ABSTRACT
Antisense agents are powerful tools to inhibit gene expression in a sequence-specific manner. They are used for functional genomics, as diagnostic tools and for therapeutic purposes. Three classes of antisense agents can be distinguished by their mode of action: single-stranded antisense oligodeoxynucleotides; catalytic active RNA/DNA such as ribozymes, DNA- or locked nucleic acid (LNA)zymes; and small interfering RNA molecules known as siRNA. The selection of target sites in highly structured RNA molecules is crucial for their successful application. This is a difficult task, since RNA is assembled into nucleoprotein complexes and forms stable secondary structures in vivo, rendering most of the molecule inaccessible to intermolecular base pairing with complementary nucleic acids. In this review, we discuss several selection strategies to identify potential target sites in RNA molecules. In particular, we focus on combinatorial library approaches that allow high throughput screening of sequences for the design of antisense agents.
Subject(s)
RNA/therapeutic use , Animals , Gene Library , Humans , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , RNA/chemistry , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Catalytic/therapeutic use , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic useABSTRACT
We isolated srp2, a gene encoding a protein composed of two RNA binding domains (RBDs) at the N-terminus followed by an arginine-rich region that is flanked by two short SR (serine/arginine) elements. The RBDs contain the signatures RDADDA and SWQDLKD found in RBD1 and RBD2 of all typical metazoan SR proteins. srp2 is essential for growth. We have analyzed in vivo the role of the modular domains of Srp2 by testing specific mutations in a conditional strain for complementation. We found that RBD2 is essential for function and determines the specificity of RBD1 in Srp2. Replacement of the first RBD with RBD1 of Srp1 of fission yeast does not change this specificity. The two SR elements in the C-terminus of Srp2 are also essential for function in vivo. Cellular distribution analysis with green fluorescence protein fused to portions of Srp2 revealed that the SR elements are necessary to target Srp2 to the nucleus. Furthermore, overexpression of modular domains of Srp2 and Srp1 show different effects on pre-mRNA splicing activity of the tfIId gene. Taken together, these findings are consistent with the notion that the RBDs of these proteins may be involved in pre-mRNA recognition.
Subject(s)
Fungal Proteins/genetics , RNA-Binding Proteins/genetics , Schizosaccharomyces pombe Proteins , Schizosaccharomyces/genetics , Alleles , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Molecular Sequence Data , Mutation , RNA, Fungal/metabolism , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Schizosaccharomyces/metabolism , Sequence AlignmentABSTRACT
The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process.
Subject(s)
RNA Splicing , RNA-Binding Proteins/genetics , RNA/metabolism , Schizosaccharomyces pombe Proteins , Schizosaccharomyces/genetics , Amino Acid Sequence , Arginine , Binding Sites , Gene Expression , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Precursors/genetics , RNA Splicing Factors , RNA, Messenger/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Schizosaccharomyces/growth & development , Serine , Structure-Activity RelationshipABSTRACT
The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain.
