Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics

Abstract

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.

Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites

“…These FRET states were static (Figure S2A; Table S2), precluding direct analysis of substrate dynamics but still allowing assessment of substrate conformation in distinct spliceosomal subpopulations. Consistent with previous studies and thereby validating our approach (Blanco et al, 2015; Crawford et al, 2013; Krishnan et al, 2013), these increasing FRET states indicate stepwise juxtaposition of the branching reactants.…”

“…To test the possibility that Prp16 antagonizes splice site juxtaposition, we monitored splice site proximity by smFRET (Abelson et al, 2010; Blanco et al, 2015; Krishnan et al, 2013), which enables an assessment of substrate conformation in crude preparations with limiting amounts of substrate in multiple conformations. We assembled spliceosomes on UBC4 splicing substrates labeled with a fluorescent acceptor just upstream of the 5’ splice site and a fluorescent donor just downstream of the branch site (BS-labeled; Figure 2A; Table S1; Krishnan et al, 2013).…”

Methodologies for studying the spliceosome’s RNA dynamics with single-molecule FRET

“…7F). This result agrees well with data reported by Blanco et al as well as recent cryo-EM structures of activated yeast spliceosomes that were determined subsequently [10, 11, 13, 47]. In these NTC-containing structures, the branchsite and 5′ splice site are separated from one another by ∼50 Å [10, 11, 13].…”

“…An excellent review of HMM and software available for smFRET data analysis is given in Blanco and Walter [44]. The Walter lab has more recently developed a cluster-based analysis method, SiMCAn, with which they were able to quickly assign FRET states with improved accuracy [45, 47]. …”

“…In these NTC-containing structures, the branchsite and 5′ splice site are separated from one another by ∼50 Å [10, 11, 13]. These sequences only become juxtaposed following catalytic activation of the spliceosome by the Prp2 ATPase [47, 61-63]. …”