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Summary 2 Haering, C.H. et al. (2002) Molecular architecture differential regulation along arms versus the The recent structural studies have of SMC proteins and the yeast cohesin complex. centric region. Cell 98, 249–259 narrowed down the number of possible Mol. Cell 9, 773–788 13 Laloraya, S. et al. (2000) Chromosomal addresses 3 Losada, A. et al. (2000) Identification and of the cohesin component Mcd1p. J. Cell Biol. 151, models for sister chromatid cohesion and characterization of SA/Scc3p subunits in the 1047–1056 provide a clearer view of how the Xenopus and human cohesin complexes. J. Cell 14 Ciosk, R. et al. (2000) Cohesin’s binding to components of the cohesin complex Biol. 150, 405–416 chromosomes depends on a separate complex interact. Nonetheless, several key 4 Sumara, I. et al. (2000) Characterization of consisting of Scc2 and Scc4 proteins. Mol. Cell 5, questions regarding the mechanism of vertebrate cohesin complexes and their 243–254 regulation in prophase. J. Cell Biol. 151, 15 Hartman, T. et al. (2000) Pds5p is an essential sister chromatid cohesion still need to be 749–762 chromosomal protein required for both sister addressed: can cohesins form higher-order 5 Pasierbek, P. et al. (2001) A Caenorhabditis chromatid cohesion and condensation in structures, how is the cohesin complex elegans cohesion protein with functions in meiotic Saccharomyces cerevisiae. J. Cell Biol. 151, recruited to specific regions of the chromosome pairing and disjunction. Genes Dev. 613–626 15, 1349–1360 16 Panizza, S. et al. (2000) Pds5 cooperates with chromosome, how does the passage of the 6 Hirano, T. (2002) The ABCs of SMC proteins: cohesin in maintaining sister chromatid cohesion. replication fork lead to the linkage of sister two-armed ATPases for chromosome Curr. Biol. 10, 1557–1564 chromatids and what role does ATP condensation, cohesion, and repair. Genes Dev. 16, 17 Skibbens, R.V. et al. (1999) Ctf7p is essential for hydrolysis play in sister chromatid 399–414 sister chromatid cohesion and links mitotic cohesion. Undoubtedly, the establishment 7 Hirano, M. et al. (2001) Bimodal activation of chromosome structure to the DNA replication SMC ATPase by intra- and inter-molecular machinery. Genes Dev. 13, 307–319 and maintenance of sister chromatid interactions. EMBO J. 20, 3238–3250 18 Ross, K.E. et al. (2002) Separase: a conserved cohesin is a dynamic process and many of 8 Cohen-Fix, O. (2001) The making and protease separating more than just sisters. its moving parts still need to be elucidated. breaking of sister chromatid cohesion. Cell 106, Trends Cell Biol. 12, 1–3 137–140 19 Dej, K.J. et al. (2000) Separation anxiety at the Acknowledgements 9 Toth, A. et al. (1999) Yeast cohesin complex centromere. Trends Cell Biol. 10, 392–399 requires a conserved protein, Eco1p(Ctf7), 20 Cohen-Fix, O. (2000) Sister chromatid separation: We thank Ann Corsi, April Robbins, to establish cohesion between sister falling apart at the seams. Curr. Biol. 10, Ritu Agarwal, Paul Megee, chromatids during DNA replication. Genes Dev. R816–819 Doug Koshland and Tatsuya Hirano for 13, 320–333 stimulating and helpful discussions. 10 Tanaka, T. et al. (1999) Identification of cohesin association sites at centromeres and along Joseph L. Campbell chromosome arms. Cell 98, 847–858 Orna Cohen-Fix* References 11 Megee, P.C. et al. (1999) The centromeric sister The Laboratory of Molecular and Cellular 1 Anderson, D.E. et al. (2002) Condensin and chromatid cohesion site directs Mcd1p binding to cohesin display different arm conformations with adjacent sequences. Mol. Cell 4, 445–450 Biology, NIDDK, NIH 8 Center Drive, characteristic hinge angles. J. Cell Biol. 156, 12 Blat, Y. et al. (1999) Cohesins bind to preferential Bethesda, MD 20892, USA. 419–424 sites along yeast chromosome III, with *e-mail: [email protected]

Protein Sequence Motif YTH: a new domain in nuclear proteins Peter Stoilov, Ilona Rafalska and Stefan Stamm

