A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex.

The SNF1 protein kinase is required for the regulatory response to glucose starvation in Saccharomyces cerevisiae. SNF1 is a protein serine/threonine kinase that has been widely conserved in both plants and mammals. Previously, we identified SIP1 and SIP2 as proteins that interact with SNF1 in vivo by the two-hybrid system. We have cloned the SIP2 gene and the encoded protein is homologous to SIP1 and to GAL83, which affects glucose repression of the GAL genes. We show that SIP2 and GAL83, like SIP1, co-immunoprecipitate with SNF1 and are phosphorylated in vitro. An 80 amino acid sequence, designated the ASC domain, is highly conserved at the C-termini of all three proteins. We show that this small domain can mediate protein-protein interaction with the SNF1 kinase complex. Thus, SIP1, SIP2 and GAL83 define a family of homologous proteins that are tightly associated with the SNF1 kinase, probably in alternative forms of the complex. Genetic evidence suggests that the three proteins have distinct, but related, functions in the SNF1 pathway, and deletion of GAL83 dramatically reduces SNF1 activity in immune complex assays. We propose that SIP1, SIP2 and GAL83 act as adaptors that promote the activity of SNF1 towards specific targets.     

Functional analysis of SNF, the Drosophila U1A/U2B" homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.     

Solution structure of the PABC domain from wheat poly (A)-binding protein: an insight into RNA metabolic and translational control in plants

In animals, the PABC domain from poly (A)-binding protein recruits proteins containing a specific interacting motif (PAM-2) to the mRNP complex. These proteins include Paip1, Paip2, and eukaryotic release factor 3 (eRF3), all of which regulate PABP function in translation. The following reports the solution structure of PABC from Triticum avestium (wheat) poly (A)-binding protein determined by NMR spectroscopy. Wheat PABC (wPABC) is an alpha-helical protein domain, which displays a fold highly similar to the human PABC domain and contains a PAM-2 peptide binding site. Through a bioinformatics search, several plant proteins containing a PAM-2 site were identified including the early response to dehydration protein (ERD-15), which was previously shown to regulate PABP-dependent translation. The plant PAM-2 proteins contain a variety of conserved sequences including a PABP-interacting 1 motif (PAM-1), RNA binding domains, an SMR endonuclease domain, and a poly (A)-nuclease regulatory domain, all of which suggest a function in either translation or mRNA metabolism. The proteins identified are well conserved throughout plant species but have no sequence homologues in metazoans. We show that wPABC binds to the plant PAM-2 motif with high affinity through a conserved mechanism. Overall, our results suggest that plant species have evolved a distinct regulatory mechanism involving novel PABP binding partners.     

Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54

SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.     

Regulation of trypanosome DNA glycosylation by a SWI2/SNF2-like protein

Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.     

Chromosomal assignment of LASP1 and LASP2 genes and organization of the LASP2 gene in chicken

Lasp-1 and lasp-2 are actin-binding proteins that contain a LIM domain, two nebulin repeats and an SH3 domain with significant identity. We determined the chromosomal locations of the LASP1 and LASP2 genes in chicken by fluorescence in situ hybridization. The LASP1 gene was localized to a pair of microchromosomes and the LASP2 gene was localized to chromosome 2p3.1, indicating that the chromosomal locations of the LASP1 and LASP2 genes are highly conserved between chicken and human. The comparison of genomic and cDNA sequences of chicken lasp-2 and nebulette, a nebulin-related protein in muscle, suggested that both the corresponding mRNAs shared exons in the same manner as their human homologues. When compared with the domain structure of nebulette, another nebulin repeat was predicted for lasp-2, and all the nebulin repeats of lasp-2 were better conserved than those in nebulette. We also found the exon boundaries in nebulin repeats of lasp-2 were similar to those of other nebulin-related proteins.     

Resurrection of an Urbilaterian U1A/U2B″/SNF Protein

The U1A/U2B″/SNF family of proteins found in the U1 and U2 spliceosomal small nuclear ribonucleoproteins is highly conserved. In spite of the high degree of sequence and structural conservation, modern members of this protein family have unique RNA binding properties. These differences have necessarily resulted from evolutionary processes, and therefore, we reconstructed the protein phylogeny in order to understand how and when divergence occurred and how protein function has been modulated. Contrary to the conventional understanding of an ancient human U1A/U2B″ gene duplication, we show that the last common ancestor of bilaterians contained a single ancestral protein (URB). The gene for URB was synthesized, the protein was overexpressed and purified, and we assessed RNA binding to modern snRNA sequences. We find that URB binds human and Drosophila U1 snRNA SLII and U2 snRNA SLIV with higher affinity than do modern homologs, suggesting that both Drosophila SNF and human U1A/U2B″ have evolved into weaker binders of one RNA or both RNAs.     

