RNA biochemistry. Determination of in vivo target search kinetics of regulatory noncoding RNA

Abstract

Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes.

Figure 1. Super-resolution imaging and analysis

(A) 3D Super-resolution images of SgrS and ptsG mRNA labeled by smFISH projected in 2D planes. (B) Diffraction-limited fluorescent images of SgrS and ptsG mRNA. Cell boundaries imaged by DIC in (A) and (B) are depicted by white solid lines. (C) Examples of clustering analysis with comparison of raw data (left) and clustered data (right). (D) Comparison of average RNA copy number per cell measured by super-resolution imaging and qPCR. (E) Kinetic scheme of SgrS-induced ptsG mRNA degradation. Kinetic steps are described in the main text. [p], [S] and [Sp] are the concentrations of ptsG mRNA, SgrS and their complex, respectively, in the mass-action equations.

Figure 2. Colocalization analysis of SgrS-ptsG complex

(A) Example of colocalization under various conditions. (B) Quantification of colocalized fraction of ptsG mRNA in cases (ii), (iii) (at 10 minutes post-SgrS induction) and (iv) (at 10 minutes post-SgrS induction). Error bars are standard deviations from three to eight images. (C) Time-course changes in the fraction of colocalized ptsG mRNA with SgrS. Error bars are standard errors from 200–600 cells from two independent measurements.

Figure 3. Estimation of kinetic parameters

(A) Modeling of time-course changes of SgrS, ptsG mRNA and SgrS-ptsG complexes. Average copy numbers per cell are plotted as a function of time. Rate constants and weighted R² for modeling are reported in Tables S4 and S5. (B) Extraction of K_D for SgrS-mRNA complex formation. The ratio of mRNA in complex with SgrS to free mRNA is plotted against average SgrS copy number and the slope of the linear fitting reports 1/K_D. Error bars in (A) and (B) report standard errors from 200–600 cells from two independent measurements.