Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability1. Causal mutations have been found in approximately 90 X-linked genes2; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD Finger 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of ~7,000 refseq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in up-regulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish developing brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signaling and developmental pathways3. Our findings suggest that an imbalance of histone methylation dynamics plays a critical role in XLMR.

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.

Progesterone stimulation of Xenopus oocyte maturation requires the cytoplasmic polyadenylation-induced translation of mos and cyclin B mRNAs. One cis element that drives polyadenylation is the CPE, which is bound by the protein CPEB. Polyadenylation is stimulated by Aurora A (Eg2)-catalyzed CPEB serine 174 phosphorylation, which occurs soon after oocytes are exposed to progesterone. Here, we show that insulin also stimulates Aurora A-catalyzed CPEB S174 phosphorylation, cytoplasmic polyadenylation, translation, and oocyte maturation. However, these insulin-induced events are uniquely controlled by PI3 kinase and PKC-, which act upstream of Aurora A. The intersection of the progesterone and insulin signaling pathways occurs at glycogen synthase kinase 3 (GSK-3), which regulates the activity of Aurora A. GSK-3 and Aurora A interact in vivo, and overexpressed GSK-3 inhibits Aurora A-catalyzed CPEB phosphorylation. In vitro, GSK-3 phosphorylates Aurora A on S290/291, the result of which is an autophosphorylation of serine 349. GSK-3 phosphorylated Aurora A, or Aurora A proteins with S290/291D or S349D mutations, have reduced or no capacity to phosphorylate CPEB. Conversely, Aurora A proteins with S290/291A or S349A mutations are constitutively active. These results suggest that the progesterone and insulin stimulate maturation by inhibiting GSK-3, which allows Aurora A activation and CPEB-mediated translation.[Keywords: Insulin; GSK-3; Xenopus oocytes; Aurora A; cytoplasmic polyadenylation; CPEB] Fully grown Xenopus oocytes arrested at the end of prophase I are stimulated to re-enter into the meiotic divisions (oocyte maturation) by progesterone. Although the initial signaling event that is propagated by progesterone is unclear, it involves an immediate but transient decrease in cyclic AMP (cAMP; Sadler and Maller 1989; for review, see Ferrell 1999) and the activation of Aurora A (Eg2), a member of the Aurora family of protein kinases (Andresson and Ruderman 1998). The most proximal known substrate of Aurora A is CPEB, a sequence-specific RNA binding protein that stimulates cytoplasmic polyadenylation and translational activation (Hake and Richter 1994;Mendez et al. 2000a). CPEB interacts with the cytoplasmic polyadenylation element (CPE), a cis element present in the 3Ј untranslated regions (UTRs) of several mRNAs including those that encode mos and cyclin B. The translation of mos mRNA is necessary to induce the MAP kinase cascade that indirectly activates M-phase promoting factor (MPF), a heterodimer of cyclin B and cdc2. MPF is responsible for many manifestations of oocyte maturation such as germinal vesicle breakdown (GVBD). Aurora phosphorylation of CPEB serine 174 enhances the association of CPEB with CPSF (cleavage and polyadenylation specificity factor), possibly helping to stabilize this group of proteins on the AAUAAA hexanucleotide, a second cis element essential for polyadenylation (Mendez et al. 2000a,b). CPSF is probably responsible for recruiting poly(A) polymerase to the end of the mRNA.Polyadenylat...

We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(w a ) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5 splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5 splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire II-ICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors.Recent studies have identified several genes important in regulating alternative mRNA splicing. Members of the SR protein family, characterized by an arginine/serine (R/S)-rich domain in the carboxyl terminus and an RNA-recognition motif in the amino terminus were originally defined by their activity complementing HeLa cell S100 extracts for constitutive splicing, but SR proteins also have distinctive activities in regulating alternative splicing (1-3). ASF/SF2 (4 -6) and SC35 (7), the two SR proteins most completely characterized as splicing regulators, can regulate alternative 5Ј and 3Ј splice site selection (7) and exon inclusion/exclusion (8). SR proteins likely differentially activate splice sites due to different affinity interactions with either 5Ј splice sites or exon enhancers (9) and may similarly affect 3Ј splice site selection (10). In addition to interacting with RNA, SR proteins interact with other constitutive splicing factors containing R/S-rich domains, including U2AF and the U1-70K protein through the R/S-rich region of each protein (11,12). Several heterogeneous nuclear ribonuclear proteins (hnRNPs) 1 can also influence alternative mRNA splicing. hnRNP A1 directly antagonizes the effect of ASF/SF2 on 5Ј splice site selection both in vitro and in vivo: ASF/SF2 (as well as other SR proteins) increases splicing to proximal 5Ј splice sites, while hnRNP A1 increases splicing to distal 5Ј splice sites (8,(13)(14)(15). In some cases it also antagonizes the effect of ASF/SF2 on exon inclusion/exclusion (8). PTB also has activity in regulating alternative mRNA splicing (16). It binds to certain pol...

CPEB is an mRNA-binding protein that stimulates poly-adenylation-induced translation of maternal mRNA once it is phosphorylated on Ser 174 or Thr 171 (species-dependent). Disruption of the CPEB gene in mice causes an arrest of oogenesis at embryonic day 16.5 (E16.5), when most oocytes are in pachytene of prophase I. Here, we show that CPEB undergoes Thr 171 phosphorylation at E16.5, but dephosphorylation at the E18.5, when most oocytes are entering diplotene. Although phosphoryla-tion is mediated by the kinase aurora, the dephosphory-lation is due to the phosphatase PP1. The temporal control of CPEB phosphorylation suggests a mechanism in which CPE-containing mRNA translation is stimulated at pachytene and metaphase I.

CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein iso-forms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/ serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA + and CD45RO + T cells. Activation of CD45RA + T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO + memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO.

CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein isoforms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA+ and CD45RO+ T cells. Activation of CD45RA+ T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO+ memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO.