AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.

The underlying cause of mental retardation remains unknown in up to 80% of patients. As chromosomal aberrations are the most common known cause of mental retardation, several new methods based on FISH, PCR, and array techniques have been developed over recent years to increase detection rate of subtle aneusomies initially of the gene rich subtelomeric regions, but nowadays also genome wide. As the reported detection rates vary widely between different reports and in order to compare the diagnostic yield of various investigations, we analyzed the diagnostic yield of conventional karyotyping, subtelomeric screening, molecular karyotyping, X‐inactivation studies, and dysmorphological evaluation with targeted laboratory testing in unselected patients referred for developmental delay or mental retardation to our cytogenetic laboratory (n = 600) and to our genetic clinic (n = 570). In the cytogenetic group, 15% of patients showed a disease‐related aberration, while various targeted analyses after dysmorphological investigation led to a diagnosis in about 20% in the genetic clinic group. When adding the patients with a cytogenetic aberration to the patient group seen in genetic clinic, an etiological diagnosis was established in about 40% of the combined study group. A conventional cytogenetic diagnosis was present in 16% of combined patients and a microdeletion syndrome was diagnosed in 5.3%, while subtelomeric screening revealed only 1.3% of causes. Molecular karyotyping with a 10 K SNP array in addition revealed 5% of underlying causes, but 29% of all diagnoses would have been detectable by molecular karyotyping. In those patients without a clear diagnosis, 5.6% of mothers of affected boys showed significant (>95%) skewing of X‐inactivation suggesting X‐linked mental retardation. The most common diagnoses with a frequency of more than 0.5% were Down syndrome (9.2%), common microdeletion 22q11.2 (2.4%), Williams–Beuren syndrome (1.3%), Fragile‐X syndrome (1.2%), Cohen syndrome (0.7%), and monosomy 1p36.3 (0.6%). From our data, we suggest the following diagnostic procedure in patients with unexplained developmental delay or mental retardation: (1) Clinical/dysmorphological investigation with respective targeted analyses; (2) In the remaining patients without an etiological diagnosis, we suggest conventional karyotyping, X‐inactivation screening in mothers of boys, and molecular karyotyping, if available. If molecular karyotyping is not available, subtelomeric screening should be performed. © 2006 Wiley‐Liss, Inc.

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Here we report that splicing inhibition of msl-2 requires a binding site for SXL at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by SXL. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that SXL can no longer displace it from the poly(Y) tract. Our results reveal a novel function for U2AF35, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35-AG interaction to regulate intron removal.

Transcription factors of the Sox family arose around the advent of multicellularity in animals, arguing that their ability to regulate the expression of extracellular matrix, cell adhesion and signaling molecules may have been instrumental in the generation of metazoans. In particular, during vertebrate evolution, the Sox family experienced a phase of expansion that led to the appearance of groups of highly homologous Sox proteins and the division of existing Sox protein functions among group members. It furthermore allowed Sox transcription factors to acquire numerous novel functions. These past events of subfunctionalization and neofunctionalization can still be recognized today in all groups of the Sox family. They have led to partial functional redundancies, but also to interesting species-specific variations in the developmental roles of Sox proteins as shown here for the SoxB and the SoxE groups.

35 and a novel function for this factor in pre-mRNA splicing.The first ATP-dependent step in the assembly of splicing complexes is the stable association of U2 snRNP with the 3Ј part of the intron (reviewed in reference 12), which includes the branch point region, the polypyrimidine (Py) tract, and the conserved dinucleotide AG at the 3Ј splice site. The branch point region establishes base-pairing interactions with U2 snRNA that are critical for catalysis of the splicing reaction. The Py tract, particularly important in higher eukaryotes, is a pyrimidine-rich sequence located between the branch point and the AG dinucleotide that serves as the binding site for the U2 snRNP auxiliary factor (U2AF).Human U2AF is an essential splicing factor purified as a heterodimer composed of 65-kDa (U2AF 65 ) and 35-kDa (U2AF 35 ) subunits (39). U2AF 65 binds directly to Py tracts (41), while U2AF 35 is tethered to the pre-mRNA through its interaction with U2AF 65 (42). When bound to the Py tract, the amino-terminal arginine-serine-rich (RS) domain of U2AF 65 contacts the branch point region, and it has been proposed that its positively charged surface can promote the otherwise unstable base pairing between U2 snRNA and the poorly conserved branch point sequence (7, 34). Other mechanisms, including the recruitment of splicing factors involved in prespliceosome formation (5) as well as direct protein-protein interactions with components of U2 snRNP (8), are likely to contribute to U2AF activity.The role of U2AF 35 remains controversial. In vivo analyses of Drosophila U2AF have shown that both subunits, as well as the interaction between them, are essential for viability (14,22,23). In contrast, biochemical complementation experiments performed with extracts chromatographically depleted of U2AF have indicated that U2AF 65 alone is able to provide U2AF activity when tested with model splicing substrates (40,41). Similar results were obtained with nuclear extracts immunodepleted with a monoclonal antibody against U2AF 65 (6, 13). Results obtained with a U2AF 65 mutant protein deficient in its interaction with U2AF 35 also indicated that U2AF 35 is dispensable for in vitro splicing of various pre-mRNAs, including substrates whose splicing depends on the presence of exonic splicing enhancers (13). These sequences are often found downstream of weak 3Ј splice sites (31, 37) and stimulate early events in spliceosome assembly (16, 28), including U2AF 65 binding (36,43).In contrast with these results, immunodepletion with antibodies against U2AF35 resulted in nuclear extracts that required addition of both U2AF 65 and U2AF 35 for efficient splicing of constitutive and exon enhancer-dependent substrates (43). Also supporting an essential role for U2AF 35 were results obtained with a U2AF 65 mutant defective in interaction with U2AF 35 , which was inactive in those assays (43). Based on results obtained in a reconstituted system using limiting amounts of recombinant proteins, and also based on previous data reporting protein-protein interac...