A novel 100–150-residue domain has been intron sequences by the spliceosome is In addition, the alternative splicing identified in the human splicing factor crucial for gene expression. However, the patterns of the splicing factor SRp20 YT521-B and its Drosophila and yeast sequences of the two splice sites and the and hTra2-β pre-mRNAs are altered homologues. Homology searches show branch point that are recognized by by YT521-B [6]. YT521 does not belong that the domain is typical for the spliceosome components are clearly to any of the known splicing factor eukaryotes and is particularly abundant in insufficient to identify the exons in the families. The only sequence feature plants. It is predicted to adopt a mixed pre-mRNA sequence. Correct recognition that it shares with some of the splicing α-helix–β-sheet fold and to bind to RNA. of exons is achieved by the cooperative factors is an RE/D repeat. Here we We propose the name YTH (for YT521-B action of multiple splicing factors report the identification of a new homology) for the domain. auxiliary to the spliceosome [2]. These domain in splicing factor YT521-B factors bind in a sequence-specific and a number of proteins of unknown Published online: 5 September 2002 manner to the pre-mRNA [3,4] and function that could be involved in recruit the spliceosome components to RNA binding. One of the most prominent features of the splice sites. The nuclear protein eukaryotic genes is their discontinuity. YT521 has been identified in Domain characterization Analysis of the working draft of the two-hybrid screens with splicing During BLAST searches to identify human genome has shown that, on factors [5,6]. It interacts with several YT521-B homologues, a conserved part of average, introns account for 95% of the splicing factors, both in two-hybrid and the protein was identified between pre-mRNA [1]. The precise removal of co-immunoprecipitation assays [5,6]. residues 356 and 499 of the rat YT521-B

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Fig. 1. Multiple alignment of sequences containing YT homology (YTH) domains. The sequences are denoted with the closely related vertebrate homologues GeneBank identifier of the corresponding nucleotide sequence followed by the species name. The GeneBank identifier of YT521. for the Saccharomyces cerevisiae refers to protein entry in the database. Translations of partial EST sequences are indicated with an asterisk. The sequences were identified using searches of the translated EST and the nonredundant We used SMART [9,10] to search the nucleotide databases at NCBI using rat YT521-B (shown in italic). Wherever available, the conceptual translation SMART and PFAM databases for provided in the database was used for the alignment. If no conceptual translation was provided (i.e. for the EST additional known domains in the sequences), the reading frame identified by the BLAST search was used if it did not contain stop codons or unknown proteins that contain YTH. The search residues. Redundant sequences originating from the same species were omitted. The similar residues that are present in all sequences are shaded (green) using the PAM250 matrix. The identical residues are shaded in yellow. The failed to identify any known domains secondary structure, as predicted by the PHD program, is shown on top. E denotes extended (β-strands) structure and except for three C–C–C–H-type zinc H denotes the predicted α-helices. The multiple sequence alignment (accession number ALIGN_000432) has been finger domains in GI:18397519, an deposited at the European Bioinformatics Institute (ftp://ftp.ebi.ac.uk/pub/databases/embl/align/). A. thaliana predicted protein of unknown function. Therefore, we protein. We therefore performed a The sequences from these searches conclude that the YTH domain defines a PSI-BLAST [7] search with this portion of that aligned to the full length of the query new protein family. the YT521-B protein, using an exclusion and had an E value <10−6 were aligned The putative secondary structure was threshold of 0.005. After four iterations, using CLUSTALW [8] after the determined using the PHD program [11]. multiple proteins from Arabidopsis redundancies had been removed (Fig. 1). The domain is predicted to have a mixed thaliana, Oryza sativa, Homo sapiens, Additional BLAST searches against the αβ-fold, with four α-helices and six Mus musculus, Drosophila melanogaster, genome databases confirmed that the β-strands. The conservation pattern Plasmodium falciparum and conserved region is present exclusively follows the predicted secondary Saccharomyces cerevisiae where found to in eukaryotic genomes. structure, with three blocks of conserved have similar regions, with E values in the The conserved region appears to sequence separated by loops of variable range 10−11–10−60. Many of these protein define a new domain in these proteins, size. Notable features of the domain are sequences were derived from automated which we termed the YT homology (YTH) the highly conserved aromatic residues gene prediction and were not confirmed domain. The YTH domain is usually located in the β-sheet. by mRNA or EST sequences. To identify located in the middle of the protein Most of the proteins identified in the sequences of existing proteins, we sequence. The domain shows remarkable BLAST searches are of plant origin, with performed BLAST [7] searches with conservation across a wide species 13 distinct sequences coming from a the conserved region against the range, with 14 invariant and 19 single species (A. thaliana). It is unclear translated nonredundant nucleotide highly conserved residues. The proteins whether this protein family is more database at NCBI (filtered using present in the alignment do not share widespread in plants or whether the ‘biomol_mrna[PROP]’ as a keyword) and significant similarity outside the observed species distribution is because the translated EST database. YTH domain, with the exception of the of bias in the databases.

