The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
Michael R Green - One of the best experts on this subject based on the ideXlab platform.
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An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal
Nature communications, 2016Co-Authors: Anant A. Agrawal, Michael R Green, Enea Salsi, Rakesh Chatrikhi, Steven Henderson, Jermaine L. Jenkins, Dmitri N. Ermolenko, Clara L. KielkopfAbstract:How the essential pre-mRNA Splicing Factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 A. These structures together with RNA binding and Splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.
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U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs
Nucleic acids research, 2013Co-Authors: Jermaine L. Jenkins, Michael R Green, Anant A. Agrawal, Ankit Gupta, Clara L. KielkopfAbstract:Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA Splicing Factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65).
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structural basis for polypyrimidine tract recognition by the essential pre mrna Splicing Factor U2AF65
Molecular Cell, 2006Co-Authors: Allen E Sickmier, Michael R Green, Haihong Shen, Katherine E Frato, Shanthi R Paranawithana, Clara L. KielkopfAbstract:Summary The essential pre-mRNA Splicing Factor, U2AF 65 , guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3′ splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF 65 is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF 65 RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF 65 and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF 65 structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF 65 proteins using surface plasmon resonance.
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a pathway of sequential arginine serine rich domain Splicing signal interactions during mammalian spliceosome assembly
Molecular Cell, 2004Co-Authors: Haihong Shen, Michael R GreenAbstract:Abstract Serine-arginine (SR) proteins are general Splicing Factors and can function through binding to exonic Splicing enhancers (ESEs). SR proteins and several other mammalian Splicing Factors contain an arginine-serine-rich (RS) domain required to promote Splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential Splicing Factor U2AF 65 and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5′ splice site specifically in the mature spliceosome. We propose that direct contact with Splicing signals is a general mechanism by which RS domains promote Splicing.
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Functional Analysis of Splicing Factors and Regulators
mRNA Formation and Function, 1997Co-Authors: Juan Valcarcel, Concepcion Martinez, Michael R GreenAbstract:Publisher Summary This chapter discusses about the functional analysis of Splicing Factors and regulators. Biochemical identification and analysis of components of the Splicing machinery requires the development of functional assays to test their associated activities. Chromatographic and immunological procedures are utilized to generate Splicing-deficient nuclear extracts whose activity depends on the addition of purified Factors. These procedures are valuable in analyzing mechanisms of alternative Splicing regulation. This chapter reviews biochemical methods used for the depletion of non-snRNP proteins from HeLa nuclear extracts, and provides detailed protocols for the preparation of extracts in which the essential Splicing Factor U2AF has been depleted. The chapter also explains concepts related to in vitro depletion systems. Analysis of regulatory mechanisms in pre- messenger ribonucleic acids (mRNA) Splicing is also discussed. The chapter presents an overview of tissue- or stage-specific Splicing regulators. Changes in the levels of general Splicing Factors are elaborated in depth. The chapter discusses about the analysis of spliced products in complexmixtures by primer extension using splice junction oligos. The chapter also reviews biochemical assays that are utilized to study mechanisms of alternative Splicing in vitro.
Donald C. Rio - One of the best experts on this subject based on the ideXlab platform.
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Evolution of a tissue-specific Splicing network.
Genes & development, 2011Co-Authors: J. Matthew Taliaferro, Nehemiah Alvarez, Richard E. Green, Marco Blanchette, Donald C. RioAbstract:Alternative Splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan Splicing Factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila Splicing Factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general Splicing Factor U2AF (U2-associated Factor). The quickly evolving LS2 gene has diverged from the Splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a Splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new Splicing Factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner.
