Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
Biochemistry ; 59(37): 3463-3472, 2020 09 22.
Article in English | MEDLINE | ID: mdl-32856901

ABSTRACT

There are few methods available for the rapid discovery of multitarget drugs. Herein, we describe the template-assisted, target-guided discovery of small molecules that recognize d(CTG) in the expanded d(CTG·CAG) sequence and its r(CUG) transcript that cause myotonic dystrophy type 1. A positive cross-selection was performed using a small library of 30 monomeric alkyne- and azide-containing ligands capable of producing >5000 possible di- and trimeric click products. The monomers were incubated with d(CTG)16 or r(CUG)16 under physiological conditions, and both sequences showed selectivity in the proximity-accelerated azide-alkyne [3+2] cycloaddition click reaction. The limited number of click products formed in both selections and the even smaller number of common products suggests that this method is a useful tool for the discovery of single-target and multitarget lead therapeutic agents.


Subject(s)
DNA/antagonists & inhibitors , Myotonic Dystrophy/drug therapy , Myotonic Dystrophy/genetics , RNA/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Trinucleotide Repeat Expansion/drug effects , Cells, Cultured , DNA/genetics , DNA/metabolism , Humans , Myotonic Dystrophy/pathology , RNA/genetics , RNA/metabolism , Trinucleotide Repeat Expansion/genetics
2.
RNA ; 26(10): 1400-1413, 2020 10.
Article in English | MEDLINE | ID: mdl-32518066

ABSTRACT

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 carboxy-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.


Subject(s)
RNA Stability/genetics , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Binding Sites/genetics , Protein Binding/genetics , RNA/genetics , RNA Cap-Binding Proteins/genetics , RNA Splicing/genetics , RNA, Messenger/genetics , RNA, Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/genetics , Saccharomyces cerevisiae/genetics , Spliceosomes/genetics
3.
Nucleic Acids Res ; 48(3): 1423-1434, 2020 02 20.
Article in English | MEDLINE | ID: mdl-31832688

ABSTRACT

U6 snRNA undergoes post-transcriptional 3' end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3' end of U6 and, in humans, leaves a 2',3' cyclic phosphate that is recognized by the Lsm2-8 complex. Saccharomycescerevisiae Usb1 has additional 2',3' cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3' phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5'-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n - 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.


Subject(s)
Evolution, Molecular , Mitochondrial Proteins/genetics , Phosphoric Diester Hydrolases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Binding Sites/genetics , Catalytic Domain/genetics , Humans , Kluyveromyces/chemistry , Mitochondrial Proteins/chemistry , Models, Molecular , Nucleic Acid Conformation , Nucleotides/chemistry , Nucleotides/genetics , Phosphates/metabolism , Phosphoric Diester Hydrolases/chemistry , RNA Splicing/genetics , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Spliceosomes/chemistry , Spliceosomes/genetics
SELECTION OF CITATIONS
SEARCH DETAIL