Autoregulation of Fox protein expression to produce dominant negative splicing factors

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxΔRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxΔRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.     

Adaptive Evolution of Mus Apobec3 Includes Retroviral Insertion and Positive Selection at Two Clusters of Residues Flanking the Substrate Groove

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.     

High Expression Level of Tra2-β1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn^−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.     

CD72几种新的剪切形式的发现及它们在红斑狼疮模型鼠中的差异表达

CD72是一个重要的B细胞特异性受体,它以多种选择性剪切形式存在.在小鼠脾细胞中发现并鉴定了8种新的CD72选择性剪切形式,这些剪切形式中包含有2种独特的插入片段,一种选择性剪切保留了一个内含子(intron1),而这个内含子被翻译成氨基酸序列后并没有改变前后外显子的读码框,另一种使用了一个位于内含子之内的3′剪切位点,从而产生移码,提前终止了蛋白质的开放读码框,称为3′AS(3′alternative splicingsite).比较了CD72所有剪切形式在BALB/C小鼠和NZB/W小鼠中的差异表达,发现:a.含有3′AS的剪切形式的表达都很少;b.WT, In1, In1-Ex3和-Ex3的表达在BLAB/C小鼠中比在NZB/W小鼠中高;c.没有ITIM2的-Ex2-Ex3剪切形式在NZB/W小鼠中有特异性高表达.这些结果提示,CD72的多种选择性剪切形式在调控B细胞受体信号转导过程中可能发挥着不同的作用,并与系统性红斑狼疮的发病密切相关.     

Identification of Two APOBEC3F Splice Variants Displaying HIV-1 Antiviral Activity and Contrasting Sensitivity to Vif

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2–4 (A3FΔ2–4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2–4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics.