Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines

Abstract

Spliceosomes are multi-megadalton RNA-protein complexes responsible for the faithful removal of non-coding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition and versatile structural dynamics. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues towards studying the mechanisms of biomolecular machines isolated directly from complex biological specimens such as cell extracts. Here we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy in exemplary single molecule pull-down FRET (SiMPull-FRET) studies of the yeast spliceosome and discuss the broad application potential of this technique.

Single-molecule pull-down for investigating protein–nucleic acid interactions

“…Hoskins et al revealed the order of spliceosome assembly during pre-mRNA maturation in cell extract via single-molecule multi-color fluorescence [22,23]. Single-molecule pull-down FRET allowed Nils et al to visualize in real time the splicing of pre-mRNA by the spliceosome [24,25]. Lee et al used a single-molecule co-immunoprecipitation approach to investigate weak interactions between different proteins [26,27].…”

Defective Ribonucleoproteins, Mistakes in RNA Processing, and Diseases

“…Single-molecule pull down (SiMPull) and its derivatives SNAP-SiMPull and SiMPull-FRET fuse conventional pull-down assays with single-molecule fluorescence microscopy or single-molecule fluorescence resonance energy transfer (FRET) to allow direct visualization of individual protein complexes. − Using these methods, studies have demonstrated the ability to detect single molecules of U1 snRNP and to investigate the dynamic nature of the mammalian target of rapamycin (mTOR)-containing complex (mTORC) dimerization and the selectivity of rapamycin on mTORC stoichiometry. , The ability to understand the dynamics of mTORC1 and mTORC2 is promising as mTOR is a regulatory protein associated with many cellular pathways like mRNA translation, ribosome biogenesis, autophagy, and metabolism. Additionally, mTOR deregulation has been linked to several types of cancer .…”

“…Additionally, mTOR deregulation has been linked to several types of cancer . SiMPull-FRET was utilized to investigate pre-mRNA splicing dynamics and revealed that the spliceosome achieves an accurate initial splice through a biased Brownian ratcheting mechanism. , Because deregulated alternative splicing has been identified in ALS, SMA, AD, and even cancer, the capability to monitor spliceosome dynamics is a strong first step toward understanding the beginning of splicing defects and their proliferation through the cell.…”

Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World

“…On the basis of these results, the authors concluded that spliceosome activation proceeds via a Brownian ratchet mechanism, in which ATP hydrolysis by Prp2 unlocks reversible fluctuations in the conformation of the pre-mRNA (and likely other components of the spliceosome), with Cwc25 acting as a pawl that facilitates the first step of splicing by trapping the conformation in which the BP and 5′SS are in close proximity. 143,260…”

“…Further addition of Cwc25 yielded a population of complexes that remained stably in the high-FRET state, which was confirmed by additional experiments to result from C complexes that have completed the first chemical reaction of splicing. On the basis of these results, the authors concluded that spliceosome activation proceeds via a Brownian ratchet mechanism, in which ATP hydrolysis by Prp2 unlocks reversible fluctuations in the conformation of the pre-mRNA (and likely other components of the spliceosome), with Cwc25 acting as a pawl that facilitates the first step of splicing by trapping the conformation in which the BP and 5′SS are in close proximity. , …”