Function and Dynamics of the LSm2-8 Protein Ring during Spliceosome Activation

Abstract

LSm2-8, a heteroheptameric protein ring, decorates the 3′ end of the U6 snRNA and is a stable component of U6 snRNP, U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and the spliceosome B complex. However, it is released during spliceosome activation, during which the active site for pre-mRNA splicing is created. While Lsm2-8 is known to stabilize the U6 snRNA in the cell, any potential function during spliceosome assembly or activation is unclear. Here, we characterize Lsm2-8 ejection during spliceosome activation using Colocalization Single Molecule Spectroscopy (CoSMoS). Our CoSMoS data reveals that LSm ring leaves after both release of U4 snRNP and arrival of Nine Teen Complex (NTC) on pre-mRNA. This indicates that a conformational change must occur within the NTC after spliceosome binding since available cryo-EM structures indicate that Lsm2-8 and NTC binding are mutually exclusive with one another. Thus, our data provides evidence for an intermediate spliceosome complex between B and Bact that has not been yet observed by cryo-EM. This unexpected late release of the Lsm2-8 ring also suggests an extended role during spliceosome activation. Based on sequence homology to bacterial Hfq proteins, it is possible that Lsm2-8 may be promoting formation of a RNA duplex between the U2 and U6 snRNAs. Using in vitro annealing assay, we observed that LSm ring forms a ternary complex in the presence U6 and U2 RNAs, which is dependent on base pairing. These results indicate that LSm ring may help in stabilizing U2/U6 helix II during activation and is retained on spliceosome until U4 unwinding is complete. After U4 is unwound from U6, LSm is replaced by NTC components that takeover stabilization of helix II during later stages of activation and splicing.