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rna binding activity of heterodimeric Splicing Factor U2AF at least one rs domain is required for high affinity binding
Molecular and Cellular Biology, 1998Co-Authors: David Z. Rudner, Roland Kanaar, Kevin S. Breger, Melissa D Adams, Donald C. RioAbstract:The generation of functional mRNA in eukaryotes requires the accurate removal of noncoding regions (introns) from pre-mRNA by a process termed pre-mRNA Splicing (14, 25). Splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise manner on the pre-mRNA (10, 14). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) Factors. The recognition of exon/intron boundaries, the splice sites, by the Splicing apparatus is a critical step in processing of both constitutively and alternatively spliced pre-mRNAs. U1 snRNP defines the 5′ splice site, and U2 snRNP defines the branchpoint sequence (3, 10, 14, 17). Since in most cases the first AG dinucleotide downstream of the branchpoint is used as the 3′ splice site, by defining the branchpoint, U2 snRNP defines the 3′ splice site (18, 27). Targeting of U2 snRNP to the branch site requires the extrinsic Splicing Factor U2 snRNP auxiliary Factor (U2AF). U2AF binds site specifically to the intron pyrimidine tract between the branchpoint sequence and 3′ splice site at an early step in spliceosome assembly and recruits U2 snRNP to the branch site (22, 29, 42). Regulation of 3′ splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (33, 35, 45). Because U2AF is a major determinant in 3′ splice site selection, it has been the subject of extensive biochemical and genetic investigation. Human U2AF is a heterodimer composed of a 65-kDa large subunit (hU2AF65) and a 35-kDa small subunit (hU2AF35) (41). Both subunits are conserved in other organisms (40), and U2AF homologs have been identified in Drosophila melanogaster (9, 21), Schizosaccharomyces pombe (16, 36), and Caenorhabditis elegans (3a, 39). The Drosophila U2AF large (dU2AF50)- and small (dU2AF38)-subunit homologs are 50 and 38 kDa, respectively (9, 21). The U2AF large subunit contains three RNA recognition motifs (RRMs) and an amino-terminal arginine-serine-rich (RS) domain (42). The small subunit contains a highly degenerate RRM (pseudo-RRM) (2), two Zn2+ binding motifs (37), and a carboxyl-terminal RS domain and glycine-rich region (43). Both U2AF subunits are involved in recognition of the intron pyrimidine tract. The large subunit (hU2AF65 and dU2AF50) is required for site-specific pyrimidine tract binding (9, 42). The small subunit acts as a coFactor to stabilize the large subunit on the pyrimidine tract, apparently through protein-protein interactions with constitutive and alternative Splicing Factors (38, 45). While it has been firmly established that all three RRMs on hU2AF65 are necessary for high-affinity RNA binding, a role for the large-subunit RS domain in RNA binding remains unresolved (11, 42). In one study removal of the hU2AF65 RS domain had a modest effect on RNA binding (42). In a second study, the RS domain was found to be absolutely required for RNA binding (11). In vitro Splicing assays using U2AF-depleted extracts prepared by two independent methods have identified independent and essential roles for the two U2AF RS domains: the large-subunit RS domain is required to target U2 snRNP to the branch site (34, 42), and in the immunodepleted extracts under certain conditions, the small-subunit RS domain is apparently necessary for protein-protein interactions with constitutive and alternative Splicing Factors to stabilize hU2AF65 on the pyrimidine tract (38, 45). In contrast to the essential roles assigned to the two U2AF RS domains in vitro, molecular genetic analysis of the Drosophila U2AF RS domains indicates that either one of the RS domains is dispensable in vivo (19). Importantly, at least one RS domain on U2AF is essential for viability (19). The observation that the dU2AF38 RS domain is not essential in vivo (19) refocused our attention on domains present in the U2AF small subunit that are phylogenetically conserved. In an exhaustive database search for proteins containing RRMs, some of the signature sequences of this motif were identified in hU2AF35 (2). These sequences are also present in the Drosophila and S. pombe small-subunit homologs (21, 36). Although some of the most conserved residues in the RNA recognition motif are present in the U2AF small subunits, the RNP-1 octamer is highly degenerate and the RNP-2 hexamer is absent. Since these defining submotifs and other conserved residues are not present in the U2AF small-subunit RRM, it was termed a degenerate RRM or pseudo-RRM (2). Degenerate RRMs have been identified in a collection of RNA binding proteins, including several of the SR proteins, the pyrimidine tract binding protein, and the large subunit of U2AF (10). The degenerate RRMs in SRp30a (ASF/SF2) (4, 46), pyrimidine tract-binding protein (15), and hU2AF65 (42) were all found to be required for high-affinity RNA binding. Two putative Zn2+ binding domains, one on either side of the pseudo-RRM, were recently identified in hU2AF35 in a