ABSTRACT
Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.
Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , DNA/metabolism , MutS Homolog 2 Protein/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Trinucleotide Repeat Expansion , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , DNA/genetics , DNA Ligase ATP , DNA Ligases/genetics , DNA Ligases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , Humans , Huntington Disease/genetics , Huntington Disease/metabolism , MutS Homolog 2 Protein/genetics , MutS Homolog 3 Protein , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Pseudouridines are the most abundant and highly conserved modified nucleotides identified in spliceosomal small nuclear RNAs (snRNAs). Most pseudouridines are also clustered in functionally important regions of spliceosomal snRNAs. Experiments carried out in several independent experimental systems show that the pseudouridines in spliceosomal snRNAs are functionally important for pre-messenger RNA (mRNA) splicing. Experimental data also indicate that spliceosomal snRNA pseudouridylation can be catalyzed by both RNA-dependent (box H/ACA Ribonucleoproteins) and RNA-independent (protein-only enzymes) mechanisms.
Subject(s)
Pseudouridine/metabolism , RNA, Small Nuclear/metabolism , Spliceosomes/metabolism , Animals , Base Sequence , Female , Humans , Models, Molecular , Molecular Sequence Annotation , Nucleic Acid Conformation , Oocytes/metabolism , Pseudouridine/chemistry , RNA Processing, Post-Transcriptional , RNA Splicing , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , XenopusABSTRACT
Accuracy in the flow of genetic information from DNA to protein, or gene expression, is essential to the viability of an organisms. Pre-mRNA splicing and protein translation are two major steps in eukaryotic gene expression that necessitate the production of accurate gene products. Both processes occur in large complexes, consisting of both proteins and noncoding RNAs. Interestingly, the RNA components contain a large number of posttranscriptional modifications, including 2'-O-methylation and pseudouridylation, which are functionally important. In this chapter, we highlight the functional aspects of the modifications of spliceosomal snRNA and rRNA and provide a framework for understanding how posttranscriptional modifications are capable of influencing gene expression.
Subject(s)
RNA Processing, Post-Transcriptional/genetics , Animals , Base Sequence , Humans , Molecular Sequence Data , RNA Splicing/genetics , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , Spliceosomes/metabolismABSTRACT
RNA modifications impact numerous cellular processes such as pre-mRNA splicing and protein synthesis. The elucidation of the mechanisms by which these modifications impact cellular processes necessitates the ability to both detect and quantify the presence of these modifications within RNA molecules. Here, we present a detailed procedure that allows the detection and quantification of RNA base modifications. This procedure involves a number of techniques, including oligonucleotide-affinity selection, site-specific cleavage and radiolabeling, nuclease digestion, and thin layer chromatography.
