The rapid accumulation of genome sequences and protein structures during the last decade has been paralleled by major advances in sequence database search methods. The powerful Position-Specific Iterating BLAST (PSI-BLAST) method developed at the NCBI formed the basis of our work on protein motif analysis. In addition, Hidden Markov Models (HMM), protein profile-against-profile comparison implemented in the HHSearch method, protein structure comparison methods, homology modeling of protein structure and genome context analysis were extensively applied. Over the last year, we made further progress in the study of the classification, evolution, and functions of several classes of proteins and domains. Specifically, we analyzed the evolution and functions of protein domains that are involved in virus-host interactions, from both the host and the virus sides. The CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold). Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and """"""""effector"""""""" domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death. Polintons (also known as Mavericks) and Tlr elements of Tetrahymena thermophila represent two families of large DNA transposons widespread in eukaryotes. We performed a detailed analysis of protein sequences encoded by these transposable elements and showed that both Polintons and Tlr elements encode two key virion proteins, the major capsid protein with the double jelly-roll fold and the minor capsid protein, known as the penton, with the single jelly-roll topology. This observation along with the previously noted conservation of the genes for viral genome packaging ATPase and adenovirus-like protease strongly suggests that Polintons and Tlr elements combine features of bona fide viruses and transposons. We proposed the name 'Polintoviruses'to denote these putative viruses that could have played a central role in the evolution of several groups of DNA viruses of eukaryotes. These ongoing studies reveal new aspects on the remarkably diverse repertoire of protein domains involved in virus-host interactions. As part of our ongoing investigation of the evolution of protein domain architectures, we analyzed the contributions of alternative splicing (AS),and alternative transcription initiation (ATI) and alternative transcription termination (ATT) to the evolution of mammalian proteins. Together, AS, ATI and ATT create the extraordinary complexity of transcriptomes and make key contributions to the structural and functional diversity of mammalian proteomes. Analysis of mammalian genomic and transcriptomic data shows that contrary to the traditional view, the joint contribution of ATI and ATT to the transcriptome and proteome diversity is quantitatively greater than the contribution of AS. Although the mean numbers of protein-coding constitutive and alternative nucleotides in gene loci are nearly identical, their distribution along the transcripts is highly non-uniform. On average, coding exons in the variable 5'and 3'transcript ends that are created by ATI and ATT contain approximately four times more alternative nucleotides than core protein-coding regions that diversify exclusively via AS. Short upstream exons that encompass alternative 5'-untranslated regions and N-termini of proteins evolve under strong nucleotide-level selection whereas in 3'-terminal exons that encode protein C-termini, protein-level selection is significantly stronger. The groups of genes that are subject to ATI and ATT show major differences in biological roles, expression and selection patterns. These studies enhance the existing understanding of the evolutionary plasticity of protein domain architecture.
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