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Predicted function 3 Lui, H.-X. et al. (1998) Identification of signaling domains. Proc. Natl. Acad. Sci. U. S. A. Biochemically and functionally, YT521-B functional exonic splicing enhancer motifs 95, 5857–5864 has been extensively characterized as a recognized by individual SR proteins. Genes Dev. 10 Letunic, I. et al. (2002) Recent improvements 12, 1998–2012 to the SMART domain-based sequence pre-mRNA splicing factor [6,12]. In 4 Lui, H.-X. et al. (2000) Exonic splicing enhancer annotation resource. Nucleic Acids Res. 30, contrast to the other known splicing motif recognized by human SC35 under splicing 242–244 factors, it lacks a recognizable domain that conditions. Mol. Cell. Biol. 20, 1063–1071 11 Rost, B. (1996) PHD: predicting one- can confer RNA binding. The conservation 5 Imai, Y. et al. (1998) Cloning of a gene, YT521, for dimensional protein structure by profile-based of aromatic residues in the β-sheets of the a novel RNA splicing-related protein induced by neural networks. Methods Enzymol. 266, hypoxia/reoxygenation. Brain Res. Mol. Brain 525–539 YTH domain is similar to the RNA Res. 53, 33–40 12 Nayler, O. et al. (2000) The ER-repeat protein recognition motif (RRM) domain. In the 6 Hartmann, A.M. et al. (1999) The interaction and YT521-B localizes to a novel subnuclear RRM domain conserved aromatic residues colocalization of Sam68 with the splicing- compartment. J. Cell Biol. 150, 949–961 located in the β-sheet are crucial for RNA associated factor YT521-B in nuclear dots is 13 Hoffman, D.W. et al. (1991) RNA-binding domain binding [13]. In addition, experimental regulated by the Src family kinase p59(fyn). of the A protein component of the U1 small Mol. Biol. Cell 10, 3909–3926 nuclear ribonucleoprotein analyzed by NMR evidence shows that the YTH domain is 7 Altschul, S.F. et al. (1997) Gapped BLAST and spectroscopy is structurally similar to ribosomal not involved in protein–protein PSI-BLAST: a new generation of protein database proteins. Proc. Natl. Acad. Sci. U. S. A. 88, interactions [6]. Based on this, we predict search programs. Nucleic Acids Res. 25, 2495–2499 that the biological function of the YTH 3389–3402 domain is to bind to RNA. 8 Thompson, J.D. et al. (1994) CLUSTAL W: improving the sensitivity of progressive multiple Peter Stoilov References sequence alignment through sequence Ilona Rafalska 1 Lander, E.S. et al. (2001) Initial sequencing and weighting, position-specific gap penalties and Stefan Stamm analysis of the human genome. Nature 409, 860–921 weight matrix choice. Nucleic Acids Res. 22, 2 Smith, C.W. and Valcarcel, J. (2000) Alternative 4673–4680 Institute of Biochemistry, Friedrich- pre-mRNA splicing: the logic of combinatorial 9 Schultz, J. et al. (1998) SMART, a simple modular Alexander-University Erlangen-Nuremberg, control. Trends Biochem. Sci. 25, 381–388 architecture research tool: identification of Fahrstrasse 17, 91054 Erlangen, Germany.

BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms Mark Gomelsky and Gabriele Klug

A novel FAD-binding domain, BLUF, dinucleotide (FAD) noncovalently with Cyanobacteria (Fig. 1). Bacterial genomes exemplified by the N-terminus of the an apparent 1:1 stoichiometry [4]. At contain up to three BLUF domains per AppA protein from Rhodobacter the time of publication of Ref. [4], two genome. No BLUF domains are encoded sphaeroides, is present in various proteins, additional bacterial proteins showing by the currently available genomes of primarily from Bacteria. The BLUF domain sequence similarity to the N-terminus of Archaea. Four BLUF domains are found is involved in sensing blue-light (and AppA had been identified [4]. One of in Eukarya, all from the unicellular possibly redox) using FAD and is similar these, YcgF from Escherichia coli (also flagellate Euglena gracilis [5] (Fig. 1). to the flavin-binding PAS domains and known as F403 and b1163; accession cryptochromes. The predicted secondary number P75990), has been purified and Involvement of the BLUF domain in sensory structure reveals that the BLUF domain is a shown to bind FAD [4]. transduction novel FAD-binding fold. Using the region most conserved To our knowledge, the functions of only between AppA and YcgF (residues 16–108 two proteins containing BLUF domains The prototype and identification of the of AppA), we performed a BLAST search have been tested experimentally. Similar BLUF domain of the nonredundant protein database and to the BLUF domain in R. sphaeroides AppA is a multidomain protein from the a TBLASTN search of the microbial AppA, the BLUF domains from the phototrophic proteobacterium genome database at NCBI, as well as a recently described photoactivated Rhodobacter sphaeroides (accession TBLASTN search of the individual adenylyl cyclase (PAC) from E. gracilis are number L42555). The N-terminus of the unfinished microbial genomes at the also involved in the blue-light-dependent AppA protein is involved in the regulation sequencing centers listed in our control of enzyme activity. Two BLUF of photosynthesis gene expression, Acknowledgements. Our searches domains belong to the α-subunit of the although its mechanism of action is not revealed a variety of uncharacterized enzyme, PACα, and two to the well understood [1–4]. The N-terminus of proteins containing domains with PACβ subunit [5] (Fig. 1). To gain an the AppA protein was identified as being significant similarity to the N-terminus insight into the putative role of the BLUF involved in repression of photosynthesis of AppA. We designated these domains domains in other proteins we analyzed genes by blue-light [1], and Gomelsky and BLUF, for ‘sensors of blue-light using their domain architecture using the Kaplan showed that the N-terminal FAD’. Most of these proteins are from two SMART [6] and Pfam [7] databases. Based ~120 residues bind flavin adenine branches of Bacteria, Proteobacteria and on the deduced domain structures, all

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