identification-rna-fragments-resulting-from-enzymatic-degradation

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

University of Denver

Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5'-3' progression of cellular exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome's 3'-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that 'brace' against an enzyme's surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies.

Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs.

Messenger RNA degradation is an important component of overall gene expression. During the final step of eukaryotic mRNA degradation, exoribonuclease 1 (Xrn1) carries out 5' → 3' processive, hydrolytic degradation of RNA molecules using divalent metal ion catalysis. To initiate studies of the 5' → 3' RNA decay machinery in our lab, we expressed a C-terminally truncated version of  Xrn1 and explored its enzymology using a second-generation, time-resolved fluorescence RNA degradation assay. Using this system, we quantitatively explored Xrn1's preference for 5'-monophosphorylated RNA substrates, its pH dependence, and the importance of active site mutations in the molecule's conserved catalytic core. Furthermore, we explore Xrn1's preference for RNAs containing a 5' single-stranded region both in an intermolecular hairpin structure and in an RNA-DNA hybrid duplex system. These results both expand and solidify our understanding of Xrn1, a centrally important enzyme whose biochemical properties have implications in numerous RNA degradation and processing pathways.

Biochemical Characterization of Yeast Xrn1.

Understanding how oxidatively damaged RNA is handled intracellularly is of relevance due to the link between oxidized RNA and the progression/development of some diseases as well as aging. Among the ribonucleases responsible for the decay of modified (chemically or naturally) RNA is the exonuclease Xrn-1, a processive enzyme that catalyzes the hydrolysis of 5'-phosphorylated RNA in a 5'→3' direction. We set out to explore the reactivity of this exonuclease towards oligonucleotides (ONs, 20-nt to 30-nt long) of RNA containing 8-oxo-7,8-dihydroguanosine (8-oxoG), obtained  solid-phase synthesis. The results show that Xrn-1 stalled at sites containing 8-oxoG, evidenced by the presence of a slower moving band ( electrophoretic analyses) than that observed for the canonical analogue. The observed fragment(s) were characterized  PAGE and MALDI-TOF to confirm that the oligonucleotide fragment(s) contained a 5'-phosphorylated 8-oxoG. Furthermore, the yields for this stalling varied from app. 5-30% with 8-oxoG located at different positions and in different sequences. To gain a better understanding of the decreased nuclease efficiency, we probed: 1) H-bonding and spatial constraints; 2) anti-syn conformational changes; 3) concentration of divalent cation; and 4) secondary structure. This was carried out by introducing methylated or brominated purines (mG, mA, or 8-BrG), probing varying [Mg], and using circular dichroism (CD) to explore the formation of structured RNA. It was determined that spatial constraints imposed by conformational changes around the glycosidic bond may be partially responsible for stalling, however, the results do not fully explain some of the observed higher stalling yields. We hypothesize that altered π-π stacking along with induced H-bonding interactions between 8-oxoG and residues within the binding site may also play a role in the decreased Xrn-1 efficiency. Overall, these observations suggest that other factors, yet to be discovered/established, are likely to contribute to the decay of oxidized RNA. In addition, Xrn-1 degraded RNA containing mG, and stalled mildly at sites where it encountered mA, or 8-BrG, which is of particular interest given that the former two are naturally occurring modifications.

Erich G. Chapman

Department of Chemistry & Biochemistry

University of Denver

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Erich G. Chapman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry & Biochemistry

University of Denver

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Department of Chemistry & Biochemistry