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The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF 65 ) binds to the polypyrimidine (Py) tract preceding the 3 splice site, while the 35-kDa subunit (U2AF 35 Intron removal from mRNA precursors (pre-mRNA splicing) is an essential step of gene expression in eukaryotes. The precise recognition of the intron boundaries, the 5Ј and 3Ј splice sites, is achieved by small nuclear RNPs (snRNPs) and non-snRNP proteins. The 5Ј splice site is initially recognized by U1 snRNP, and the 3Ј splice site region is recognized by U2 snRNP. Subsequent addition of the U4/U6/U5 tri-snRNP forms the spliceosome, the macromolecular complex within which splicing catalysis takes place (reviewed in references 6 and 23).Several sequence elements help to define the 3Ј splice site region in higher eukaryotes (reviewed in reference 35) : the branchpoint (BP) sequence, usually followed by a pyrimidinerich sequence (the polypyrimidine tract or Py tract), and a conserved AG dinucleotide at the 3Ј end of the intron. The BP contains an adenosine residue that forms a 2Ј to 5Ј phosphodiester bond with the 5Ј end of the intron during the first catalytic step of the splicing reaction (39). U2 snRNP binds to the BP through base pairing interactions between this sequence and U2 snRNA (31,33,50,56). U2 snRNP binding requires auxiliary factors, including SF1/mBBP and U2AF (22,24,40). SF1/mBBP has been shown to specifically recognize the BP (2, 34) and play a kinetic role in spliceosome assembly (17, 41). U2AF is a heterodimer of 65 and 35-kDa subunits (52). U2AF 65 binds specifically to the Py tract via its RNA recognition motifs (RRMs) (53) and contacts the BP via its RS domain (11, 44), whereas U2AF 35 contacts the AG dinucleotide at the 3Ј splice site (30,51,58).The 3Ј splice site AG marks the 3Ј intron boundary and is involved in exon ligation, the second catalytic step of the splicing reaction. For some AG-dependent substrates, however, this dinucleotide is already required for early steps of spliceosome assembly prior to catalysis (36). AG-dependent substrates typically contain weak Py tracts, and substrates with strong Py tracts generally do not require the presence of the 3Ј splice site AG before the second catalytic step and are considered AG independent. Interaction between U2AF35 and the 3Ј splice site AG dinucleotide was shown to stabilize U2AF 65 binding to a weak Py tract and to be essential for splicing of AG-dependent substrates (51).An alternative set of interactions has been proposed for U2AF 35 . The arginine-serine (RS) region of U2AF 35 has been shown to establish protein-protein interactions with splicing factors of the SR family (49) (reviewed in references 10, 13, 29, and 45). One type of sequences bound by SR proteins are purine-rich exonic splicing enhancers (ESE), which stimulate splicing of pre-mRNAs containing weak 3Ј splice sites (reviewed in references 7 and 42). Based upon experiments using

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.The expression of eukaryotic genes requires the accurate removal of intervening sequences (introns) and the concomitant fusion of the flanking exons via RNA splicing. The correct recognition of 5Ј and 3Ј splice sites occurs in the spliceosome, a large and dynamic macromolecular complex of small nuclear ribonucleoprotein particles (snRNPs) 1 and non-snRNP proteins that assembles in a stepwise manner on the pre-mRNA (1, 2). Five snRNPs play a role in removal of canonical GU/AG introns, each composed of a different U snRNA, a set of polypeptides common to most spliceosomal snRNPs and a set of proteins specific for each snRNP (reviewed in Refs. 3 and 4).The first ATP-dependent step in spliceosome assembly is the stable association of U2 snRNP with the 3Ј part of the intron (reviewed in Refs. 5 and 6). This part of the pre-mRNA contains three sequence elements important for the splicing process as follows: the branch point region (BP), the polypyrimidine tract (Py tract), and the conserved dinucleotide AG at the 3Ј splice site. The BP is highly conserved in yeast (UACUAAC) and is more degenerate in higher eukaryotes (consensus YNCURAY, Y is pyrimidine, R is purine, N is any nucleotide). The BP establishes base pairing interactions with a specific sequence of U2 snRNA, bulging out the nucleotide, usually adenosine, that forms a 2Ј-5Ј phosphodiester bond with the 5Ј end of the intron (7-10). The Py tract, particularly important for splicing in higher eukaryotes, is a pyrimidine-rich sequence located immediately downstream of the BP and upstream of the AG dinucleotide (1).Biochemical fractionation of mammalian nuclear extracts indicated that stable binding of purified U2 snRNP to the pre-mRNA requires four ...