scholarly journals Characterization of U2AF6, a Splicing Factor Related to U2AF35

2002 ◽  
Vol 22 (1) ◽  
pp. 221-230 ◽  
Author(s):  
Jeremiah Shepard ◽  
Martin Reick ◽  
Sara Olson ◽  
Brenton R. Graveley
Keyword(s):  
Sequence Similarity ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Physical Interaction ◽  
U2 Auxiliary Factor ◽  
Auxiliary Factor ◽  
Strong Sequence Similarity ◽  
Splicing Enhancer

ABSTRACT The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits. U2AF35 has multiple functions in pre-mRNA splicing. First, U2AF35 has been shown to function by directly interacting with the AG at the 3′ splice site. Second, U2AF35 is thought to play a role in the recruitment of U2AF65 by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF35 and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF35, designated U2AF26. The N-terminal 187 amino acids of U2AF35 and U2AF26 are nearly identical. However, the C-terminal domain of U2AF26 lacks many characteristics of the U2AF35 RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF26 can associate with U2AF65 and can functionally substitute for U2AF35 in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF26 functions by enhancing the binding of U2AF65 to weak 3′ splice sites. These studies identify U2AF26 as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF35, U2AF26 may play a role in regulating alternative splicing.

scholarly journals Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

1999 ◽  
Vol 19 (3) ◽  
pp. 1705-1719 ◽  
Author(s):  
Thomas D. Schaal ◽  
Tom Maniatis
Keyword(s):  
In Vitro Selection ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Sequence Motifs ◽  
Rna Sequences ◽  
Sr Protein ◽  
Enhancer Sequence ◽  
Splicing Enhancer

ABSTRACT Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

scholarly journals Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.

1996 ◽  
Vol 16 (10) ◽  
pp. 5400-5408 ◽  
Author(s):  
W J Zhang ◽  
J Y Wu
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Sr Protein ◽  
Protein Protein Interaction ◽  
Small Nuclear Ribonucleoprotein ◽  
S100 Extract ◽  
U2 Auxiliary Factor ◽  
Auxiliary Factor

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner.

Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract

1991 ◽  
Vol 11 (7) ◽  
pp. 3425-3431
Author(s):  
P Delannoy ◽  
M H Caruthers
Keyword(s):  
Heat Treatment ◽  
Hela Cell ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Nuclear Extracts ◽  
Small Nuclear Rnas ◽  
A Minor ◽  
Cell Nuclear Extract

Mild heat treatment of HeLa cell nuclear extracts (NE) selectively inhibits pre-mRNA splicing. Heat-inactivated extracts can be complemented by a small amount of untreated NE. Utilizing this complementation assay and a combination of ion-exchange, affinity, and hydrophobic chromatography, a heat reversal factor (HRF) was purified from NE that is required to rescue pre-mRNA splicing from a heat-inactivated extract. This activity in its most purified form consistently copurified in a fraction containing two 70-kDa proteins and a minor polypeptide of approximately 100 kDa. It was free of the major small nuclear RNAs, sensitive to protease, and required to rescue spliceosome formation from a heat-inactivated nuclear extract. These results suggest that this factor is a protein that may be an important component in pre-mRNA splicing, or alternatively, it may be involved in renaturation of a heat-sensitive splicing factor.

scholarly journals Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7347-7356 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Michel Popielarz ◽  
Georges Hildwein ◽  
James Stevenin
Keyword(s):  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Adenovirus E1a ◽  
Differential Involvement ◽  
Splicing Enhancer

ABSTRACT The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

scholarly journals Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing

1999 ◽  
Vol 19 (8) ◽  
pp. 5352-5362 ◽  
Author(s):  
W. Hayes McDonald ◽  
Ryoma Ohi ◽  
Natalia Smelkova ◽  
David Frendewey ◽  
Kathleen L. Gould
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fission Yeast ◽  
Genetic Interaction ◽  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Immunoaffinity Purification ◽  
Snrnp Protein

ABSTRACT Myb-related cdc5p is required for G2/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiaesplicing factor Prp19p. cwf1 + is allelic to theprp5 + gene defined by the S. pombesplicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 andprp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lackingcdc5 + function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.

scholarly journals Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

2001 ◽  
Vol 12 (8) ◽  
pp. 2328-2340 ◽  
Author(s):  
Yaron Shav-Tal ◽  
Michal Cohen ◽  
Smadar Lapter ◽  
Billy Dye ◽  
James G. Patton ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Nuclear Organization ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Regulatory Proteins ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Nuclear Structures ◽  
Differential Phosphorylation ◽  
Antigenic Epitopes

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1–70K and SR proteins and therefore may acquire new functions.

scholarly journals Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract.

1991 ◽  
Vol 11 (7) ◽  
pp. 3425-3431 ◽  
Author(s):  
P Delannoy ◽  
M H Caruthers
Keyword(s):  
Heat Treatment ◽  
Hela Cell ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Nuclear Extracts ◽  
Small Nuclear Rnas ◽  
A Minor ◽  
Cell Nuclear Extract

Mild heat treatment of HeLa cell nuclear extracts (NE) selectively inhibits pre-mRNA splicing. Heat-inactivated extracts can be complemented by a small amount of untreated NE. Utilizing this complementation assay and a combination of ion-exchange, affinity, and hydrophobic chromatography, a heat reversal factor (HRF) was purified from NE that is required to rescue pre-mRNA splicing from a heat-inactivated extract. This activity in its most purified form consistently copurified in a fraction containing two 70-kDa proteins and a minor polypeptide of approximately 100 kDa. It was free of the major small nuclear RNAs, sensitive to protease, and required to rescue spliceosome formation from a heat-inactivated nuclear extract. These results suggest that this factor is a protein that may be an important component in pre-mRNA splicing, or alternatively, it may be involved in renaturation of a heat-sensitive splicing factor.

scholarly journals Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

1998 ◽  
Vol 18 (2) ◽  
pp. 676-684 ◽  
Author(s):  
Wan-Jiang Zhang ◽  
Jane Y. Wu
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Interacting Protein

ABSTRACT Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.

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