The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. Recombinant dU2AF50 protein complements mammalian splicing extracts depleted of U2AF activity. Germline transformation of Drosophila with the dU2AF50 complementary DNA rescues a lethal mutation, establishing that the dU2AF50 gene is essential for viability. R/S domains have been found in numerous metazoan splicing factors, but their function is unknown. The mutation in Drosophila U2AF will allow in vivo analysis of a conserved R/S domain-containing general splicing factor (Kanaar, 1993).
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, a genetic approach was undertaken 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 (Rudner, 1996).
The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF(50). Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs (Blanchette, 2004).
A fully penetrant recessive lethal deletion of the gene coding for dU2AF50 has been characterized and can be rescued by dU2AF50 cDNA transgenes under the control of the dU2AF50 genomic promoter. S. pombe temperature-sensitive alleles in highly conserved residues of the large U2AF subunit have been identified and documented in vivo (Potashkin, 1993 and Romfo, 1999). The yeast mutations were individually introduced into the cDNA coding for dU2AF50 (Rudner, 1998b; Rudner, 1998c), and transgenic flies were successfully recovered for all four mutations tested. The transgenes were then assayed for their ability to genetically complement a deletion of dU2AF50 (XR15). Females carrying the XR15 deletion over a balancer chromosome were mated with the different transgenic males. A functional rescue allele was scored by the presence of non-Binsinscy males (males without the balancer chromosome) in the progeny resulting from flies carrying the XR15 deletion allele and rescued by the transgene. Of the four mutations tested only two, D204N and S284Y, successfully rescued the dU2AF50 XR15 deletion (Blanchette, 2004).
The two mutant dU2AF50 alleles were then tested for a temperature-sensitive phenotype by repeating the crosses at 25°C and 29°C and by counting the ratio of rescued males to the nonbalanced females that eclosed at the two temperatures. The wild-type dU2AF50 transgene efficiently rescued the dU2AF50 deletion with a similar rescue percentage at both 25°C and 29°C. By contrast, flies carrying the mutant transgenes efficiently rescued the dU2AF50 deletion at 25°C, but a marked reduction in rescued male viability was observed upon growth of the two mutant transgenic strains at 29°C. This strong temperature-sensitive phenotype was observed for each mutation in two independent transgenic strains. The temperature-dependent phenotype was not due to differences in either dU2AF50 expression or stability in the mutant transgenic strains since similar levels of dU2AF50 protein were observed in rescued males from the wild-type and from the two mutant transgenic strains at all temperatures tested. Homozygous males and females carrying the dU2AF50 XR15 deletion rescued by the S284Y transgene were successfully engineered. This homozygous mutant strain displayed the same temperature-sensitive phenotype when grown at the restrictive temperature (30°C), the homozygous females laid eggs with abnormal eggshell morphology, and the mutant embryos failed to developed into larvae. Taken together, these results confirm that the S. pombe temperature-sensitive mutations are transferable to the Drosophila dU2AF50 gene to generate new temperature-sensitive alleles (Blanchette, 2004).
dU2AF50 binds to the intron polypyrimidine tract upstream of the 3′ splice site and U2AF is required for 3′ splice site recognition of all characterized pre-mRNAs. The identified temperature-sensitive mutations D204N and S284Y flank the second RRM and thus might affect dU2AF50/dU2AF38 RNA binding. Using a bicistronic system to simultaneously express both dU2AF subunits in bacteria, soluble wild-type and mutant recombinant dU2AF50/dU2AF38 heterodimers (hereafter referred to as U2AF) were efficiently produced and tested for their ability to bind RNA (Rudner, 1998a). All three recombinant protein preparations were similar in composition, with dU2AF50 being expressed as a single species and dU2AF38 being slightly proteolysed, as observed previously (Rudner, 1998a). Wild-type dU2AF efficiently forms specific RNA-protein complexes with an RNA oligonucleotide carrying the polypyrimidine tract and AG of the 3′ splice site of the first intron of the adenovirus major late pre-mRNA (MINX) when incubated at different temperatures, ranging from 20°C to 35°C. However, both the D204N and S284Y mutants are defective in forming specific RNA-protein complexes at all temperatures tested. Interestingly, both dU2AF mutants formed apparent protein-RNA aggregates at the top of the gel similar to what was seen with the wild-type protein at 4°C. Those large complexes are attributed to nonspecific RNA-protein complexes which are displaced upon specific interaction between the RNA and dU2AF. At 20°C, small amounts of specific RNA-protein complexes can be seen with the D204N and S284Y U2AF mutants. By titrating the amount of protein, specific complexes can be formed at 20°C with the D204N and S284Y dU2AF, although less efficiently than with the wild-type dU2AF (apparent KD of 0.6 microM, 2.3 microM, and 7.8 microM for the wild-type, D204N and S284Y dU2AF, respectively). Incubating the binding reaction at 35°C does not affect wild-type dU2AF (apparent KD of 1.0 microM), but completely abrogates binding of both the D204N and S284Y mutant dU2AFs (apparent KD > 50 microM). These results demonstrate that the D204N and S284Y temperature-sensitive dU2AF mutations affect RNA binding in a temperature-dependent manner (Blanchette, 2004).
