Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[32]  D. Helfman,et al.  Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA. , 1990, Genes & development.

[33]  P. Bingham,et al.  On/off regulation of gene expression at the level of splicing. , 1988, Trends in genetics : TIG.

[34]  T. Maniatis,et al.  The role of the mammalian branchpoint sequence in pre-mRNA splicing. , 1988, Genes & development.

[35]  P. Zamore,et al.  Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Rosenfeld,et al.  Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor , 1989, Nature.

[37]  J. Steitz,et al.  Functions of the Abundant U-snRNPs , 1988 .

[38]  J. Mertz,et al.  Exon mutations that affect the choice of splice sites used in processing the SV40 late transcripts. , 1985, Nucleic acids research.

[39]  T. Cooper,et al.  Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing. , 1989, Nucleic acids research.

[40]  I. Mattaj.  A binding consensus: RNA-protein interactions in splicing, snRNPs, and sex , 1989, Cell.

[41]  C. Prives,et al.  In vitro splicing of simian virus 40 early pre mRNA. , 1986, Nucleic acids research.

[42]  J. Manley,et al.  Factors influencing alternative splice site utilization in vivo , 1987, Molecular and cellular biology.

[43]  D. Helfman,et al.  Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo. , 1988, Genes & development.

[44]  B. S. Baker,et al.  Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides , 1989, Cell.

[45]  L. Bell,et al.  Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins , 1988, Cell.

[46]  Carol Prives,et al.  Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites , 1987, Cell.

[47]  Christopher W. J. Smith,et al.  Mutually exclusive splicing of α-tropomyosin exons enforced by an unusual lariat branch point location: Implications for constitutive splicing , 1989, Cell.

[48]  M. Green.  Pre-mRNA splicing. , 1986, Annual review of genetics.

[49]  S. Leff,et al.  Complex transcriptional units: diversity in gene expression by alternative RNA processing. , 1986, Annual review of biochemistry.

[50]  A. Weiner,et al.  The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA. , 1987, Molecular and cellular biology.

[51]  C. Guthrie,et al.  5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. , 1988, Genes & development.

[52]  M. Mckeown,et al.  Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene , 1987, Cell.

[53]  F. Rottman,et al.  Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences , 1989, Molecular and cellular biology.

[54]  Michael G. Rosenfeld,et al.  Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products , 1982, Nature.

[55]  Michael R. Green,et al.  A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly , 1988, Cell.

[56]  M. Mckeown,et al.  Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage , 1989, Cell.

[57]  A. Krainer,et al.  Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for Pre-mRNA splicing in vitro , 1985, Cell.

[58]  B. Séraphin,et al.  A U1 snRNA:pre‐mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. , 1988, The EMBO journal.

[59]  Michael R. Green,et al.  Cryptic branch point activation allows accurate in vitro splicing of human β-globin intron mutants , 1985, Cell.

[60]  J. Steitz,et al.  A protein associated with small nuclear ribonucleoprotein particles recognizes the 3′ splice site of premessenger RNA , 1986, Cell.

[61]  P. Keohavong,et al.  Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro , 1987, Cell.

[62]  A. Weiner,et al.  UACUAAC is the preferred branch site for mammalian mRNA splicing. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[63]  H. Amrein,et al.  The sex-determining gene tra-2 of Drosophila encodes a putative RNA binding protein , 1988, Cell.

[64]  J. Steitz,et al.  The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein. , 1985, Science.

[65]  A. Weiner,et al.  A compensatory base change in human U2 snRNA can suppress a branch site mutation. , 1989, Genes & development.

[66]  C. W. Smith,et al.  The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing , 1988, Molecular and cellular biology.