Small Nuclear Ribonucleoprotein Auxiliary Factor Research Articles

Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition.

Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the intron–exon border of some 3′ss and (ii) mediates enhancer-binding splicing activators’ interactions with the spliceosome. Alternative mechanisms for 3′ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3′ss recognition where the intron–exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3′ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3′ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons’ inclusion. Most probably, both factors stochastically bind the 3′ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3′ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants’ effects on splicing.

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6

JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344–403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.

Arabidopsis U2AF65 Regulates Flowering Time and the Growth of Pollen Tubes.

During pre-mRNA splicing, U2 small nuclear ribonucleoprotein auxiliary factor 65 (U2AF65) interacts with U2AF35 and splicing factor 1 (SF1), allowing for the recognition of the 3′-splice site by the ternary complex. The functional characterization of U2AF65 homologs has not been performed in Arabidopsis thaliana yet. Here, we show that normal plant development, including floral transition, and male gametophyte development, requires two Arabidopsis U2AF65 isoforms (AtU2AF65a and AtU2AF65b). Loss-of-function mutants of these two isoforms displayed opposite flowering phenotypes: atu2af65a mutants showed late flowering, whereas atu2af65b mutants were characterized by slightly early flowering, as compared to that in the wild-type (Col-0) plants. These abnormal flowering phenotypes were well-correlated with the expression patterns of the flowering time genes such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). However, the two atu2af65 mutants did not display any morphological abnormalities or alterations in abiotic stress tests. Double mutation of the AtU2AF65a and AtU2AF65b genes resulted in non-viable seeds due to defective male gametophyte. In vitro pollen germination test revealed that mutations in both AtU2AF65a and AtU2AF65b genes significantly impaired pollen tube growth. Collectively, our findings suggest that two protein isoforms of AtU2AF65 are differentially involved in regulating flowering time and display a redundant role in pollen tube growth.

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1–2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.

Jumonji Domain Containing Protein 6 Is Decreased in Human Preeclamptic Placentas and Regulates sFLT-1 Splice Variant Production.

The anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1), plays a central role in preeclamptic pathophysiology. A splice variant of FLT-1 (VEGF receptor 1), sFLT-1 is released in excessive amounts from the preeclamptic placenta into the maternal circulation, where it causes endothelial dysfunction manifesting as end-organ disease. However, the mechanisms regulating its production within the placenta remain poorly understood. Recently it was shown in endothelial cells that Jumonji domain containing protein 6 (JMJD6) hydroxylates U2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65, a component of the splicesome). The hydroxylation by JMJD6 is oxygen dependent. Under hypoxia, JMJD6 is less able to hydroxylate U2AF65, and this unhydroxylated form of U2AF65 biases splicing of FLT-1 to sFLT-1. We assessed whether oxygen-sensing JMJD6 is differentially expressed in preeclamptic placenta and regulates sFLT-1 splicing in placenta via U2AF65. JMJD6 protein expression was significantly reduced in preterm preeclamptic placenta (P < 0.0001; n = 21) relative to preterm controls (n = 10). Exposing both placental and endothelial cells to hypoxia significantly reduced JMJD6 mRNA and increased sFLT-1 mRNA and protein expression. Silencing JMJD6 in primary endothelial and trophoblast cells significantly increased sFLT-1 secretion. Next, we examined whether these molecules may be directly interacting. We demonstrated that placental U2AF65 colocalized with JMJD6. In turn, we found JMJD6 directly interacts with U2AF65, which in turn produces sFLT-1 mRNA transcripts. Taken together, our findings provide evidence that JMJD6 may play a role in regulating the production of sFLT-1 in the preeclamptic placenta. Decreased placental JMJD6 expression may be an important component to the pathophysiology of preeclampsia.

Association between pancreatic cancer risk and the interaction of U2AF65 gene polymorphisms and smoking

To determine the association between U2 small nuclear ribonucleoprotein auxiliary factor 35/65 (U2AF35 and U2AF65) and pancreatic cancer (PC). A two-stage analysis case-control study was conducted. Four candidate tag single nucleotide polymorphisms (tagSNPs) were genotyped by Taqman Openarray assay in a screening population living in Central China (298 PC cases and 525 controls). Thereafter, rs310445 in U2AF65 was genotyped by TaqMan real-time polymerase chain reaction (RT-PCR) in a validation Chinese Han population from Beijing (413 cases and 557 controls). rs310445 in U2AF65 gene was significantly associated with PC in both screened population and combined population. Subjects with C allele had a higher risk of PC compared to those with the TT genotype, with OR of 1.31 (95%CI:1.07-1.60, P = 0.010) for the combined population. A synergic effect of smoking and C allele of rs310445 was also observed in the combined population, with Synergic Index of 2.08 (95% CI:1.37-2.78) in the combined population. Our findings suggested the interaction between smoking and U2AF65 might play a role in PC. These findings should be confirmed by further independently large-scale population studies.

The splicing factor U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis

The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure proper telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essentialpre-mRNA splicingfactor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.

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.

