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.     

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.     

Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine–serine-rich (RS) domains of SR- and SR-like proteins

The Fe(II) and 2-oxoglutarate dependent oxygenase Jmjd6 has been shown to hydroxylate lysine residues in the essential splice factor U2 auxiliary factor 65 kDa subunit (U2AF65) and to act as a modulator of alternative splicing. We describe further evidence for the role of Jmjd6 in the regulation of pre-mRNA processing including interactions of Jmjd6 with multiple arginine–serine-rich (RS)-domains of SR- and SR-related proteins including U2AF65, Luc7-like protein 3 (Luc7L3), SRSF11 and Acinus S′, but not with the bona fide RS-domain of SRSF1. The identified Jmjd6 target proteins are involved in different mRNA processing steps and play roles in exon dependent alternative splicing and exon definition. Moreover, we show that Jmjd6 modifies splicing of a constitutive splice reporter, binds RNA derived from the reporter plasmid and punctually co-localises with nascent RNA. We propose that Jmjd6 exerts its splice modulatory function by interacting with specific SR-related proteins during splicing in a RNA dependent manner.     

Prp40 pre-mRNA processing factor 40 homolog B (PRPF40B) associates with SF1 and U2AF65 and modulates alternative pre-mRNA splicing in vivo

The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5′ splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3′ splice site. The 5′ and 3′ splice site complexes are thought to be joined together by protein–protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF⁶⁵. Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5′ and 3′ splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival.     

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.     

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.     

The splicing factor U2AF65 is functionally conserved in the thermotolerant deep-sea worm Alvinella pompejana

Due to their inherent stability, thermophilic bacteria and archaea serve as important resources for biochemical and biophysical analyses of many biological processes. Unfortunately, scientists characterizing eukaryote-specific processes, such as nuclear pre-mRNA splicing, are unable to take advantage of these sources of thermostable proteins. To identify and provide a source of thermostable eukaryotic proteins, we are characterizing splicing factors in the thermotolerant deep-sea vent polychaete, Alvinella pompejana. This worm, also known as the Pompeii worm, is found in the extreme environment of deep-sea hydrothermal vents, and is one of the most thermotolerant eukaryotic organisms known. We report on detailed analyses of U2AF65, the large subunit of the U2 small nuclear ribonucleoprotein auxiliary factor, an essential splicing factor important for intron definition and alternative splicing. The cloning and characterization of Pompeii U2AF65 show it is highly similar to human U2AF65 in sequence and function and is more thermostable than the human protein when bound to RNA in vitro. Notably, Pompeii U2AF65 can restore splicing in a human extract depleted of human U2AF. We also determine that the general splicing mechanisms and signal sequences are conserved in the Pompeii worm, an annelid which has previously been uncharacterized in terms of splicing factors and signals.     

Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF

Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF(65) binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF(65), arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF(65) binding.     

Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal

The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine > adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.     

Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron

Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.     

Cancer-relevant Splicing Factor CAPERα Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex

U2AF homology motifs (UHMs) mediate protein-protein interactions with U2AF ligand motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERα regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERα UHM is unknown. Here we present structures of the CAPERα UHM bound to a representative SF3b155 ULM at 1.7 Å resolution and, for comparison, in the absence of ligand at 2.2 Å resolution. The prototypical UHM/ULM interactions authenticate CAPERα as a bona fide member of the UHM family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERα in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERα UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERα molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously identified, UHM/ULM-mediated complexes of U2AF⁶⁵ and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.