G C Conway

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States

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Publications (5)55.15 Total impact

  • A R Krainer, G C Conway, D Kozak
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    ABSTRACT: SF2 is a 33 kd protein factor required for 5' splice site cleavage and lariat formation during pre-mRNA splicing in HeLa cell extracts. In addition to its essential role in constitutive splicing, SF2 can strongly influence 5' splice site selection. When pre-mRNAs containing multiple cis-competing 5' splice sites are spliced in vitro, high concentrations of purified SF2 promote the use of the 5' splice site closest to the 3' splice site. However, SF2 discriminates properly between authentic and cryptic splice sites. These effects of SF2 on splice site selection may reflect the cellular mechanisms that prevent exon skipping and ensure the accuracy of splicing. In addition, alterations in the concentration or activity of SF2, and of other general splicing factors, may serve to regulate alternative splicing in vivo.
    Cell 08/1990; 62(1):35-42. · 31.96 Impact Factor
  • A R Krainer, G C Conway, D Kozak
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    ABSTRACT: SF2, an activity necessary for 5' splice site cleavage and lariat formation during pre-mRNA splicing in vitro, has been purified to near homogeneity from HeLa cells. The purest fraction contains only two related polypeptides of 33 kD. This fraction is sufficient to complement an S100 fraction, which contains the remaining splicing factors, to splice several pre-mRNAs. The optimal amount of SF2 required for efficient splicing depends on the pre-mRNA substrate. SF2 is distinct from the hnRNP A1 and U1 snRNP a polypeptides, which are similar in size. Endogenous hnRNA copurifies with SF2, but this activity does not appear to have an essential RNA component. SF2 appear to be necessary for the assembly or stabilization of the earliest specific prespliceosome complex, although in the absence of other components, it can bind RNA in a nonspecific manner. SF2 copurifies with an activity that promotes the annealing of complementary RNAs. Thus, SF2 may promote specific RNA-RNA interactions between snRNAs and pre-mRNA, between complementary snRNA regions, and/or involving intramolecular pre-mRNA helices. Other purified proteins with RNA annealing activity cannot substitute for SF2 in the splicing reaction.
    Genes & Development 08/1990; 4(7):1158-71. · 12.44 Impact Factor
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    ABSTRACT: Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.
    Molecular and Cellular Biology 01/1990; 9(12):5273-80. · 5.37 Impact Factor
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    ABSTRACT: An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins A1 and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.
    Molecular and Cellular Biology 08/1988; 8(7):2884-95. · 5.37 Impact Factor
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    ABSTRACT: complex. Atleast twosplicing factors other thanthesnRNPswere also associated withthese large structures. Uponincubation withATP, these splicing factors aswell asUlandU2snRNPswere released fromthese complexes. Thepresenceof multiple splicing factors suggests thatthese complexes may beendogenous spliceosomes released fromnuclei during preparation ofsplicing extracts. Theremoval ofthese structures fromextracts thathadbeen preincubated withATPyielded a splicing extract devoid oflarge structures. Thisextract should proveuseful inthefractionation ofsplicing factors andtheisolation ofnative spliceosomes formed on exogenously added pre-mRNA. Several factors havebeenidentified that arenecessaryfor pre-mRNAsplicing. Themostextensively characterized of these components arethemajornucleoplasmic small nu- clear ribonucleoprotein

Publication Stats

512 Citations
174 Views
55.15 Total Impact Points

Institutions

  • 1990
    • Cold Spring Harbor Laboratory
      Cold Spring Harbor, New York, United States
  • 1988
    • Vanderbilt University
      • Department of Molecular Physiology and Biophysics
      Nashville, MI, United States