database search (37). These Cys3His Zn2+ binding motifs are conserved in all three small subunit homologs. Though sequence-specific RNA binding has not been described for proteins that contain this type of Zn2+ binding motif, several proteins that have this domain are involved in RNA metabolism (37). The evolutionary conservation of the pseudo-RRM and the two Zn2+ binding motifs in all three U2AF small subunit homologs suggested to us that these domains are important for function. The lack of requirement for the dU2AF38 RS domain in vivo prompted us to search for novel biochemical activities associated with the small subunit. The phylogenetically conserved, degenerate RRM (2) and two Zn2+ binding motifs (37) in the small subunit suggested that it might participate in RNA binding. While we detected weak RNA binding activity for the Drosophila small subunit on its own, we found that when complexed with the large subunit, dU2AF pyrimidine tract binding affinity increased 20-fold. This increase in RNA binding activity was not specific to Drosophila U2AF; the human U2AF heterodimer bound RNA with 15-fold-higher affinity than hU2AF65. Surprisingly, removal of the dU2AF38 RS domain abolished the increase in binding activity of the dU2AF heterodimer, indicating that the RS domain is necessary for high-affinity binding. Deletion of the dU2AF50 RS domain (dU2AF50ΔRS) dramatically reduced RNA binding activity of the large-subunit monomer. High-affinity binding was restored when the dU2AF38 RS domain was supplied in trans to dU2AF50ΔRS. These data suggest that high-affinity RNA binding activity requires at least one RS domain on U2AF, which is consistent with the requirement for at least one RS domain in vivo.
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Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo
Molecular and cellular biology, 1998Co-Authors: David Z. Rudner, Roland Kanaar, Kevin S. Breger, Donald C. RioAbstract:The heterodimeric pre-mRNA Splicing Factor, U2AF (U2 snRNP auxiliary Factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in Splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in Splicing in vitro.
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Molecular genetic analysis of the heterodimeric Splicing Factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo.
Genes & development, 1998Co-Authors: David Z. Rudner, Kevin S. Breger, Donald C. RioAbstract:The pre-mRNA Splicing Factor U2AF (U2 snRNP auxiliary Factor) has an essential role in 3′ splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3′ splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine–serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro—the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein–protein interactions with constitutive and alternative Splicing Factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent Splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50ΔRS and dU2AF38ΔRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo.
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Mutations in the small subunit of the Drosophila U2AF Splicing Factor cause lethality and developmental defects
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: David Z. Rudner, Roland Kanaar, Kevin S. Breger, Donald C. RioAbstract:Abstract The essential eukaryotic pre-mRNA Splicing Factor U2AF (U2 small nuclear ribonucleoprotein auxiliary Factor) is required to specify the 3' splice at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in Splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in Splicing only indirectly by its interaction with known Splicing Factors. In the absence of a biochemical assay, we have taken a genetic approach to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine- and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in Splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit.
Juan Valcarcel - One of the best experts on this subject based on the ideXlab platform.
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Intron Removal Requires Proofreading of U2AF/3' Splice Site Recognition by DEK
Science (New York N.Y.), 2006Co-Authors: Luis Miguel Mendes Soares, Michael Sattler, Katia Zanier, Cameron D. Mackereth, Juan ValcarcelAbstract:Discrimination between splice sites and similar, nonsplice sequences is essential for correct intron removal and messenger RNA formation in eukaryotes. The 65- and 35-kD subunits of the Splicing Factor U2AF, U2AF 65 and U2AF 35 , recognize, respectively, the pyrimidine-rich tract and the conserved terminal AG present at metazoan 3′ splice sites. We report that DEK, a chromatin- and RNA-associated protein mutated or overexpressed in certain cancers, enforces 3′ splice site discrimination by U2AF. DEK phosphorylated at serines 19 and 32 associates with U2AF 35 , facilitates the U2AF 35 -AG interaction and prevents binding of U2AF 65 to pyrimidine tracts not followed by AG. DEK and its phosphorylation are required for intron removal, but not for Splicing complex assembly, which indicates that proofreading of early 3′ splice site recognition influences catalytic activation of the spliceosome.
