The brace for a growing scaffold: Mss116 protein promotes RNA folding by stabilizing an early assembly intermediate

Abstract

The ai5γ group II intron requires a protein cofactor to facilitate native folding in the cell. Yeast protein Mss116 greatly accelerates intron folding under near-physiological conditions both in vivo and in vitro. Although the effect of Mss116 on the kinetics of ai5γ ribozyme folding and catalysis has been extensively studied, the precise structural role and interaction sites of Mss116 have been elusive. Using Nucleotide Analog Interference Mapping to study the folding of splicing precursor constructs, we have identified specific intron functional groups that participate in Mss116-facilitated folding and we have determined their role in the folding mechanism. The data indicate that Mss116 stabilizes an early, obligate folding intermediate within intron domain 1, thereby laying the foundation for productive folding to the native state. In addition, the data reveal an important role for the IBS2 exon sequence and for the terminus of domain 6, during the folding of self-splicing group IIB intron constructs.

Fig. 1

(a) A general schematic of the NAIM experiment, in which the selection step is RNA folding in the presence or in the absence of Mss116. (b) Nucleotide analog phosphorothioates used in the NAIM experiment to identify functional groups important for protein-assisted group II intron folding. (c) A schematic of expected NAIM effects consistent with the destabilizing or stabilizing function of the protein.

Fig. 1

(a) A general schematic of the NAIM experiment, in which the selection step is RNA folding in the presence or in the absence of Mss116. (b) Nucleotide analog phosphorothioates used in the NAIM experiment to identify functional groups important for protein-assisted group II intron folding. (c) A schematic of expected NAIM effects consistent with the destabilizing or stabilizing function of the protein.

Fig. 2

Representative native gels showing the formation of the compact species (NAIM selection step) in the presence and in the absence of Mss116 by the SE RNA (a) and SEΔ2Δ4 RNA (b). Both RNAs were transcribed in the presence of 2,6-DAP.

Fig. 3

(Top) Representative sequencing gels showing functional groups in the 5′-exon, which interfere with compaction. On each gel, lane U corresponds to unfolded species, I corresponds to extended intermediate species, and C corresponds to compact species. Symbols on the left of each gel indicate nucleotide positions that interfere with the formation of the compact (red) or intermediate (green) species. (Bottom) A bar graph showing average value and standard deviation for interference effects observed in the 5′-exon upon formation of the compact and intermediate states in the absence of Mss116 and compact state in the presence of Mss116.

Fig. 4

Functional groups important for compaction of the SEΔ2Δ4 RNA in the absence (a) and in the presence (b) Mss116 and ATP under near-physiological conditions. Insets, functional groups in full-length domains 2 and 4, which important for compaction of SEΔ4 and SE RNAs, respectively, in the absence (a) and in the presence (b) of Mss116 and ATP.

Fig. 4

Functional groups important for compaction of the SEΔ2Δ4 RNA in the absence (a) and in the presence (b) Mss116 and ATP under near-physiological conditions. Insets, functional groups in full-length domains 2 and 4, which important for compaction of SEΔ4 and SE RNAs, respectively, in the absence (a) and in the presence (b) of Mss116 and ATP.

Fig. 5

(a) Representative sequencing gels showing functional groups in the 5′-end of D1, which interfere with intron compaction. On each gel, lane U corresponds to unfolded species, I corresponds to extended intermediate species, and C corresponds to compact species. Symbols on the left of each gel indicate nucleotide positions that interfere with the formation of the compact (red) or intermediate (green) species. Respective substructures in D1 are denoted in gray on the left. (b) A bar graph showing average value and standard deviation for interference effects observed in the 5′-end of D1 upon formation of the compact state in the absence and in the presence of Mss116.

Fig. 6

(Top) Representative sequencing gels showing functional groups interfering with compaction at adenosine (a) and guanosine (b) positions in the κ–ζ element. On each gel, lane U corresponds to unfolded species, I corresponds to extended intermediate species, and C corresponds to compact species. Symbols on the left of each gel indicate nucleotide positions that interfere with (filled symbols) or enhance (open symbols) the formation of the compact (red) or intermediate (green) species. Black arrows indicate positions where NAIM effects are increased in the presence of Mss116. Positions where NAIM effects appear only in the presence of Mss116 are denoted by blue arrows. Respective substructures in D1 are denoted in gray on the left. Bar graphs on the bottom (a) and on the right (b) show average value and standard deviation for interference and enhancement effects observed in the κ–ζ element upon formation of the compact state in the absence and in the presence of Mss116. (c) A schematic of functional groups exhibiting increased interference or enhancement effects in the presence of Mss116.

Fig. 6

(Top) Representative sequencing gels showing functional groups interfering with compaction at adenosine (a) and guanosine (b) positions in the κ–ζ element. On each gel, lane U corresponds to unfolded species, I corresponds to extended intermediate species, and C corresponds to compact species. Symbols on the left of each gel indicate nucleotide positions that interfere with (filled symbols) or enhance (open symbols) the formation of the compact (red) or intermediate (green) species. Black arrows indicate positions where NAIM effects are increased in the presence of Mss116. Positions where NAIM effects appear only in the presence of Mss116 are denoted by blue arrows. Respective substructures in D1 are denoted in gray on the left. Bar graphs on the bottom (a) and on the right (b) show average value and standard deviation for interference and enhancement effects observed in the κ–ζ element upon formation of the compact state in the absence and in the presence of Mss116. (c) A schematic of functional groups exhibiting increased interference or enhancement effects in the presence of Mss116.

Fig. 7

(Top) Representative sequencing gels showing functional groups in D2, D3, and D4, which affect the intron compaction. On each gel, lane U corresponds to unfolded species, I corresponds to extended intermediate species, and C corresponds to compact species. Symbols on the left of each gel indicate nucleotide positions that interfere with (filled symbols) or enhance (open symbols) the formation of the compact (red) or intermediate (green) species. (Bottom) A bar graph showing average value and standard deviation for interference effects observed in the D2, D3, and D4 upon formation of the compact state in the absence and in the presence of Mss116.

Fig. 8

Comparison of NAIM effects in D6 in the full-length (SE) and truncated (SEΔ2Δ4) RNAs. (Top) Representative sequencing gels showing functional groups in D6, which affect the intron compaction. On each gel, lane U corresponds to unfolded species, I corresponds to extended intermediate species, and C corresponds to compact species. Symbols on the left of each gel indicate nucleotide positions that interfere with the formation of the compact species. (Bottom) A bar graph showing average value and standard deviation for interference effects observed in D6 upon formation of the compact state in the absence and in the presence of Mss116 in the full-length and truncated self-splicing intron RNAs.

Fig. 9

(a) Scheme illustrating the mechanism of Mss116-promoted folding of the ai5γ group II intron. (b) Scheme of a hypothetical free energy diagram for the ai5γ intron folding in the presence of Mss116.