Diversity of human U2AF splicing factors

U2 snRNP auxiliary factor (U2AF) is an essential heterodimeric splicing factor composed of two subunits, U2AF(65) and U2AF(35). During the past few years, a number of proteins related to both U2AF(65) and U2AF(35) have been discovered. Here, we review the conserved structural features that characterize the U2AF protein families and their evolutionary emergence. We perform a comprehensive database search designed to identify U2AF protein isoforms produced by alternative splicing, and we discuss the potential implications of U2AF protein diversity for splicing regulation.     

Characterization of U2AF6, a Splicing Factor Related to U2AF35

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) 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 U2AF(35) 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 U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) 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 U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) 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 U2AF(35), U2AF(26) may play a role in regulating alternative splicing.     

FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35

We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.     

Molecular characterization and phylogeny of U2AF35 homologs in plants

U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is an essential splicing factor with critical roles in recognition of the 3'-splice site. In animals, the U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 small nuclear RNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit. Two copies of U2AF35-encoding genes were identified in Arabidopsis (Arabidopsis thaliana; atU2AF35a and atU2AF35b). Both are expressed in all tissues inspected, with atU2AF35a expressed at a higher level than atU2AF35b in most tissues. Differences in the expression patterns of atU2AF35a and atU2AF35b in roots were revealed by a promoter::beta-glucuronidase assay, with atU2AF35b expressed strongly in whole young roots and root tips and atU2AF35a limited to root vascular regions. Altered expression levels of atU2AF35a or atU2AF35b cause pleiotropic phenotypes (including flowering time, leaf morphology, and flower and silique shape). Novel slicing isoforms were generated from FCA pre-mRNA by splicing of noncanonical introns in plants with altered expression levels of atU2AF35. U2AF35 homologs were also identified from maize (Zea mays) and other plants with large-scale expressed sequence tag projects. A C-terminal motif (named SERE) is highly conserved in all seed plant protein homologs, suggesting it may have an important function specific to higher plants.     

Nucleocytoplasmic shuttling of heterodimeric splicing factor U2AF

The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is a heterodimeric splicing factor composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) 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 U2AF(35) 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 U2AF(65) and U2AF(35) 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 U2AF(65) and U2AF(35) 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 U2AF(65) or U2AF(35) 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.     

剪接因子U2AF65相关蛋白的研究进展

U2核糖核蛋白小体辅助因子(U2AF)65是参与前体mRNA剪接的重要辅助因子,前体RNA生成之初,U1核糖核蛋白小体(snRNP)结合到内含子的5'剪接位点,U2AF65和U2AF35分别结合到多聚嘧啶序列和3'剪接位点,剪接因子1(SF1)结合到分支位点是剪接体形成的第一步。U2AF的存在又辅助U2snRNP代替SF1结合到分支位点,使剪接反应顺利进行。最近几年,发现基因组中存在一些U2AF65的旁系同源基因序列。这些旁系同源基因由祖先基因经连续复制而横向形成,复制出的基因副本经历了各自的进化途径,最终它们在结构和功能上有相似之处,又各有独特之处。我们简要讨论了U2AF65、PUF60、CAPERα和CAPERβ这4种同源蛋白的发现过程、结构特征、自身的多样性、基因的进化和生物学功能。     

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

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.     

U2AF participates in the binding of TAP (NXF1) to mRNA.

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates.     

Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65)

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.     

The role of U2AF35 and U2AF65 in enhancer-dependent splicing.

Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.     

Interactome for auxiliary splicing factor U2AF65 suggests diverse roles

U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is an essential component of the splicing machinery that is composed of two protein subunits, the 35 kDa U2AF³⁵ (U2AF1) and the 65 kDa U2AF⁶⁵ (U2AF2). U2AF interacts with various splicing factors within this machinery. Here we expand the list of mammalian splicing factors that are known to interact with U2AF⁶⁵ as well as the list of nuclear proteins not known to participate in splicing that interact with U2AF⁶⁵. Using a yeast two-hybrid system, we found fourteen U2AF⁶⁵-interacting proteins. The validity of the screen was confirmed by identification of five known U2AF⁶⁵-interacting proteins, including its heterodimeric partner, U2AF³⁵. In addition to binding these known partners, we found previously unrecognized U2AF⁶⁵ interactions with four splicing-related proteins (DDX39, SFRS3, SFRS18, SNRPA), two zinc finger proteins (ZFP809 and ZC3H11A), a U2AF⁶⁵ homolog (RBM39), and two other regulatory proteins (DAXX and SERBP1). We report which regions of U2AF⁶⁵ each of these proteins interacts with and we discuss their potential roles in regulation of pre-mRNA splicing, 3′-end mRNA processing, and U2AF⁶⁵ sub-nuclear localization. These findings suggest expanded roles for U2AF⁶⁵ in both splicing and non-splicing functions.     

