U8 variants on the brain: a small nucleolar RNA and human disease.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs vital for ribosomal RNA (rRNA) maturation. The U8 snoRNA, encoded by the SNORD118 gene in humans, is an atypical C/D box snoRNA as it promotes rRNA cleavage rather than 2'-O-methylation and is unique to vertebrates. The U8 snoRNA is critical for cleavage events that produce the mature 5.8S and 28S rRNAs of the large ribosomal subunit. Unexpectedly, single nucleotide polymorphisms (SNPs) in the SNORD118 gene were recently found causal to the neurodegenerative disease leukoencephalopathy, brain calcifications, and cysts (LCC; aka Labrune syndrome), but its molecular pathogenesis is unclear. Here, we will review current knowledge on the function of the U8 snoRNA in ribosome biogenesis, and connect it to the preservation of brain function in humans as well as to its dysregulation in inherited white matter disease.

Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin.

Long noncoding RNAs (lncRNAs), defined as nontranslated transcripts greater than 200 nucleotides in length, are often differentially expressed throughout developmental stages, tissue types, and disease states. The identification, visualization, and suppression/overexpression of these sequences have revealed impacts on a wide range of biological processes, including epigenetic regulation. Biochemical investigations on select systems have revealed striking insight into the biological roles of lncRNAs and lncRNA:protein complexes, which in turn prompt even more unanswered questions. To begin, multiple protein- and RNA-centric technologies have been employed to isolate lncRNA:protein and lncRNA:chromatin complexes. LncRNA interactions with the multi-subunit protein complex PRC2, which acts as a transcriptional silencer, represent some of the few cases where the binding affinity, selectivity, and activity of a lncRNA:protein complex have been investigated. At the same time, recent reports of full-length lncRNA secondary structures suggest the formation of complex structures with multiple independent folding domains and pave the way for more detailed structural investigations and predictions of lncRNA three-dimensional structure. This review will provide an overview of the methods and progress made to date as well as highlight new methods that promise to further inform the molecular recognition, specificity, and function of lncRNAs.

Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide.

The bacterial repair enzyme MutT hydrolyzes the damaged nucleotide OdGTP (the 5'-triphosphate derivative of 8-oxo-2'-deoxyguanosine; OdG), which is a known mutagen and has been linked to antibacterial action. Previous work has indicated important roles for the C8-oxygen, N7-hydrogen, and C2-exocyclic amine during OdGTP recognition by MutT. In order to gain a more nuanced understanding of the contribution of these three sites to the overall activity of MutT, we determined the reaction parameters for dGTP, OdGTP, and nine of their analogues using steady state kinetics. Our results indicate that overall high reaction efficiencies can be achieved despite altering any one of these sites. However, altering two or more sites leads to a significant decrease in efficiency. The data also suggest that, similar to another bacterial OdG repair enzyme, MutM, a specific carbonyl in the enzyme can not only promote activity by forming an active site hydrogen bond with the N7-hydrogen of OdGTP, but can also hinder activity through electrostatic repulsion with the N7-lone pair of dGTP.

Biochemical investigations into the mutagenic potential of 8-oxo-2'-deoxyguanosine using nucleotide analogues.

8-Oxo-2'-deoxyguanosine (OdG) is an abundant DNA lesion produced during oxidative damage to DNA. It can form relatively stable base pairs with both dC and dA that mimic natural dG:dC and dT:dA base pairs, respectively. Thus, when in the template strand, OdG can direct the insertion of either dCTP or dATP during replication, the latter of which can lead to a dG → T transversion. The potential for OdG to cause mutation is dependent on the preference for dCTP or dATP insertion opposite OdG, as well as the ability to extend past the resulting base pairs. The C2-amine and C8-oxygen could play major roles during these reactions since both would lie outside the Watson-Crick cognate base pairs shape in the major groove when OdG base pairs to dA and dC, respectively, and both have the ability to form strong interactions, like hydrogen bonds. To gain a more generalized understanding of how the C2-amine and C8-oxygen of OdG affect its mutagenic potential, the incorporation opposite and extension past seven analogues of dG/OdG that vary at C2 and/or C8 were characterized for three DNA polymerases, including an exonuclease-deficient version of the replicative polymerase from RB69 (RB69), human polymerase (pol) ß, and polymerase IV from Sulfolobus solfataricus P2 (Dpo4). Based on the results from these studies, as well as those from previous studies with RB69, pol ß, Dpo4, and two A-family polymerases, the influence of the C2-amine and C8-oxygen during each incorporation and extension reaction with each polymerase is discussed. In general, it appears that when the C2-amine and the C8-oxygen are in the minor groove, they allow OdG to retain interactions that are normally present during insertion and extension. However, when the two groups are in the major groove, they each tend to form novel active site interactions, both stabilizing and destabilizing, that are not present during insertion and extension with natural DNA.

Importance of the C2, N7, and C8 positions to the mutagenic potential of 8-Oxo-2'-deoxyguanosine with two A family polymerases.

8-Oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion produced from the reaction of 2'-deoxyguanosine (dG) with reactive oxygen species. While dG directs the insertion of only dCTP during replication, OdG can direct the insertion of either dCTP or dATP, allowing for the production of dG → dT transversions. When replicated by Klenow fragment-exo (KF-exo), OdG preferentially directs the incorporation of dCTP over dATP, thus decreasing its mutagenic potential. However, when replicated by a highly related polymerase, the large fragment of polymerase I from Bacillus stearothermophilus (BF), dATP incorporation is preferred, and a higher mutagenic potential results. To gain insight into the reasons for this opposite preference and the effects of the C2, N7, and C8 positions on OdG mutagenicity, single-nucleotide insertions of dCTP and/or dATP opposite dG, OdG, and seven of their analogues were examined by steady state kinetics with both KF-exo and BF. Results from these studies suggest that the two enzymes behave similarly and are both sensitive not only to steric and electronic changes within the imidazole ring during both dCTP and dATP incorporation but also to the presence of the C2-exocyclic amine during dATP incorporation. The difference in incorporation preference opposite OdG appears to be due to a somewhat increased sensitivity to structural perturbations during dCTP incorporation with BF. Single-nucleotide extensions past the resulting base pairs were also studied and were not only similar between the two enzymes but also consistent with published ternary crystallographic studies with BF. These results are analyzed in the context of previous biochemical and structural studies, as well as stability studies with the resulting base pairs.