Although the RNA-affinity of the mutant protein is greatly reduced, in vitro splicing using a U2AF depletion-reconstitution system failed to show any defect of the mutant dU2AF recombinant proteins. Different pre-mRNAs at all temperatures tested were spliced as efficiently in the presence of the mutant proteins as they were spliced in the presence of the wild-type dU2AF. The use of efficiently spliced in vitro models might have impaired the capacity to observe splicing defect with the mutant dU2AF50 proteins. However, in vivo, more sensitive dU2AF50 targets might be less efficiently spliced, retained in the nucleus because they still contain an intron, and then degraded in the mutant dU2AF50 flies at the restrictive temperature. Individual gene expression using high-density microarrays was quantified from RNA extracted from both wild-type and mutant homozygous adult grown at the permissive and restrictive temperatures. Temperature-dependent variations in expression of individual genes were obtained by pairwise comparison of the triplicate experimental samples (nine comparisons). Significant variations in gene expression were determined using the Affymetrix statistical algorithm, and a positive score was attributed to genes that were significantly affected in at least seven out of nine pairwise comparisons (highly stringent). The analysis revealed that, at the restrictive temperature, 88 and 53 genes were downregulated in the wild-type and the mutant flies, respectively and that 35 genes were specifically downregulated only in the mutant flies. By contrast, 143 and 426 genes were significantly upregulated in the wild-type and mutant flies grown at the restrictive temperature, out of which 374 were specifically upregulated in the S284Y mutant flies. The differential expression of some genes were confirmed by semiquantitative RT-PCR (Blanchette, 2004).
While the upregulated genes from the mutant flies do not display any striking functional clustering, the downregulated genes cluster into two major categories. Eight genes (23% of the downregulated genes) are known or predicted to be trypsin-like proteases, while seven genes (20% of the downregulated genes) are known to be involved in oogenesis. The observation that several genes involved in oogenesis were downregulated fits well with the observed abnormal eggshell phenotype in the mutant strains (Blanchette, 2004).
Since U2AF is known to be a splicing factor, the intron characteristics were examined in the up- and downregulated genes in the S284Y mutant flies shifted to the restrictive temperature. In the latest release of the Drosophila genome annotation (v. 3.1), the average number of introns per gene ranges from 0 introns (17.9% of all genes) to 49 introns (CG17150) and the distribution of the average number of introns per gene is centered around 1 intron per gene (21%). Interestingly, the upregulated genes from the S284Y mutant flies grown at the restrictive temperature show a consistent overrepresentation for genes containing multiple introns with a distribution centered around three introns per gene. By contrast, the identified downregulated genes show predominantly zero or one intron per gene (37% and 45.7%, respectively). The differences in the intron number distributions are specific for the differentially expressed genes in the mutant flies since no significant differences were observed for the genes that were up- and down-regulated in the wild-type flies grown at 30°C. The observation that the distribution of introns for the specifically affected genes in the S284Y dU2AF50 mutant flies is significantly different from the average genomic distribution suggests that the dU2AF50 mutation might have a direct effect on those genes. In Drosophila, the modal intron length is 75 nt and does not vary significantly for the genes up- and down-regulated in the dU2AF50 mutant S284Y flies grown at the restrictive temperature. These results indicate that a mutation which reduces the RNA binding affinity of dU2AF50 can directly affect the expression of both intronless, as well as, intron-containing genes (Blanchette, 2004).
