Association of nuclear matrix antigens with exon-containing splicing complexes (original) (raw)
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A coactivator of pre-mRNA splicing
Genes & Development, 1998
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.
In Vitro Assay of Pre-mRNA Splicing in Mammalian Nuclear Extract
Methods in molecular biology, 2014
The in vitro splicing assay is a valuable technique that can be used to study the mechanism and machinery involved in the splicing process. The ability to investigate various aspects of splicing and alternative splicing appears to be endless due to the flexibility of this assay. Here, we describe the tools and techniques necessary to carry out an in vitro splicing assay. Through the use of radiolabeled pre-mRNA and crude nuclear extract, spliced mRNAs can be purified and visualized by autoradiography for downstream analysis.
The EMBO Journal, 1991
In previous studies we have shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in the form of large nuclear ribonucleoprotein (InRNP) particles. By electron microscopy, these particles appeared as compact composite structures, 50 nm in diameter, which invariably sedimented at the 200S region in sucrose gradients. In order to identify putative protein splicing factors associated with the 200S hnRNP particles, a panel of monoclonal antibodies directed against these particles were screened for their ability to inhibit splicing of pre-mRNA in vitro. In this study we have focused on a nuclear protein of 88 kd in molecular weight, which is an integral component of the InRNP complex and is recognized by monoclonal antibodies from a specific clone. This protein has been identified here as a novel splicing factor by, (i) antibody inhibition of splicing in vitro and (ii) depletion of splicing activity from HeLa cell nuclear extract after removing the 88 kd polypeptide by immunoadsorption, and complementation of the depleted activity with an affinity-purified 88 kd antigen. This splicing factor has further been shown to be required for the assembly of an active splicing complex.
Methods in molecular biology, 2017
The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.
Biochemical Journal, 2002
We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72\74 kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72\74 kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72\74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in itro, the antigenic 72\74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72\74(M) with both the initially formed H assembly and the first splicing-committed E
Molecular Cell, 2000
have been identified as effectors of mRNA export (Gö rlich and Kutay, 1999; Nakielny and Dreyfuss, 1999). The mechanism of HIV-1 Rev-mediated RNA export has been studied extensively (Pollard and Malim, 1998). Rev binds to unspliced or partially spliced viral mRNAs via the Rev-responsive element (RRE), and the export factor University of Pennsylvania School of Medicine CRM1, a member of nuclear transport receptor (NTR) Philadelphia, Pennsylvania 19104 family (Gö rlich and Kutay, 1999; Nakielny and Dreyfuss, 1999), binds to the leucine-rich nuclear export signals (NES) of Rev and mediates export of this complex in a Summary
Nucleic Acids Research, 1995
The monoclonal antibody CC3 recognizes a phosphorylated epitope present on an interphase protein of 255 kDa. Previous work has shown that p255 is localized mainly to nuclear speckles and remains associated with the nuclear matrix scaffold following extraction with non-ionic detergents, nucleases and high salt. The association of p255 with splicing complexes is suggested by the finding that mAb CC3 can inhibit in vitro splicing and immunoprecipitate pre-messenger RNA and splicing products. Small nuclear RNA immunoprecipitation assays show that p255 is a component of the U5 small nuclear ribonucleoprotein (snRNP) and the [U4AU6.U5] tri-snRNP complex. In RNase protection assays, mAb CC3 immunoprecipitates fragments containing branch site and 3' splice site sequences. As predicted for a [U4WU6.U5]-associated component, the recovery of the branch site-protected fragment requires binding of U2 snRNP and is inhibited by EDTA. p255 may correspond to the previously identified p220 protein, the mammalian analogue of the yeast PRP8 protein. Our results suggest that changes in the phosphorylation of p255 may be part of control mechanisms that interface splicing activity with nuclear organization.
The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein
Science (New York, N.Y.), 1985
A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region. Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease. The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease. This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.
Gene Analysis Techniques, 1988
A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 × 10 7 mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class 11 prorooters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous Jbr biochemical analysis of transcription and RNA processing in mammalian cells. An understanding of transcription and pre-mRNA processing at the biochemical level depends on the ability to study these processes in vitro. This has been made possible by the development of methods for preparing soluble extracts from cultured mammalian cells that support transcription (for review, see [I]) and RNA processing (for reviews, see [2] and [3]). The conventionally prepared extracts referred to above support the basic transcription [4-6] and RNA processing reactions [7-9] However, it is