Essential Splicing Factor Research Articles

Alternative Modes of Binding by U2AF65 at the Polypyrimidine Tract

During initial recognition of an intron in pre-mRNA, the 3' end of the intron is bound by essential splicing factors. Notably, the consensus RNA sequences bound by these proteins are highly degenerate in humans. This raises the question of 3' splicing factor function in introns lacking canonical binding sites. Investigating the introns of the model organism Neurospora crassa revealed a different organization at the 3' end of the intron compared to most eukaryotic organisms. The predicted branch point sequences of Neurospora introns are much closer to the 3' splice site compared to those in human introns. In addition, Neurospora introns lack the canonical polypyrimidine tract found at the end of introns in most eukaryotic organisms. The large subunit of the U2 snRNP associated factor (U2AF65), which is essential for splicing of human introns and specifically recognizes the polypyrimidine tract, is also present in Neurospora. We show that Neurospora U2AF65 binds RNA with low affinity and specificity, apparently evolving with its disappearing binding site. The arginine/serine rich domain at the N-terminus of Neurospora U2AF65 regulates its RNA binding. We find that this regulated binding can be recapitulated in human U2AF65 which has been mutated to decrease both affinity and overall charge. Finally, we show that the addition of the small U2AF subunit (U2AF35) to U2AF65 with weakened RNA binding affinity significantly enhances the affinity of the resulting U2AF heterodimer.

Mutation in the splicing factor Hprp3p linked to retinitis pigmentosa impairs interactions within the U4/U6 snRNP complex

Mutations in PRPF3, a gene encoding the essential pre-mRNA splicing factor Hprp3p, have been identified in patients with autosomal dominant retinitis pigmentosa type 18 (RP18). Patients with RP18 have one of two single amino acid substitutions, Pro493Ser or Thr494Met, at the highly conserved Hprp3p C-terminal region. Pro493Ser occurs sporadically, whereas Thr494Met is observed in several unlinked RP families worldwide. The latter mutation also alters a potential recognition motif for phosphorylation by casein kinase II (CKII). To understand the molecular basis of RP18, we examined the consequences of Thr494Met mutation on Hprp3p molecular interactions with components of the U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) complex. Since numerous mutations causing human diseases change pre-mRNA splice sites, we investigated whether Thr494Met substitution affects the processing of PRPF3 mRNA. We found that Thr494Met does not affect PRPF3 mRNA processing, indicating that the mutation may exert its effect primarily at the protein level. We used small hairpin RNAs to specifically silence the endogenous PRPF3 while simultaneously expressing HA-tagged Thr494Met. We demonstrated that the C- but not N-terminal region of Hprp3p is indeed phosphorylated by CKII in vitro and in cells. CKII-mediated Hprp3p phosphorylation was significantly reduced by Thr494Met mutation. Consequently, the Hprp3p C-terminal region is rendered partially defective in its association with itself, Hprp4p, and U4/U6 snRNA. Our findings provide new insights into the biology of Hprp3p and suggest that the loss of Hprp3p phosphorylation at Thr494 is a key step for initiating Thr494Met aberrant interactions within U4/U6 snRNP complex and that these are likely linked to the RP18 phenotype.

A Conditional Role of U2AF in Splicing of Introns with Unconventional Polypyrimidine Tracts

Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3' splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3' splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3' splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.

Deletion of the N-terminus of SF2/ASF Permits RS-Domain-Independent Pre-mRNA Splicing

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.

ZRANB2: Structural and functional insights into a novel splicing protein

ZRANB2 was identified originally in a differential display experiment on 2-day and 10-day primary cultures of rat juxtaglomerular cells. During prolonged culture it was found to undergo down-regulation in concert with renin, the archetypical constituent of these cells. ZRANB2 has two zinc fingers that form a novel fold and show striking homology to Ran-binding protein domains. Human ZRANB2 mRNA is alternatively spliced to give two variants with different 3′ ends. ZRANB2 has homologues across a range of species, the N-terminal end being particularly conserved. ZRANB2 is present in the nucleus of human cells. It binds to mRNA, as well as the essential splicing factors U170K and U2AF 35 and the novel splicing component SFRS17A (formerly known as XE7). ZRANB2 is one of 20 genes up-regulated in grade III ovarian serous papillary carcinoma. Here, we review current knowledge surrounding ZRANB2.

Biochemical Characterization of the ATPase and Helicase Activity of UAP56, an Essential Pre-mRNA Splicing and mRNA Export Factor

DEXD/H-box protein UAP56 is an essential pre-mRNA splicing factor required for the first ATP-dependent spliceosome assembly step. UAP56 is also essential for the export of the majority of mRNAs from the nucleus to the cytoplasm. We performed biochemical characterization of UAP56's ATPase and helicase activity, which is important for further understanding the role of these activities in UAP56's function. We showed that UAP56 is an RNA-stimulated ATPase that can only hydrolyze ATP. We demonstrated that UAP56 is an ATP-dependent RNA helicase that can unwind substrates with 5' or 3' overhangs or blunt ends in vitro. We showed that U2AF(65) and Aly, two proteins known to interact with UAP56, do not influence UAP56's ATPase or helicase activity. We also demonstrated that several mutants in the conserved helicase motifs I, II, and III abolish UAP56's ATPase and/or helicase activity, providing tools for future investigation of the role of UAP56's ATPase and helicase activity in spliceosome assembly and mRNA export.