Whether splicing was affected was tested for some of the genes downregulated in the S284Y dU2AF50 mutant flies grown at the restrictive temperature. Using oligonucleotide primer pairs flanking the single intron of two genes (CG4783 and Ag5r2, which are the fourth and second most downregulated genes in the S284Y flies, respectively), RT-PCR was performed on RNA extracted from wild-type and mutant flies grown at both the permissive and restrictive temperatures. Splicing of CG4783 is efficient and similar in wild-type flies grown at both 25°C and 30°C temperatures. However, in the S284Y mutant flies at 25°C, the splicing efficiency of CG4783 is reduced and, moreover, is even more reduced in the S284Y flies grown at 30°C. Additionally, splicing of Ag5r2 is efficient and similar in both wild-type and mutant flies grown at the permissive temperature (25°C). However, when the flies are shifted to the restrictive temperature (30°C), the splicing efficiency of Ag5r2 is reduced in the wild-type and the reduction in splicing is even more evident in the S284Y mutant flies. This confirms that the S284Y dU2AF50 mutation affects, in a temperature-sensitive fashion, the splicing efficiency of some target genes in vivo (Blanchette, 2004).
RT-PCR assays were also performed on two genes that were upregulated in the dU2AF50 mutant flies shifted to the restrictive temperature (30°C). Oligonucleotide primer pairs flanking their multiple introns, two and six introns in CG7036 and transportin, respectively, were used to analyze splicing defects. The splicing efficiency of both pre-mRNAs was similar in both wild-type and mutant flies grown at either the permissive or restrictive temperatures. Taken together, these results suggest that the temperature-sensitive S284Y mutation in dU2AF50 reduces splicing of some of the downregulated pre-mRNAs in vivo in the mutant flies grown at the restrictive temperature but does not appear to affect the splicing of the upregulated genes that contain multiple introns (Blanchette, 2004).
It was reasoned that the reduction in the level of some intronless mRNA in the dU2AF50 mutant flies might result from nuclear retention and degradation as has been observed when RNA export factor expression is knocked down (Herold, 2003). The expression of dU2AF50 was efficiently knocked down by RNAi in cultured Drosophila SL2 cells to less than 20% of the endogenous level, and nuclear and cytoplasmic RNA was isolated from control and dU2AF50 knocked-down cells. High-density oligonucleotide microarrays were used to measure the RNA level of 13,738 genes in both the nuclear and cytoplasmic fractions isolated from control and dU2AF50 knocked-down cells. Four thousand, seven hundred, and thirty-six genes show consistent expression in L2 cells, and for each expressed gene, the nucleo-cytoplasmic ratio from the control and dU2AF50- knocked-down sample was used to calculated a nuclear retention index describing the effect of the dU2AF50 RNAi. Using an arbitrary retention index greater than 0.1, more than 28% of the analyzed genes (1334 genes) were predicted to be retained in the nucleus in the dU2AF50 knocked-down cells with 12% of the retained mRNAs (166 genes) being intronless. The microarray analysis was validated by RT-PCR with eight individual genes showing variable retention indices. The RpL32 pre-mRNA contains two introns while the other genes are intronless. The nucleo-cytoplasmic distribution predicted by the microarray and measured by RT-PCR nicely correlates for RpL32, as well as the intronless genes CG15784, CyCB3, Gip, Mpp6, and slp1, and confirms their nuclear retention in the dU2AF50 knocked-down cells. In addition, the number of genes predicted to be retained in the nucleus by the microarray analyses is likely to be an underestimate of the real number due to the normalization process. The normalization is done using the calculated population average, and this normalization process is based on the ad hoc assumption that only a small proportion of genes are significantly different between the compared samples. As an example, the mRNAs coding for CG30342 and noi, which have slightly negative retention index (−0.39 and −0.07 respectively) and thus were not predicted to be retained in the nucleus, nonetheless showed nuclear retention when assayed by RT-PCR. These results indicate that the essential pre-mRNA splicing factor dU2AF50 also plays a significant and unexpected role in the nuclear export of a large number of intronless mRNAs (Blanchette, 2004).
The previous results predict that dU2AF50 should associate with intronless mRNAs, either directly or indirectly, as part of a multicomponent RNP complex in order to promote their nuclear export. In order to test this prediction, a specific immunopurification of nuclear RNP complexes from a 0-12 hr Drosophila embryonic RNP preparation was performed using affinity-purified anti-dU2AF50 antibody (Rudner, 1998c). Genome-wide identification of the associated RNAs was performed using spotted Drosophila cDNA microarrays containing approximately 6000 different EST PCR fragments. Five independent experiments were performed, and RNAs consistently present in at least three assays were used for further analyses. In the top 200 RNAs, the dU2AF50-associated genes show an overrepresentation for pre-mRNAs with multiple introns, with a distribution centered around two introns per gene. Surprisingly, but consistent with the hypothesis, four of the first 200 dU2AF50-bound RNAs were intronless, confirming that dU2AF50 can be found stably associated with intronless RNAs. Semiquantitative RT-PCR was used to confirm that those RNAs were immunoaffinity purified together with dU2AF50. Moreover, three of them (CG30342, Slp1, and noi) are expressed in L2 cells and show nuclear retention when the expression of dU2AF50 is knocked down (Blanchette, 2004).
