RNA modifications and Prp24 coordinate Lsm2–8 binding dynamics during S. cerevisiae U6 snRNP assembly

Figure 1. Fluorophore-labeled yeast Lsm2–8 binds 3′-end modified RNAs.A, cartoon structure of Lsm2–8fSNAP_DY549. The SNAP tag is attached to the C terminus of Lsm8 via a flexible linker. B, crystal structure of the yeast U6 snRNP. U6 snRNA is shown in dark gray. Prp24 is depicted in a surface representation in light gray and yellow-orange colors. The SNFFL-box domain of Prp24 is shown in red, bound to the Lsm2–8 complex. The flexible linker connecting this domain to rest of Prp24 could not be modeled based on the crystallographic data and is represented as red dashes. The Lsm2–8 ring is shown in surface representation in blue–green colors with Lsm8 in dark blue (Protein Data Bank ID: 5VSU). C, EMSA analysis of Lsm2–8fSNAP and Lsm2–8fSNAP_DY549 binding ability relative to WT protein to U6 RNAs. D, fluorescence polarization binding data comparing Lsm2–8WT and Lsm2–8fSNAP binding to U6 RNA fragments (nt 95–112 or −113) with either diol or 3′ phosphate-end modifications. Each point represents the average of three technical replicates, and bars represent ±SD. snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein.

Figure 2. U6 RNA 3′-end modification impacts Lsm2–8fsnap binding kinetics. A, schematic representation of the CoSMoS assay for monitoring Lsm2–8fSNAP DY549 binding to immobilized, Cy5-labeled U6 RNAs. B, left, representative micrographs showing individual U6 snRNA molecules tethered to the slide surface in a field of view (FOV). Right, zoom-in of a portion of the FOV showing individual U6 RNA molecules (red boxes on the left) and colocalized Lsm2–8fSNAP molecules (bright spots inside the green boxes on the right). C, chemical structures of the RNAs used in the CoSMoS assays highlighting the structure of the RNA 3′ ends. D, representative fluorescence trajectories showing Lsm2–8fSNAP DY549 binding events to the RNAs. E–G, rastergrams illustrating the binding of Lsm2–8 to 90 different molecules of U6 1–113-OH (E), U6 1–112-P (F), and U6 1–112-OH (G), sorted based on the time of first Lsm2–8fSNAP DY549 binding event detection. H, average fraction of time U6 molecules spend colocalized with Lsm2–8fSNAP DY549. Bar heights represent the average from three technical replicates (dots), and bars represent ±SD. I and J, violin plots representing the dwell time distributions for unbound (I) and bound…

Figure 3. Prp24 enhances Lsm2–8fSNAP binding kinetics to unmodified RNAs.A, schematic representation of the CoSMoS assay for monitoring Lsm2–8fSNAP DY549 binding in the presence of unlabeled Prp24. B, representative fluorescence trajectories showing single Lsm2–8fSNAP DY549 (top traces) or double (bottom traces) to RNAs in the presence of Prp24. C–E, rastergrams illustrating the binding of Lsm2–8 to 90 different molecules of U6 1–113-OH (C), U6 1–112-P (D), and U6 1–112-OH (E), sorted based on the time of first Lsm2–8fSNAP DY549 binding event detection. F, average fraction of time U6 molecules spend colocalized with Lsm2–8fSNAP DY549 in the presence of Prp24. Bar heights represent the average from three technical replicates (dots), and bars represent ±SD. Data for the absence of Prp24 are replicated from Figure 2H to facilitate comparison. G and H, violin plots representing the dwell time distributions for unbound (G) and bound (H) states across three different RNAs. Each violin plot is overlaid with a box plot that indicates the median (horizontal line), interquartile range (IQR, box), and whiskers extending to 1.5 times the IQR. The numbers above each violin plot denote the number of…

Figure 4. The Prp24 SNFFL box is required for recruitment of Lsm2–8fSNAP to unmodified U6 RNAs.A, EMSA analysis of Lsm2–8–Prp24–U6 complex formation using EMSA using Prp24 containing (lanes 3 and 4) or lacking (lanes 6 and 7) the SNFFL-box domain. The concentrations of Cy5-labeled U6 oligos and Lsm2–8 were maintained at a constant 2 nM and 100 nM, respectively. B, average fractions of time U6 RNAs are simultaneously bound by two Lsm2–8fSNAP DY549 complexes. C, chemical structures of the U6 1–113-OH and 1–113 + 5U-OH RNAs, highlighting the RNA 3′ ends. D, relative numbers of Lsm2–8fSNAP DY549 binding events per U6 RNA in the presence or absence of Prp24 and Prp24ΔSNFFL. In B and D, bar heights represent the average from three technical replicates (dots), and bars represent ±SD.

Figure 5. U6 RNA 3′-end modification permits an alternate assembly route for the U6 snRNP. The unmodified U6 snRNA depends on interaction with Prp24 for Lsm2–8 recruitment, whereas U6 modification (3′ phosphate modification is denoted by the black star) permits Prp24-dependent and -independent Lsm2–8 recruitment. snRNP, small nuclear ribonucleoprotein.