SMN Transcript Stability: Could Modulation of Messenger RNA Degradation Provide a Novel Therapy for Spinal Muscular Atrophy?

Proximal spinal muscular atrophy is caused by deletion or mutation of the survival motor neuron 1 gene, SMN1. Rentention of a nearly identical copy gene, SMN2, enables survival but is unable to fully compensate for the loss of SMN1. The SMN1 and SMN2 genes differ by a single nucleotide that results in alternative splicing of SMN2 exon 7 due to the disruption of a binding site for an essential splicing factor. This alternatively spliced form encodes a partially functional truncated protein. Because SMN2 is present in patients with spinal muscular atrophy, it is an ideal therapeutic target. Some of the current approaches to increase SMN protein levels are aimed at increasing the transcription from SMN2 or at preventing exon 7 skipping. One area that has yet to be investigated is the stability of messenger ribonucleic acid (RNA) transcripts produced from SMN2. We postulated that transcripts derived from SMN2 may be less stable because alternative splicing, recruitment of RNA-binding proteins, and alteration of stop codons have been associated with changes in rates of messenger RNA decay; these features are all characteristic of SMN2. Accordingly, transcript degradation was examined within primary fibroblast cells that exclusively contained SMN1 or SMN2 by treating cultures with a transcriptional inhibitor to observe messenger RNA stability. The results indicate that SMN transcript instability does not play a role in the disease mechanism, suggesting that therapeutic modulation of messenger RNA degradation would not target a molecular defect in patients with spinal muscular atrophy, although it could provide general benefits by increasing total pools of SMN2 transcripts.

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

Segregation distortion in Arabidopsis gametophytic factor 1 (gfa1) mutants is caused by a deficiency of an essential RNA splicing factor

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle.

Identification of RNA binding sequences of Drosophila SR protein DX16

Dxl6 is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. To get more insight of Dx16 function, we generated the monoclonal antibody against Dx16 and determined its expression pattern and subcellular location. It is mainly expressed in the nucleus of CNS in Drosophila embryos. In order to investigate the RNA-binding specificity of Dxl6, Dxl6-binding RNAs were identified by SELEX screen by using recombinant Dxl6 N-terminus protein as the target. These RNAs contained a consensus motif. Some pre-mRNAs from the corresponding genes showed splicing defects in the Dxl6-P-element insertional mutant fly. These results indicate that Dxl6 has unique functions in the removal of some introns during development.

Human immunodeficiency virus type 1 Vpr interacts with spliceosomal protein SAP145 to mediate cellular pre-mRNA splicing inhibition

Vpr, an accessory gene product of human immunodeficiency virus type 1 (HIV-1), affects both viral and cellular proliferation by mediating long terminal repeat activation, cell cycle arrest at the G2 phase, and apoptosis. We previously found that Vpr plays a novel role as a regulator of pre-mRNA splicing both in vivo and in vitro. However, the cellular target of Vpr, as well as the mechanism of cellular pre-mRNA splicing inhibition by Vpr, is unknown. Here, we show clearly that Vpr inhibits the splicing of cellular pre-mRNA, such as β-globin pre-mRNA and immunoglobulin (Ig) M pre-mRNA and that the third α-helical domain and arginine-rich region are important its ability to inhibit splicing. Additionally, using mutants with specific substitutions in two domains of Vpr, we demonstrated that the interaction between Vpr and SAP145, an essential splicing factor, was indispensable for splicing inhibition. Finally, co-immunoprecipitation and in vitro competitive binding assays indicated that Vpr associates with SAP145 and interferes with SAP145–SAP49 complex formation. Thus, these results suggest that cellular expression of Vpr may block spliceosome assembly by interfering with the function of the SAP145–SAP49 complex in host cells.

Alternative Conformations at the RNA-binding Surface of the N-terminal U2AF65 RNA Recognition Motif

The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF65) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF65 must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF65 variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF65. Here, the X-ray structure of the N-terminal RRM domain of U2AF65 (RRM1) is described at 1.47 Å resolution in the absence of RNA. Notably, RNA-binding by U2AF65 selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the β2/β3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF65 to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.

Novel Model of Stimulation-Responsive Splicing for Generation of Immunogenic Isoforms of Tumor Antigens and Autoantigens.