A statistical model of all known U2AF binding sites (3′ splice sites) was generated and used to search for binding sites in intronless genes. This approach found that more than one third of all intronless mRNAs contain at least one site that matches this U2AF model as well as the average 3′ splice site. However, no enrichment for strong U2AF binding sites was observed in the set of intronless genes as a whole. The four intronless mRNAs found by microarray analysis of affinity-selected dU2AF50-containing RNP complexes all contain putative U2AF binding sites that match the model as well as the average splice site (Blanchette, 2004).
The binding assay supports the notion that dU2AF50 can associate with intronless RNAs, and the bioinformatic analyses are consistent with this observation. Together this suggests that dU2AF50 participates in the export of a large number of intronless genes. While only a small fraction of the top mRNAs found in stable U2AF-containing nuclear RNP particles were intronless, the vast majority of intronless mRNAs are predicted to contain putative U2AF binding sites. Thus, even transient association of U2AF with these transcripts for the recruitment of RNA export factors could account for the effects of dU2AF50 mutations or depletion of the nucleo-cytoplasmic transport of intronless mRNAs (Blanchette, 2004).
Reference names in red indicate recommended papers.
Achsel, T. and Shimura, Y. (1996). Factors involved in the activation of pre-mRNA splicing from downstream splicing enhancers. J. Biochem. (Tokyo). 120(1): 53-60. 8864844
Banerjee, H., Rahn, A., Davis., W. and Singh, R. (2003). Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding. RNA 9(1): 88-99. 12554879
Banerjee, H., Rahn, A., Gawande, B., Guth, S., Valcarcel, J. and Singh, R. (2004). The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro. RNA 10(2): 240-53. 14730023
Berglund, J. A., Abovich, N. and Rosbash, M., (1998). A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev. 12: 858-867. 9512519
Blanchette, M., Labourier, E., Green, R. E., Brenner, S. E. and Rio, D. C. (2004). Genome-Wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs. Mol. Cell 14(6): 775-86. 15200955
Chaouki, A. S. and Salz, H. K. (2006). Drosophila SPF45: A bifunctional protein with roles in both splicing and DNA repair. PLoS Genet. 2(12): e178. Medline abstract: 17154718
Domon, C., Lorkovic, Z. J., Valcarcel, J. and Filipowicz, W. (1998). Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants. J. Biol. Chem. 273(51): 34603-10. 9852132
Fleckner, J., Zhang, M., Valcˇrcel, J. and Green, M. R. (1997). U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev. 11: 1864-1872. 9242493
Fong, Y. W. and Zhou, Q. (2001). Stimulatory effect of splicing factors on transcriptional elongation. Nature 414: 929-933. 11780068
Forch, P., Merendino, L., Martinez, C. and Valcarcel, J. (2001). Modulation of msl-2 5' splice site recognition by Sex-lethal. RNA 7(9): 1185-91. 11565743
Forch, P., Merendino, L., Martinez, C. and Valcarcel, J. (2003). U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5' splice sites. Biochem. J. 372(Pt 1): 235-40. 12558503
Gama-Carvalho, M., Carvalho, M. P., Kehlenbach, A., Valcarcel, J. and Carmo-Fonseca M. (2001). Nucleocytoplasmic shuttling of heterodimeric splicing factor U2AF. J. Biol. Chem. 276(16): 13104-12. 11118443
Gatfield, D., Le Hir, H., Schmitt, C., Braun, I. C., Kocher, T., Wilm, M. and Izaurralde, E. (2001). The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr. Biol. 11: 1716-1721. 11696332
Gozani, O., Potashkin, J. and Reed, R. (1998). A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol. Cell. Biol. 18: 4752-4760. 9671485
Granadino, B., Penalva, L. O., Green, M. R., Valcarcel, J. and Sanchez, L. (1997). Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal. Proc. Natl. Acad. Sci. 94(14): 7343-8. 9207093
Guth, S., Martinez, C., Gaur, R. K. and Valcarcel, J. (1999). Evidence for substrate-specific requirement of the splicing factor U2AF35 and for its function after polypyrimidine tract recognition by U2AF65. Mol. Cell. Biol. 19(12): 8263-71. 10567551
Guth, S. and Valcarcel J. (2000). Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF. J. Biol. Chem. 275(48): 38059-66. 10954700
Guth, S., Tange, T. O., Kellenberger, E. and Valcarcel, J. (2001). Dual function for U2AF35 in AG-dependent pre-mRNA splicing. Mol. Cell. Biol. 21(22): 7673-81. 11604503
