RNA-binding proteins play myriad roles in regulating RNAs and RNA-mediated functions. In bacteria, the RNA chaperone Hfq is an important post-transcriptional gene regulator. Using live-cell super-resolution imaging, we can distinguish Hfq binding to different sizes of cellular RNAs. We demonstrate that under normal growth conditions, Hfq exhibits widespread mRNA-binding activity, with the distal face of Hfq contributing mostly to the mRNA binding in vivo. In addition, sRNAs can either co-occupy Hfq with the mRNA as a ternary complex, or displace the mRNA from Hfq in a binding face-dependent manner, suggesting mechanisms through which sRNAs rapidly access Hfq to induce sRNA-mediated gene regulation. Finally, our data suggest that binding of Hfq to certain mRNAs through its distal face can recruit RNase E to promote turnover of these mRNAs in an sRNA-independent manner, and such regulatory function of Hfq can be decoyed by sRNA competitors that bind strongly at the distal face.

Hfq binds broadly to cellular mRNAs and sRNAs [13][14][15] , in line with its main biological 51 functions. Hfq can bind RNAs through multiple interfaces of its homohexameric structure. The 52 surface containing the N-terminal α-helices is referred as the "proximal face" of the Hfq hexamer, 53whereas the opposite surface is referred as the "distal face", and the outer ring as the "rim" (Figure 54 1a). The proximal face binds preferably U-rich sequences, and the distal face prefers A-rich 55 sequences, with the exact motif of the A-rich sequence depending on the species [16][17][18][19][20] . The rim 56 can also interact with UA-rich RNAs through the patch of positively charged residues 21-23 . Finally, 57 the unstructured C-terminal end of Hfq can also interact with certain RNAs to promote the 58 exchange of RNAs 18,21,24 . The most refined model describing the interactions between Hfq and 59 sRNAs/mRNAs has sorted sRNAs into two classes 25 . The proximal face of Hfq is generally 60 important for binding of the sRNAs through their poly-U tail of the Rho-independent terminator. 61Class I sRNAs (such as RyhB and DsrA) use the rim as the second binding site, whereas class II 62 sRNAs (such as ChiX and MgrR) use the distal face as the second binding site 25 . In addition, the 63 preferred target mRNAs of the two classes of the sRNA are proposed to have the complementary 64 binding sites on Hfq, i.e. class I sRNA-targeted mRNAs binding to the distal face, and class II 65 sRNA-targeted mRNA binding to the rim, in order to efficiently form sRNA-mRNA complexes 25 . 66As for many other RBPs, the functions of Hfq are facilitated by its interactions with other essential 67 protein factors. Particularly, RNase E, the key ribonuclease for processing and turnover of 68 ribosomal RNA (rRNAs) and mRNAs, is known to interact with Hfq through its C-terminal scaffold 69 region [26][27][28] . The Hfq-RNase E interactions can promote degradation of the sRNA-targeted 70 mRNA 28-31 . 71 While Hfq is an abundant RBP in bacterial cells 32,33 , it is still considered to be limiting, given 72 the abundance of cellular mRNAs and sRNAs. Particularly, in vitro studies on specific sRNAs 73 demonstrate that Hfq binds RNAs tightly with a dissociation constant of nM, and the Hfq-RNA 74

N 6 -methyladenosine (m 6 A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA (lncRNA). Although candidate sites for the m 6 A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m 6 A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m 6 A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0% to 100% and applied our method to 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 m 6 A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications. AUTHOR CONTRIBUTIONSM.B. J.Z. and J.F. designed the experiments. M.B., J.Z., and E.H. performed the experiments. M.B. analyzed the data. Q.D. synthesized the 35-40 mer synthetic oligonucleotides. M.B., J.Z. and J.F. wrote the manuscript.

Highlights d SgrS and RyhB can regulate transcripts as soon as their binding sites exit the RNAP d Co-transcriptional regulation is promoted by Rho-dependent termination d Binding to the target mRNA is the primary determinant of hierarchical regulation d Co-and post-transcriptional level regulation all contribute to overall efficiency

N 6 -methyladenosine (m 6 A) is the most prevalent modified base in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRNA). Although candidate sites for m 6 A modification are identified at the transcriptomic level, site-specific quantification methods for m 6 A 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 m 6 A 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 m 6 A fraction at specific loci, expanding the current toolkit for studying RNA modifications.