Conformational dynamics of stem II of the U2 snRNA

Abstract

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg 2+ and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg 2+ and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg 2+ may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg 2+ or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg 2+ toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg 2+ -dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.

A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex

“…smFRET experiments were carried out at room temperature on quartz microscope slides derivatized with a mixture of polyethylene glycol (PEG) and biotin-derivatized PEG and assembled into microfluidic flow chambers as previously described (18). Before use, slides were washed with 200 μl of 1× phosphate buffered saline (PBS) to remove unreacted PEG.…”

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

“…Rodgers et al utilized smFRET to study stem II conformation in vitro in the presence of Mg 2+ and Cus2, a protein known to genetically interact with stem II mutations in vivo [20, 55]. Since the stem II region is ∼100 nucleotides, a smFRET reporter RNA was designed from two smaller fragments that were labeled with fluorophores and joined via splinted ligation as described in Section 2 .…”

“…U2 snRNA dynamics analyzed by smFRET by Rodgers et al [20]. (A) Two mutually exclusive RNA structures can be adopted U2 stem II and are described by different FRET states.…”

“…(B) Histograms plotting E FRET for the smFRET reporter RNA in response to Mg 2+ (C) TODPs of E FRET for stem II molecular transitions. The structural transitions of the RNA increased and changed in E FRET in the presence of Mg 2+ The figure shown was adapted from reference [20] and used with permission.…”

Lights, camera, action! Capturing the spliceosome and pre‐mRNA splicing with single‐molecule fluorescence microscopy

“…How these two conformations interconvert has been unclear with various models proposed that include stabilization of stem IIc by the RNA‐binding protein Cus2 and unwinding of stem IIc by the DEAD‐box ATPase Prp5 . To study stem II conformational changes directly, Rodgers et al designed a model yeast U2 snRNA that allowed for real time observation of IIa/IIc interconversion by smFRET (Figure (a)) . The model RNA interconverted spontaneously between FRET states consistent with stem IIc formation (with high E FRET ) and a cluster of FRET values consistent with stem IIa formation (with low to mid E FRET ) in the absence of other factors.…”