Habara, Y., Urushiyama, S., Tani, T., and Ohshima, Y. (1998). The fission yeast prp10+ gene involved in pre-mRNA splicing encodes a homologue of highly conserved splicing factor, SAP155. Nucleic Acids Res. 26: 5662-5669. 9837997
Herold, A., Teixeira, L. and Izaurralde, E. (2003). Genome-wide analysis of nuclear mRNA export pathways in Drosophila. EMBO J. 22: 2472-2483. 12743041
Huang, Y. and Steitz, J. A. (2001). Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol. Cell 7: 899-905 . 11336712
Huang, Y., Gattoni, R., Stevenin, J. and Steitz, J. A. (2003). SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol. Cell 11: 837-843. 12667464
Ito, T., Muto, Y., Green, M.R. and Yokoyama, S. (1999). Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF65). EMBO J. 18: 4523-4534. 10449418
Jensen, T. H., Boulay, J., Rosbash, M. and Libri, D. (2001). The DECD box putative ATPase Sub2p is an early mRNA export factor. Curr. Biol. 11: 1711-1715. 11696331
Kanaar, R., Roche, S. E., Beall, E. L., Green, M. R. and Rio, D. C. (1993). The conserved pre-mRNA splicing factor U2AF from Drosophila: requirement for viability. Science 262(5133): 569-73. 7692602
Kennedy, C. F. and Berget, S. M. (1997). Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron. Mol. Cell. Biol. 17(5): 2774-80. 9111348
Kennedy, C. F., Kramer, A. and Berget, S. M. (1998). A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point. Mol. Cell. Biol. 18(9): 5425-34. 9710626
Kent, O. A., Ritchie, D. B. and Macmillan, A. M. (2005). Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway. Mol. Cell. Biol. 25(1): 233-40. 15601845
Kielkopf, C. L., Rodionova, N. A., Green, M. R. and Burley S. K. (2001). A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell 106(5): 595-605. 11551507
Kielkopf, C. L., Lucke, S. and Green, M. R. (2004). U2AF homology motifs: protein recognition in the RRM world. Genes Dev. 18(13):1513-26. 15231733
Lallena, M. J., Chalmers, K. J., Llamazares, S.., Lamond, A. I. and Valcarcel, J. (2002). Splicing regulation at the second catalytic step by Sex-lethal involves 3' splice site recognition by SPF45. Cell 109(3): 285-96. 12015979
Li, Y. and Blencowe, B. J. (1999). Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract. J. Biol. Chem. 274(49): 35074-9. 10574987
Lou, H., Helfman, D. M., Gagel, R. F. and Berget, S. M. (1999). Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon. Mol. Cell. Biol. 19: 78-85. 9858533
McKinney, R., Wentz-Hunter, K., Schmidt, H. and Potashkin, J. (1997). Molecular characterization of a novel fission yeast gene spUAP2 that interacts with the splicing factor spU2AF59. Curr. Genet. 32: 323-33. 9371883
Merendino, L., Guth, S., Bilbao, D., Martinez, C. and Valcarcel, J. (1999). Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG. Nature 402(6763): 838-41. 10617208
Moore M. (2000). Intron recognition comes of AGe. Nature 7 14-16. 10625417
Nagengast, A. A., Stitzinger, S. M., Tseng, C. H., Mount, S. M. and Salz, H. K. (2003). Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping. Development. 2003 Feb;130(3):463-71. 12490553
Pacheco, T. R.., et al. (2004). Diversity of vertebrate splicing factor U2AF35: identification of alternatively spliced U2AF1 mRNAS. J. Biol. Chem. 279(26): 27039-49. 15096518
Page-McCaw, P. S., Amonlirdviman, K. and Sharp, P. A. (1999) PUF60: a novel U2AF65-related splicing activity. RNA 5: 1548-1560. 10606266
Peled-Zehavi, H., Berglund, J. A., Rosbash, M. and Frankel, A. D. (2001). Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (Mammalian branch point binding protein) in a splicing factor complex. Mol. Cell. Biol. 21: 5232-5241. 11438677
Potashkin, J., Naik, K. and Wentz-Hunter, K. (1993). U2AF homolog required for splicing in vivo. Science 262: 573-575. 8211184
Rain, J. C., Rafi, Z., Rhani, Z., Legrain, P. and Krämer, A. (1998). Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1. RNA 4: 551-565. 9582097
Reed R. (1989). The organization of 3' splice-site sequences in mammalin introns. Genes Dev. 3: 2113-2123. 2628164
Romfo C. M., and Wise, J. A. (1997). Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast. Nucleic Acids Res. 25: 4658-4665. 9358179
Romfo, C., Lakhe-Reddy S., and Wise, J .A. (1999). Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: Differential sensitivity of introns to mutational inactivation. RNA 5: 49-65. 9917066
Rudner, D. Z., Kanaar, R., Breger, K. S. and Rio, D. C. (1996). Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects. Proc. Natl. Acad. Sci. 93(19): 10333-7. 8816800
Rudner, D. Z., Breger, K. S., Kanaar, R., Adams, M. D. and Rio, D. C. (1998a).