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U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary Factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5′ splice sites
Biochemical Journal, 2003Co-Authors: Patrik Förch, Livia Merendino, Concepcion Martinez, Juan ValcarcelAbstract:The Splicing Factor U2AF(65), U2 small nuclear ribonucleoprotein particle (snRNP) auxillary Factor of 65 kDa, binds to pyrimidine-rich sequences at 3' splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF(65) can also promote the recruitment of U1 snRNP to weak 5' splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF(65) is critical for U1 recruitment, and we discuss the role of its RNA-RNA annealing activity in this novel function of U2AF(65).
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Dual function for U2AF(35) in AG-dependent pre-mRNA Splicing.
Molecular and cellular biology, 2001Co-Authors: Sabine Guth, Thomas Ø. Tange, E Kellenberger, Juan ValcarcelAbstract:The Splicing Factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with Splicing Factors of the SR protein family bound to exonic Splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the Splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, Splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA Splicing.
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Nucleocytoplasmic Shuttling of Heterodimeric Splicing Factor U2AF
The Journal of biological chemistry, 2000Co-Authors: Margarida Gama-carvalho, Juan Valcarcel, Marcos Paulo Carvalho, Angelika Kehlenbach, Maria Carmo-fonsecaAbstract:Abstract The U2 small nuclear ribonucleoprotein auxiliary Factor (U2AF) is a heterodimeric Splicing Factor composed of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits. The large subunit of U2AF recognizes the intronic polypyrimidine tract, a sequence located adjacent to the 3′ splice site that serves as an important signal for both constitutive and regulated pre-mRNA Splicing. The small subunit U2AF35 interacts with the 3′ splice site dinucleotide AG and is essential for regulated Splicing. Like several other proteins involved in constitutive and regulated Splicing, both U2AF65 and U2AF35 contain an arginine/serine-rich (RS) domain. In the present study we determined the role of RS domains in the subcellular localization of U2AF. Both U2AF65 and U2AF35 are shown to shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. The RS domain on either U2AF65 or U2AF35 acts as a nuclear localization signal and is sufficient to target a heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm possibly represents a means to control the availability of this Factor to initiate spliceosome assembly and therefore contribute to regulate Splicing.
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Functional Analysis of Splicing Factors and Regulators
mRNA Formation and Function, 1997Co-Authors: Juan Valcarcel, Concepcion Martinez, Michael R GreenAbstract:Publisher Summary This chapter discusses about the functional analysis of Splicing Factors and regulators. Biochemical identification and analysis of components of the Splicing machinery requires the development of functional assays to test their associated activities. Chromatographic and immunological procedures are utilized to generate Splicing-deficient nuclear extracts whose activity depends on the addition of purified Factors. These procedures are valuable in analyzing mechanisms of alternative Splicing regulation. This chapter reviews biochemical methods used for the depletion of non-snRNP proteins from HeLa nuclear extracts, and provides detailed protocols for the preparation of extracts in which the essential Splicing Factor U2AF has been depleted. The chapter also explains concepts related to in vitro depletion systems. Analysis of regulatory mechanisms in pre- messenger ribonucleic acids (mRNA) Splicing is also discussed. The chapter presents an overview of tissue- or stage-specific Splicing regulators. Changes in the levels of general Splicing Factors are elaborated in depth. The chapter discusses about the analysis of spliced products in complexmixtures by primer extension using splice junction oligos. The chapter also reviews biochemical assays that are utilized to study mechanisms of alternative Splicing in vitro.
Clara L. Kielkopf - One of the best experts on this subject based on the ideXlab platform.
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An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal
Nature communications, 2016Co-Authors: Anant A. Agrawal, Michael R Green, Enea Salsi, Rakesh Chatrikhi, Steven Henderson, Jermaine L. Jenkins, Dmitri N. Ermolenko, Clara L. KielkopfAbstract:How the essential pre-mRNA Splicing Factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 A. These structures together with RNA binding and Splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.
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U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs
Nucleic acids research, 2013Co-Authors: Jermaine L. Jenkins, Michael R Green, Anant A. Agrawal, Ankit Gupta, Clara L. KielkopfAbstract:Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA Splicing Factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65).
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Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to Splicing Factors.
