6 research outputs found
Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics
The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines
Invited Review Meeting Report: SMART Timing-Principles of Single Molecule Techniques Course at the University of Michigan 2014
ABSTRACT: Four days after the announcement of the 2014 Nobe
The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies
Single Molecule Characterization of Pre-mRNA Dynamics throughout Spliceosome Assembly and Catalysis
Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics
The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines
Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing
The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns in the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used single molecule fluorescence resonance energy transfer to monitor the distance of the 5′ splice site (5′SS) and branchpoint (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately prior to the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5′SS and BP that accommodate chemistry. Addition of Cwc25 then strongly biases this equilibrium towards the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor “pawl”, a principle possibly widespread among the many helicase-driven RNPs