U2AF65蛋白表达水平影响基因UBQLN1的可变剪接

目的:研究U2AF65蛋白的表达水平对基因UBQLN1可变剪接的影响。方法:应用pSR-GFP/Neo载体构建2个U2AF65-siRNA干扰载体,转染293T细胞,通过Western印迹、QRT-PCR检测干扰效果,RT-PCR验证基因UBQLN1的可变剪接。结果:利用设计的U2AF65-siRNA能够干扰细胞中U2AF65的表达;RT-PCR结果显示U2AF65表达水平的下降促使UBQLN1第8外显子的跳跃增加。结论:U2AF65可以通过表达水平的变化参与调控基因UBQLN1的可变剪接。     

茄子SmU2AF65基因的可变剪接

摘要: U2AF(U2 snRNP auxiliary factor)是参与前体mRNA剪接的重要辅助因子, 在进化上具有较高保守性。文章根据茄子BAC 77N19(GenBank登录号: EF517791)的基因组序列信息和烟草NpU2AF⁶⁵a和NpU2AF⁶⁵b基因的全长cDNA序列, 设计特异引物, 经cDNA末端快速扩增法(RACE)获得了1 986 bp的茄子同源基因(SmU2AF⁶⁵)全长cDNA, GenBank登录号为EU543263。序列分析表明该序列包含1 665 bp的可阅读框, 编码554个氨基酸, 在氨基酸序列的C末端有3个保守的RNA识别结构域RRM。RT-PCR分析表明, SmU2AF⁶⁵基因在不同组织中均有表达,但是该基因通过可变剪接至少能够产生两个转录本, 在根中产生与其他组织中不同的剪切子     

A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer

U2 auxiliary factor (U2AF) is an essential splicing factor that recognizes the 3' splice site and recruits the U2 snRNP to the branch point. The X-ray structure of the human core U2AF heterodimer, consisting of the U2AF35 central domain and a proline-rich region of U2AF65, has been determined at 2.2 A resolution. The structure reveals a novel protein-protein recognition strategy, in which an atypical RNA recognition motif (RRM) of U2AF35 and the U2AF65 polyproline segment interact via reciprocal "tongue-in-groove" tryptophan residues. Complementary biochemical experiments demonstrate that the core U2AF heterodimer binds RNA, and that the interacting tryptophan side chains are essential for U2AF dimerization. Atypical RRMs in other splicing factors may serve as protein-protein interaction motifs elsewhere during spliceosome assembly.     

Fas splicing regulation during early apoptosis is linked to caspase-mediated cleavage of U2AF65

U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) is an essential splicing factor in the recognition of the pre-mRNA 3' splice sites during the assembly of the splicing commitment complex. We report here that U2AF65 is proteolyzed during apoptosis. This cleavage is group I or III caspase dependent in a noncanonical single site localized around the aspartic acid(128) residue and leads to the separation of the N- and C-terminal parts of U2AF65. The U2AF65 N-terminal fragment mainly accumulates in the nucleus within nuclear bodies (nucleoli-like pattern) and to a much lesser extent in the cytoplasm, whereas the C-terminal fragment is found in the cytoplasm, even in localization studies on apoptosis induction. From a functional viewpoint, the N-terminal fragment promotes Fas exon 6 skipping from a reporter minigene, by acting as a dominant-negative version of U2AF65, whereas the C-terminal fragment has no significant effect. The dominant-negative behavior of the U2AF65 N-terminal fragment can be reverted by U2AF35 overexpression. Interestingly, U2AF65 proteolysis in Jurkat cells on induction of early apoptosis correlates with the down-regulation of endogenous Fas exon 6 inclusion. Thus, these results support a functional link among apoptosis induction, U2AF65 cleavage, and the regulation of Fas alternative splicing.     

Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts

U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF(35) subunit contacts the 3'-splice site AG. Here we show that U2AF35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.     

U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR‐SREBP‐1c signalling pathway

U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and β‐estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of β‐casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP‐1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP‐1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR‐SREBP‐1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.