Journal of molecular biology, 2008Co-Authors: Valérie Manceau, André Sobel, Clara L. Kielkopf, Alexandre MaucuerAbstract:Abstract The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA Splicing Factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF65. KIS phosphorylates the Splicing Factor SF1, which in turn enhances SF1 binding to U2AF65 and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF65, the UHM-containing C-terminal domain of KIS is required for binding to the Splicing Factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF65 in pull-down assays. These results further support the functional link of KIS with Splicing Factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.
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structural basis for polypyrimidine tract recognition by the essential pre mrna Splicing Factor U2AF65
Molecular Cell, 2006Co-Authors: Allen E Sickmier, Michael R Green, Haihong Shen, Katherine E Frato, Shanthi R Paranawithana, Clara L. KielkopfAbstract:Summary The essential pre-mRNA Splicing Factor, U2AF 65 , guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3′ splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF 65 is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF 65 RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF 65 and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF 65 structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF 65 proteins using surface plasmon resonance.
Ravinder Singh - One of the best experts on this subject based on the ideXlab platform.
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genomic mrna profiling reveals compensatory mechanisms for the requirement of the essential Splicing Factor U2AF
Molecular and Cellular Biology, 2011Co-Authors: Vinod Sridharan, Joseph Heimiller, Ravinder SinghAbstract:The large subunit of the U2 auxiliary Factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3′ splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA Splicing, its requirement for Splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for Splicing in the Schizosaccharomyces pombe U2AF59 (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of Splicing defects in the mutant strain. While U2AF59 inactivation (nonpermissive) conditions inhibit Splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF59-insensitive introns have stronger 5′ splice sites and higher A/U content. Most importantly, features that contribute to U2AF59 insensitivity of an intron unexpectedly reside in its 5′-most 30 nucleotides. These include the 5′ splice site, a guanosine at position 7, and the 5′ splice site-to-branch point sequence context. A differential requirement (similar to U2AF59) for introns may also apply to other general Splicing Factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.
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A conditional role of U2AF in Splicing of introns with unconventional polypyrimidine tracts.
Molecular and cellular biology, 2007Co-Authors: Vinod Sridharan, Ravinder SinghAbstract:In animals, removal of introns from the majority (>80 to 90%) of nascent transcripts via pre-mRNA Splicing is an important step for gene regulation (19). Alternative Splicing serves important biological roles in diverse developmental contexts and provides an important mechanism to generate molecular diversity (7). The 5′ splice site (SS), the branch point sequence (BPS), and the polypyrimidine (Py) tract 3′ SS in the pre-mRNA are important Splicing signals; they are recognized by the U1 snRNP, the U2 snRNP, and the U2 snRNP auxiliary Factor U2AF, respectively, resulting in the formation of a dynamic RNA-protein complex called the spliceosome (23). In humans, the essential Splicing Factor U2AF is a heterodimer of a large protein subunit (U2AF65) and a small protein subunit (U2AF35). U2AF65 binds to the Py tract (62), and U2AF35 recognizes the 3′ SS (33, 61, 65). Both subunits of U2AF are essential for viability in many model organisms, such as the zebrafish, the fruit fly, the nematode worm, and fission yeast (U2AF59) (14, 24, 36, 43, 56, 66). However, in budding yeast the large subunit is dispensable (1) and the small subunit is absent. U2AF65 interacts with other Splicing Factors such as BBP/SF1, UAP56, SAP155 (or SF3b155), and SRp54 (1, 10, 15, 39, 64). The branch point binding protein BBP/SF1 binds to the BPS and cooperates with U2AF65 for RNA binding (2, 6). Detailed in vitro biochemical analyses using model Splicing substrates in metazoans have significantly contributed to our mechanistic view of the role of U2AF65 function in Splicing. The N terminus of U2AF65 harbors an arginine-serine-rich (RS) activation domain, and its C terminus contains three RNA recognition motifs (RRMs), each with a four-stranded antiparallel β-sheet and two α-helices (62). In metazoans, binding to the Py tract serves as the primary determinant for U2AF recruitment onto pre-mRNA. This