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

Abstract

The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.

The life of U6 small nuclear RNA, from cradle to grave

“…As observed in the U4/U6.U5 tri-snRNP structure (Agafonov et al 2016;Nguyen et al 2016;Wan et al 2016b), the telestem is unwound when U6 is paired to U4 snRNA. However, in vitro data suggest the telestem can form transiently in free U4/U6 RNA (Brow and Vidaver 1995;Rodgers et al 2016). During spliceosome activation, U4 is unwound from U4/U6 by the helicase Brr2 (Laggerbauer et al 1998;Raghunathan and Guthrie 1998a), U6 base pairs with U2 (Madhani and Guthrie 1992), and the U6 ISL reforms (Fig.…”

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

“…Rodgers et al used smFRET in combination with biochemical and genetic assays to study U6 conformation when it is paired to U4 [21]. The authors designed a U6 snRNA smFRET construct that could report on potential intramolecular basepairing, leading towards telestem formation (Fig.…”

“…6B). In addition to FRET distributions, Rodgers et al also analyzed the kinetics of the conformational switching [21]. This was achieved by measuring the dwell times, τ, for U6 in each FRET state followed by fitting the observed distribution of dwell times to a kinetic model using maximum likelihood methods (Fig.…”

“…U4/U6 snRNA dynamics analyzed by smFRET by Rodgers et al [21]. (A) 2D representation of the proposed structural states of U6 while basepaired to U4 snRNA.…”

“…Calculated lifetimes of the FRET states were obtained by fitting these distributions to maximum likelihood functions with either one (D) or two (E) kinetic parameters. The figure shown was adapted from reference [21] and used with permission.…”