Concepción Martínez

Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG.

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.

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites.

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre‐mRNA 5′ splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5′ ss is promoted by binding of the protein TIA‐1 to uridine‐rich sequences immediately downstream from the 5′ ss. Here we describe a molecular dissection of the activities of TIA‐1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre‐mRNA. The non‐ consensus RRM1 and the C‐terminal glutamine‐rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5′ ss. Co‐precipitation experiments revealed a specific and direct interaction involving the N‐terminal region of the U1 protein U1‐C and the Q‐rich domain of TIA‐1, an interaction enhanced by RRM1. The results argue that binding of TIA‐1 in the vicinity of a 5′ ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1‐C, thus providing one molecular mechanism for the function of this splicing regulator.

αTAT1 is the major α-tubulin acetyltransferase in mice

Post-translational modification of tubulin serves as a powerful means for rapidly adjusting the functional diversity of microtubules. Acetylation of the ε-amino group of K40 in α-tubulin is one such modification that is highly conserved in ciliated organisms. Recently, αTAT1, a Gcn5-related N-acetyltransferase, was identified as an α-tubulin acetyltransferase in Tetrahymena and C. elegans. Here we generate mice with a targeted deletion of Atat1 to determine its function in mammals. Remarkably, we observe a loss of detectable K40 α-tubulin acetylation in these mice across multiple tissues and in cellular structures such as cilia and axons where acetylation is normally enriched. Mice are viable and develop normally, however, the absence of Atat1 impacts upon sperm motility and male mouse fertility, and increases microtubule stability. Thus, αTAT1 has a conserved function as the major α-tubulin acetyltransferase in ciliated organisms and has an important role in regulating subcellular specialization of subsets of microtubules.

Evidence for Substrate-Specific Requirement of the Splicing Factor U2AF35 and for Its Function after Polypyrimidine Tract Recognition by U2AF65

ABSTRACT U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF65 and U2AF35. U2AF65 binds to the polypyrimidine (Py) tract upstream from the 3′ splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF35 facilitates U2AF65 binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF35 remain controversial. Here we show that recombinant U2AF65 is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF65, U2AF35, and the interaction between them are required for splicing of an immunoglobulin μ pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF35 occurs without changes in U2AF65cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF35 and a novel function for this factor in pre-mRNA splicing.

Molecular cloning and sequencing of influenza virus A/Victoria/3/75 polymerase genes: sequence evolution and prediction of possible functional domains

The influenza virus A/Victoria/3/75 (H3N2) polymerase genes encoding PB1, PB2 and PA have been cloned by cDNA synthesis and insertion into bacterial vectors. The complete sequence for each polymerase gene has been obtained from random M13 subclones and compared to other influenza virus polymerase genes. A total of 45, 74 and 78 nucleotide changes were fixed in the period 1968-1975, corresponding to 10, 12 and 9 amino acid changes, for PB1, PB2 and PA genes, respectively. The amino acid sequence of PB1 polypeptide contains motifs found in a series of positive- and negative-RNA virus polymerase genes and that of PA polypeptide share invariant residues common to DNA and presumptive RNA helicases.

Tubulin Acetyltransferase αTAT1 Destabilizes Microtubules Independently of Its Acetylation Activity

ABSTRACT Acetylation of α-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently, αTAT1, a member of the Gcn5-related N-acetyltransferase superfamily, has been identified as an α-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of αTAT1 with the aim of understanding the consequences of αTAT1-mediated microtubule acetylation. We demonstrate that α-tubulin is the major target of αTAT1 but that αTAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin. We further show that in mammalian cells, αTAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in an αTAT1 mutant with no acetyltransferase activity, suggesting that interaction of αTAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that αTAT1 has cellular functions that extend beyond its classical enzymatic activity as an α-tubulin acetyltransferase.

Regulation of the proneural gene achaete by helix-loop-helix proteins.

The Achaete (Ac) protein, a transcriptional regulator of the basic-helix-loop-helix (bHLH) type, confers upon ectodermal cells the ability to become neural precursors. Its temporally and spatially regulated expression, together with that of the related Scute (Sc) protein, helps define the pattern of Drosophila melanogaster sensory organs. We have examined the transcriptional control of the ac gene and shown, using in vivo assays, that several E-boxes, putative interacting sites for bHLH proteins, present in the ac promoter are most important for ac regulation. They most likely mediate ac self-stimulation and sc trans-activation. We also demonstrate that ac transcription is negatively regulated in vivo by the gene extramacrochaetae (emc) in a manner dependent on Ac and Sc products. emc encodes an HLH protein that lacks the basic region and presumably antagonizes Ac and Sc function by sequestering these proteins in complexes unable to interact with DNA. Our results strongly support the model of negative regulation of emc on ac and sc transcription through titration of their products. As currently thought, this seems accomplished by heterodimerization via the HLH domain, because an amino acid substitution in this region abolishes the emc antagonistic effect both in vitro and in vivo.

Evolution of the influenza virus neuraminidase gene during drift of the N2 subtype

The complete genetic information for the neuraminidase (NA) gene of influenza virus A/Bangkok/1/79 has been cloned by in vitro synthesis of dsDNA, insertion into pBR322 plasmid, and transformation of Escherichia coli. The nucleotide sequence of the NA gene has been determined by the Maxam and Gilbert method. It is 1466 nucleotides long and contains a single open reading frame with a coding capacity for 469 amino acids. When compared to the NA genes of the N2 strains A/Victoria/3/75, A/Udorn/72, A/NT/60/68, and A/RI/5-/57, 90% of the nucleotide positions and 87% of the amino acid positions remained invariant. Forty-two nucleotide changes and 14 amino acid changes accumulated in the period 1975-1979, but the general structure of the protein appeared to remain constant.

Degradation of cellular mRNA during influenza virus infection : its possible role in protein synthesis shutoff

The kinetics of cellular mRNA decay in influenza virus-infected cells have been studied by means of blot hybridization using as probes cloned cDNAs of alpha- and beta-actin, alpha- and beta-tubulin and vimentin. Both cellular mRNAs isolated from the cytoplasmic fractions as well as total cell mRNAs showed a rapid decay, with up to 50% concentration reductions at infection times at which influenza virus M1 mRNA was still not detectable. In contrast, these cellular mRNAs were stable in uninfected cells. To ascertain the possible role of mRNA degradation in the cellular protein synthesis shutoff, the kinetics of protein synthesis in infected cells were examined by two-dimensional gel electrophoresis of extracts pulse-labelled at several times after viral infection. The synthesis of the cellular proteins was reduced, showing kinetics paralleling those of mRNA decay. It is proposed that influenza virus infection induces the destabilization of mRNAs and that this mRNA degradation is, at least in part, responsible for cellular protein synthesis shutoff.

The synthesis of influenza virus negative-strand RNA takes place in insoluble complexes present in the nuclear matrix fraction

The replication of influenza virus RNA in vitro has been studied by cell fractionation of MDCK-infected cells and characterization of in vitro synthesized RNA. Analysis of the RNA product polarity by liquid hybridization to excess single-stranded DNA probes shows that only the RNP complexes present in the nuclear matrix fraction are able to synthesize negative-polarity RNA. This RNA product has been characterized as authentic vRNA by size analysis, RNase-protection by unlabelled, positive-polarity riboprobes and T1-fingerprinting. Priming the in vitro reaction with ApG stimulates preferentially the synthesis of positive-polarity RNA, while ApGpU stimulates both positive and negative-polarity RNA synthesis.