interaction positions the RS domain to engage in a series of interactions with pre-RNA during spliceosome assembly, including stabilization of the base pairing between the BPS and the U2 snRNA (46, 53). U2AF65 is also an important target for Splicing regulation, where Splicing regulators such as SXL, PTB, hnRNP A1, ASF/SF2, SC35, and TRA can facilitate or antagonize its activity (7, 48). Whereas U2AF65 is highly conserved from fission yeast to humans, its C-terminal RRM3 domain is the only recognizable portion in the budding yeast protein Mud2p (1). The human RRM3 interacts with BBP/SF1 and SAP155 (or Schizosaccharomyces pombe prp10) (1, 15), and RRM3-related domains are present in several Splicing Factors (27). Deletion of the conserved RRM3 domain of the large subunit of U2AF (U2AF59) is lethal in S. pombe (4). Intriguingly, RRM3 shows no detectable RNA binding and is not required for the Splicing of model substrates in a HeLa cell nuclear extract. RRM1 and RRM2 domains of the human U2AF65 are sufficient for Py tract recognition and in vitro Splicing (4). Previously, we proposed that the RRM3 domain might be important for the Splicing of only a subset of introns in vivo. Relative to Saccharomyces cerevisiae, S. pombe shares with mammals many more features of pre-mRNA Splicing, including the presence of degenerate Splicing signals, similarity of Splicing Factors (snRNAs and proteins), and a requirement for both subunits of U2AF (U2AF59 and U2AF23) (57, 59). Thus, by combining the power of genomics, molecular genetics, and biochemical analysis, S. pombe represents an excellent model system for analysis of the role of U2AF and Py tract in vivo. Our study of Splicing in S. pombe offers important new information on RRM3 and U2AF functions and Py tract requirements in vivo. There is a large diversity in the arrangement of intronic sequences relevant for 3′ splice site recognition, and in the requirements for U2AF subunits and domains, beyond what is known from detailed in vitro biochemical analysis of model pre-mRNAs in metazoans. These findings also help explain why deletion of RRM3 is lethal in S. pombe whereas deletion of the human RRM3 has no effect on RNA binding and on the Splicing of model substrates in vitro. Finally, whereas Splicing of introns that lack a Py tract remains dependent on U2AF59, upstream Py tracts, located between the 5′ splice site and the BPS, are required for Splicing in vivo only under conditions of U2AF59 inactivation.
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sex lethal and u2 small nuclear ribonucleoprotein auxiliary Factor U2AF65 recognize polypyrimidine tracts using multiple modes of binding
RNA, 2003Co-Authors: Hiren Banerjee, Andrew Rahn, William Davis, Ravinder SinghAbstract:The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila Splicing repressor Sex lethal (SXL), which has two RRMs, can directly compete with the essential Splicing Factor U2AF 65 , which has three RRMs, for binding to specific Py tracts. We have combined site-specific photocross-linking and chemical cleavage of the proteins to biochemically map crosslinking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF 65 shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF 65 and SXL can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. We present a model to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA–protein complexes, which could allow tuning of the binding affinity without changing sequence specificity.
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the protein sex lethal antagonizes the Splicing Factor U2AF to regulate alternative Splicing of transformer pre mrna
Nature, 1993Co-Authors: Juan Valcarcel, Ravinder Singh, Phillip D Zamore, Michael R GreenAbstract:Somatic sexual differentiation in Drosophila melanogaster involves a cascade of regulated Splicing events and provides an attractive model system for the analysis of alternative Splicing mechanisms. The protein Sex-lethal (Sxl) activates a female-specific 3' splice site in the first intron of transformer (tra) pre-mRNA while repressing an alternative non-sex-specific site. We have developed an in vitro system that recapitulates this regulation in a manner consistent with genetic, transfection and fly transformation studies. Using this system, we have determined the molecular basis of the splice site switch. Here we show that Sxl inhibits Splicing to the non-sex-specific (default) site by specifically binding to its polypyrimidine tract, blocking the binding of the essential Splicing Factor U2AF. This enables U2AF to activate the lower-affinity female-specific site. A Splicing 'effector' domain present in U2AF but absent from Sxl accounts for the different activities of these two polypyrimidine-tract-binding proteins: addition of the U2AF effector domain to Sxl converts it from a Splicing repressor to an activator and renders it unable to mediate splice-site switching.