Alternative splicing is a process that removes introns and alters exons to generate multiple isoforms from a single pre-mRNA transcript. Alternative splicing is the major mechanism by which a small number of human genes (6 × 104) can encode the larger complexity of the human proteome (1 × 106 proteins). Previously we demonstrated that alternative splicing of apoptosis-regulatory protein transcripts regulates immune responses by modulating lymphocyte survival (Immunity, 1997; Mol. Immunol. 2002; J Exp Med, 2002; Oncogene 2005; Biochem J, 2005). To examine the hypothesis that alternative splicing plays a role in selection of nonmutated self-protein isoforms for tumor antigens and autoantigens, recently, we showed that alternative splicing is a major mechanism in regulation of the immunogenicity of tumor antigen CML66 (J. Immunol. 2004). In addition, we found that alternative splicing occurs in 100% of the autoantigen transcripts. This is significantly higher than the approximately 42% rate of alternative splicing observed in the 10,000 randomly selected human gene transcripts (p<0.001) [J. Allergy Clin. Immunol., 2004 (cover article)]. Here, we report that essential alternative splicing factor ASF/SF2 expression in samples from patients with chronic inflammation is lower than that of the healthy controls (p<0.05). In addition, TNF-a significantly downregulates ASF/SF2 expression (7 folds) in cultured cells in comparison to the expression variations of b-actin control. These findings demonstrate that ASF/SF2, presumably affecting splicing of self-antigen transcripts, is downregulated in autoimmune inflammatory disease potentially via a TNF-a-mediated pathway. Collectively, we propose for the first time a novel model of “stimulation-responsive splicing”, which emphasizes that stimulation-responsive splicing plays a critical role in selection of nonmutated self-protein isoforms to become tumor antigens and autoantigens (Clin. Immunol. Invited Review, in press, 2006). The new model for the definition of immunogenic isoforms of tumor antigens and autoantigens is significant in facilitating the development of:immunogenic antigen isoform microarrays for disease diagnosis and prognosis;autoantigen-tolerizing therapy and splicing-redirection therapy for autoimmune diseases; andimmunogenic antigen isoforms-based immunotherapy for tumors.

In Vivo Requirement of the Small Subunit of U2AF for Recognition of a Weak 3′ Splice Site

The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF(65)) and a small subunit, U2AF(35). U2AF(65) binds to the polypyrimidine tract upstream from the 3' splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF(35) plays a role in assisting U2AF(65) recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3' splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF(65) triggers the downregulation of U2AF(35). We further show that the knockdown of each U2AF subunit inhibits weak 3' splice site recognition, while overexpression of U2AF(65) alone is sufficient to activate the selection of this splice site. A variant of U2AF(65) lacking the interaction domain with U2AF(35) shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3' splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF(35) regulates the selection of weak 3' splice sites in a specific subset of cellular transcripts.

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.

A specific SR protein binds preferentially to the secretory protein gene transcripts in salivary glands of Chironomus tentans

The members of the serine-arginine (SR) family of proteins play multiple roles in posttranscriptional gene expression. Initially considered as essential splicing factors confined to the nucleus and regulating constitutive and alternative splicing, SR proteins are now known to shuttle between the nucleus and the cytoplasm and to be involved in mRNA biogenesis, transport, and translation. In Chironomus tentans, hrp45 is an SR protein structurally similar to the Drosophila SRp55/B52 SR protein. We have studied how hrp45, hrp36 [a heterogenous nuclear ribonucleoprotein (hnRNP) protein], and small nuclear RNP (snRNP) proteins are distributed in the transcriptionally active loci of polytene chromosomes in C. tentans. Immunofluorescence visualization of the proteins in double-labeling experiments revealed that hrp45 preferentially associates with a small number of puffs. On the other hand, hrp36 and snRNP proteins were found distributed in a large number of loci with little quantitative difference. Remarkably, hrp45-labeled loci coincide with the sites of transcription of premessenger RNPs of secretory protein (sp) genes. Because the labeling was found sensitive to RNase A treatment, we conclude that the SR protein hrp45 preferentially binds to sp gene transcripts in salivary gland cells. The preferential association of a specific SR protein with a particular type of gene transcripts reflects substrate-specific function(s) of an SR protein, in vivo. The possible roles that hrp45 might be playing in speedy and efficient processing of sp gene transcripts are discussed.

Structural Basis for Polypyrimidine Tract Recognition by the Essential Pre-mRNA Splicing Factor U2AF65

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.

Crystallization and preliminary X-ray analysis of a U2AF65variant in complex with a polypyrimidine-tract analogue by use of protein engineering

The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF65) binds the polypyrimidine tract near the 3' splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein-nucleic acid complex. Cocrystals of a U2AF65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 angstroms resolution and contain one complex per asymmetric unit.

The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that snRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that snRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in snRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new snRNPs--and perhaps regenerated snRNPs after splicing--is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.

Alternative splicing factor ASF/SF2 is down regulated in inflamed muscle

Background: In our recent studies, alternative splicing has been shown to have a major role in inflammation and autoimmune muscle diseases. Aim: To examine the novel hypothesis that the expression...