RNA binding activity of heterodimeric splicing factor U2AF: at least one RS
domain is required for high-affinity binding. Mol. Cell. Biol. 18(7): 4004-11. 9632785
Rudner, D. Z., Breger, K. S. and Rio, D. C. (1998b). Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo Rudner, D. Z., Kanaar, R., Breger, K. S. and Rio, D. C. (1998c). Interaction between subunits of heterodimeric splicing factor U2AF is essential in vivo
Ruskin, B., Zamore, P. D. and Green, M. R. (1988). A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell 52(2): 207-19. 2963698
Selenko, P., Gregorovic, G., Sprangers, R., Stier, G., Rhani, Z., Kramer, A. and Sattler, M. (2003). Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP. Mol. Cell 11: 965-976. 12718882
Singh, R., Valcarcel, J. and Green, M. R. (1995). Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268(5214): 1173-6. 7761834
Singh, R., Banerjee, H. and Green, M. R. (2000). Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor Sex-lethal. RNA 6(6): 901-11. 10864047
Strasser, K. and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p. Nature 413: 648-652. 11675790
Strasser, K., Masuda, S., Mason, P., Pfannstiel, J., Oppizzi, M., Rodriguez-Navarro, S., Rondon, A. G., Aguilera, A., Struhl, K., Reed, R. and Hurt, E. (2002). TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417: 304-308. 11979277
Tronchere, H., Wang, J. and Fu, X. D. (1997). A protein related to splicing factor U2AF35 that interacts with U2AF65 and SR proteins in splicing of pre-mRNA. Nature 388: 397-400. 9237760
Valcarcel, J., Singh, R., Zamore, P. D. and Green, M. R. (1993). The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature 362(6416): 171-5. 7680770
Valcarcel, J., Gaur, R. K., Singh, R. and Green, M. R. (1996). Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA Science 273(5282): 1706-9. 8781232
Webb, C. J. and Wise, J. A. (2004a). The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans. Mol. Cell. Biol. 24: 4229-4240. 15121844
Webb, C. J., Lakhe-Reddy, S., Romfo, C. M. and Wise, J. A. (2004b). Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo. Mol. Biol. Cell. 16(2): 584-96. 15548596
Wu, J. Y. and Maniatis, T. (1993). Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75(6): 1061-70. 8261509
Wu, S., Romfo, C., Nilsen, T. and Green M. (1999). Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature 402: 832-835. 10617206
Zamore, P. D. and Green, M. R. (1991). Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 10(1): 207-14. 1824937
Zhang, M., Zamore, P. D., Carmo-Fonseca, M., Lamond, A. I., and Green, M. R. (1992). Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc. Natl. Acad. Sci. 89: 8769-8773. 1388271
Zolotukhin, A. S., et al. (2002). U2AF participates in the binding of TAP (NXF1) to mRNA. J. Biol. Chem. 277: 3935-3942. 11724776
Zorio, D. A., Lea, K. and Blumenthal, T. (1997). Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon. Mol. Cell. Biol. 17(2): 946-53. 9001248
Zorio, D. A. and Blumenthal, T. (1999a). Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans. Nature 402(6763): 835-8. 10617207
Zorio, D. A. and Blumenthal, T. (1999b). U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans. RNA 5(4): 487-94. 10199565
date revised: 25 May 2007
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.