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

Hu Antigen R (HuR) Functions as an Alternative Pre-mRNA Splicing Regulator of Fas Apoptosis-promoting Receptor on Exon Definition

Exclusion of exon 6 by alternative RNA splicing of the primary transcript of the apoptosis receptor Fas produces a soluble isoform that prevents programmed cell death. I report that antiapoptotic regulator Hu antigen R (HuR, ELAVL1), a member of the embryonic lethal, abnormal vision, Drosophila-like (ELAVL) family, promotes Fas exon 6 skipping by binding to an exonic splicing silencer. HuR inhibits the association of U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) with the upstream 3' splice site, without decreasing recognition of the downstream 5' splice site by U1 snRNP but by antagonizing the role of TIA-1 (T-cell intracellular antigen 1)/TIAR (TIA-1 related protein) on exon definition. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is partially required for efficient U2AF65 inhibition. Further, the silencing capacity of HuR as splicing regulator resides in the RRM1 and hinge-RRM3 domains. Taken together, these results support a functional link between HuR as repressor of alternative Fas splicing and the molecular mechanisms modulating programmed cell death.

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.

Nuclear protein profiling of Jurkat cells during heat stress‐induced apoptosis by 2‐DE and MS/MS

Heat stress causes severe constraints on numerous physiological functions of cells, such as the repression of splicing of mRNA precursors. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat cells during heat stress using 2-DE and LC-MS/MS. We found 10 protein spots whose expression had changed after heat stress at 43 degrees C for 30 min. Seven of those protein spots, periodic tryptophan protein 1 homolog (PWP1), importin beta-1 subunit, sumoylated protein, splicing factor 3a subunit 3 (SF3a3), TAR DNA-binding protein 43, U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit (U2AF35) and small ubiquitin-related modifier-1 (SUMO-1) were downregulated, while three other protein spots, Protein SET, 40S ribosomal protein SA and 60S acidic ribosomal protein P0 were upregulated by the heat stress. We focused on the downregulation of two splicing factors, which might participate in the repression of pre-mRNA processing by heat stress, leading to cell apoptosis.

Global gene transcription patterns in in vitro-cultured fertilized embryos and diploid and haploid murine parthenotes

To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked ( Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoprotein auxiliary factor 1, related sequence 1 ( U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58 h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49 h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138 h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components.

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.

Cloning and characterization of a novel splice variant of humanU2AF1L3gene

Pre-mRNA splicing allows individual genes to produce multiple protein isoforms with diverse functions. Recognition of functional splice sites in pre-mRNAs is very important in this splicing process and requires some protein auxiliary factors such as U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35, encoded by U2AF1). By its RNA binding domains, U2AF35 interacts with U2AF65 to bind 3' splice site of pre-mRNA and initiates splicing. Another protein, which is named as U2AF1-like3 (U2AF1L3), shows high similarity with U2AF35 and may have related function in pre-mRNA splicing. Here, we report a splice variant of U2AF1L3, which is 767 bp in length and has an open reading frame (ORF) coding a predicted 181 amino acids protein. Reverse transcription-PCR (RT-PCR) shows that this isoform has different expression pattern with U2AF1L3 and is highly expressed in heart, brain and lung.

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.

Diversity of Vertebrate Splicing Factor U2AF35: IDENTIFICATION OF ALTERNATIVELY SPLICED U2AF1 mRNAs

U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U2AF(35)c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF(35)b differs from the previously described U2AF(35)a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF(65). Biochemical experiments indicate that isoform U2AF(35)b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF(65), stimulates U2AF(65) binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF(35)a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF(35)a and U2AF(35)b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF(35) protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.

Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding.

The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (SXL), which has two RRMs, can directly compete with the essential splicing factor U2AF(65), which has three RRMs, for binding to specific Py tracts. We have combined site-specific photocross-linking and chemical cleavage of the proteins to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF(65) shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF(65) and SXL can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. We present a model to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity.

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.

DNA Demethylation Reactivates a Subset of Imprinted Genes in Uniparental Mouse Embryonic Fibroblasts

Although most imprinted genes show allelic differences in DNA methylation, it is not clear whether methylation regulates the expression of some or all imprinted genes in somatic cells. To examine the mechanisms of silencing of imprinted alleles, we generated novel uniparental mouse embryonic fibroblasts exclusively containing either the paternal or the maternal genome. These fibroblasts retain parent-of-origin allele-specific expression of 12 imprinted genes examined for more than 30 cell generations. We show that p57(Kip2) (cyclin-dependent kinase inhibitor protein 2) and Igf2 (insulin-like growth factor 2) are induced by inhibiting histone deacetylases; however, their activated state is reversed quickly by withdrawal of trichostatin A. In contrast, DNA demethylation results in the heritable expression of a subset of imprinted genes including H19 (H19 fetal liver mRNA), p57(Kip2), Peg3/Pw1 (paternally expressed gene 3), and Zac1 (zinc finger-binding protein regulating apoptosis and cell cycle arrest). Other imprinted genes such as Grb10 (growth factor receptor-bound protein 10), Peg1/Mest (paternally expressed gene 1/mesoderm-specific transcript), Sgce (epsilon-sarcoglycan), Snrpn (small nuclear ribonucleoprotein polypeptide N), and U2af1 (U2 small nuclear ribonucleoprotein auxiliary factor), remain inactive, despite their exposure to inhibitors of histone deacetylases and DNA methylation. These results demonstrate that changes in DNA methylation but not histone acetylation create a heritable epigenetic state at some imprinted loci in somatic cells.