ABSTRACT
Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.
Subject(s)
Carrier Proteins , Gene Expression Regulation , Genetic Vectors , Immunophilins/metabolism , Phosphotransferases (Alcohol Group Acceptor) , Retroviridae/genetics , Transcription, Genetic , Transfection/methods , 3T3 Cells , Animals , Cell Line , Dimerization , Gene Expression Regulation/drug effects , Humans , Immunophilins/genetics , Kinetics , Mice , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Tumor Cells, CulturedABSTRACT
The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.
Subject(s)
Antigens, Nuclear , Nuclear Matrix-Associated Proteins , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Splicing/genetics , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Antibodies, Monoclonal , Cell Nucleus/metabolism , Cells, Cultured , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Complementary/metabolism , Humans , Molecular Sequence Data , Nuclear Proteins/immunology , Precipitin Tests , RNA Splicing/physiology , RNA, Messenger/metabolism , Spliceosomes/metabolismABSTRACT
The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.
Subject(s)
Antigens, Nuclear , Nuclear Matrix-Associated Proteins , Nuclear Proteins/genetics , RNA Precursors/metabolism , RNA-Binding Proteins/genetics , Spliceosomes/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , HeLa Cells/metabolism , Humans , Interphase/physiology , Lymphoma, Large B-Cell, Diffuse/pathology , Metaphase/physiology , Molecular Sequence Data , Nuclear Matrix/immunology , Nuclear Proteins/immunology , RNA Precursors/genetics , RNA-Binding Proteins/immunology , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoprotein, U2 Small Nuclear/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spliceosomes/genetics , Tumor Cells, CulturedABSTRACT
A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. Using a selective precipitation method, more than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Iariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level.
Subject(s)
Arginine , RNA Splicing , RNA-Binding Proteins/chemistry , Serine , Antibodies, Monoclonal , Antigens/chemistry , Antigens/isolation & purification , Antigens, Nuclear , Chemical Precipitation , Epitopes , HeLa Cells , Humans , Immunoblotting , Nuclear Proteins/chemistry , Nuclear Proteins/immunology , Nuclear Proteins/isolation & purification , Phosphorylation , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunologyABSTRACT
mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.
