Heideman, E. M. | bioRxiv Science

bioRxiv2020

Deoxyribozyme-based Method for Site-specific Absolute Quantification of N6-methyladenosine Modification Fraction

N6-methyladenosine (m6A) is the most prevalent modified base in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRNA). Although candidate sites for m6A modification are identified at the transcriptomic level, site-specific quantification methods for m6A modifications are still limited. Herein, we present a facile method implementing deoxyribozyme that preferentially cleaves the unmodified RNA. We leverage reverse transcription and real-time quantitative PCR along with key control experiments to quantify the absolute methylation fraction of specific m6A sites. We validate the accuracy of the method using synthetic RNA with controlled methylation fraction and apply our method on several endogenous sites that were previously identified in sequencing-based studies. This method provides a time and cost-effective approach for absolute quantification of the m6A fraction at specific loci, expanding the current toolkit for studying RNA modifications.

biochemistry↗

bioRxiv2020

Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs and mRNAs in live bacterial cells

RNA binding proteins play myriad roles in controlling and regulating RNAs and RNA-mediated functions, often through simultaneous binding to other cellular factors. In bacteria, the RNA chaperone Hfq modulates post-transcriptional gene regulation. Absence of Hfq leads to the loss of fitness and compromises the virulence of bacterial pathogens. Using live-cell super-resolution imaging, we are able to distinguish Hfq binding to different sizes of cellular RNAs. We demonstrate that under normal growth conditions, Hfq exhibits widespread mRNA binding activity. Particularly, the distal face of Hfq contributes mostly to the mRNA binding in vivo. In addition, binding of Hfq to these mRNAs can recruit RNase E to promote turnover of these mRNAs in an sRNA-independent manner, providing one mechanism to release Hfq from the pre-bound mRNAs. Finally, our data indicate that sRNAs, once expressed, can either co-occupy Hfq with the mRNA or displace the mRNA from Hfq, suggesting mechanisms through which sRNAs rapidly access Hfq to induce sRNA-mediated gene regulation. Our data collectively demonstrate that Hfq dynamically changes its interactions with different RNAs in response to changes in cellular conditions.

biophysics↗