In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins

Proteins belonging to SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. The yeast Ino80, the catalytic ATPase subunit of the INO80 complex, is the most recently described member of the SNF2 family. Outside the conserved ATPase domain, it has very little similarity with other well-characterized SNF2 proteins hence it is believed to represent a new subfamily. We have identified new members of this subfamily in different organisms and have detected characteristic features of this subfamily. Using various data mining tools we have identified a new, previously undetected domain in all members of this subfamily. This domain designated DBINO is characteristic of the INO80 subfamily and is predicted to have DNA-binding function. The presence of this domain in all the INO80 subfamily proteins from different organisms suggests its conserved function in evolution.     

The HIRAN Domain and Recruitment of Chromatin Remodeling and Repair activities to Damaged DNA

Aided by sensitive sequence profile searches we identify a novel conserved domain in the Nterminalregions of the SWI2/SNF2 proteins typified by HIP116 and Rad5p (hence HIP116,Rad5p N-terminal domain: HIRAN domain). We show that the HIRAN domain is found as astandalone protein in several bacteria and prophages, or fused to other catalytic domains, suchas a nuclease of the restriction endonuclease fold and TDP1-like DNA phosphoesterases, in theeukaryotes. Based on a network of contextual connections in the form of domain architectures,conserved gene neighborhoods and functional interactions we predict that the HIRAN domain islikely to function as a DNA-binding domain that probably recognizes features associated withdamaged DNA or stalled replication forks. It might thus act as a sensor to initiate a damaged DNAcheckpoint and engage different DNA repair and chromatin remodeling or modifying activities tothese sites. In evolutionary terms, the fusion of the HIRAN domain, and the functionallyanalogous Rad18 Zn-finger and the PARP-type Zn-finger to SWI2/SNF2 ATPases appears tohave been a notable factor for recruiting these ATPases for chromatin modification andremodeling in the context of DNA repair.     

Cloning and expression of chicken CLIP-170 and Restin isoforms

We have cloned cDNA for the chicken homologues of human CLIP-170 and Restin and characterized expression of chicken CLIP-170 and Restin messages in a variety of chicken tissues. Chicken CLIP-170 and Restin, like the human homologues, differ only in a stretch of 35 amino acids present in Restin but missing from CLIP-170. This Restin-specific insert is perfectly conserved between the chicken and human sequences at both the protein and nucleotide level and contributes an additional five heptads to one of the heptad repeat regions in the central α-helical coiled-coil rod domain. Other highly conserved chicken and human CLIP-170/Restin regions confirm the importance of certain protein domains as crucial for protein function, including two CAP-Gly microtubule-binding motifs in the N-terminal globular head domain and two CCHC metal-binding motifs in the C-terminal globular tail domain. We have used Southern DNA blot analysis and PCR amplification of exon–intron junctions of chicken genomic DNA to confirm that CLIP-170 and Restin are isoforms encoded by the same gene. Semiquantitative RT-PCR analysis of CLIP-170 and Restin mRNA expression revealed expression of both isoforms in a variety of chicken tissues but in different ratios. In the tissues tested, except brain, the message for CLIP-170 was more abundant than that for Restin. Comparison of the levels of CLIP-170 and Restin messages in RNA from chicken and human intestinal epithelial cells revealed remarkably similar ratios in the two species. Our data suggest that expression of CLIP-170 and Restin is differentially regulated and that the two isoforms have distinct functions in a wide variety of cells.     

The Drosophila SNR1 (SNF5/INI1) subunit directs essential developmental functions of the Brahma chromatin remodeling complex

The Drosophila melanogaster Brahma (Brm) complex, a counterpart of the Saccharomyces cerevisiae SWI/SNF ATP-dependent chromatin remodeling complex, is important for proper development by maintaining specific gene expression patterns. The SNR1 subunit is strongly conserved with yeast SNF5 and mammalian INI1 and is required for full activity of the Brm complex. We identified a temperature-sensitive allele of snr1 caused by a single amino acid substitution in the conserved repeat 2 region, implicated in a variety of protein-protein interactions. Genetic analyses of snr1(E1) reveal that it functions as an antimorph and that snr1 has critical roles in tissue patterning and growth control. Temperature shifts show that snr1 is continuously required, with essential functions in embryogenesis, pupal stages, and adults. Allele-specific genetic interactions between snr1(E1) and mutations in genes encoding other members of the Brm complex suggest that snr1(E1) mutant phenotypes result from reduced Brm complex function. Consistent with this view, SNR1(E1) is stably associated with other components of the Brm complex at the restrictive temperature. SNR1 can establish direct contacts through the conserved repeat 2 region with the SET domain of the homeotic regulator Trithorax (TRX), and SNR1(E1) is partially defective for functional TRX association. As truncating mutations of INI1 are strongly correlated with aggressive cancers, our results support the view that SNR1, and specifically the repeat 2 region, has a critical role in mediating cell growth control functions of the metazoan SWI/SNF complexes.