BUTCHER, S. E. | bioRxiv Science

bioRxiv2025

Sequence and ionic requirements of pUG fold quadruplexes

Poly(UG) or "pUG" repeat RNA can fold into a left-handed parallel quadruplex, the pUG fold. The pUG fold directs the amplification of RNAi in C. elegans, and pUG sequences are abundant in eukaryotic transcriptomes. Here, we report the sequence and ionic requirements for pUG folding. The pUG fold requires 12 guanosines but has an otherwise flexible sequence requirement. The uridines can be substituted with other nucleotides, with some sequence variants folding better than pUG RNA. The GA repeat sequence (GA)12 also forms a pUG-like fold, albeit with lower thermodynamic stability than (GU)12. The pUG fold can tolerate multiple deoxyribose substitutions but does not fold when the backbone is entirely deoxyribose. It has a high affinity and specificity for potassium ions (K1/2 = 6 mM) and does not fold in sodium or ammonium ions. Addition of 2 mM Mg2+ does not further stabilize the pUG fold, and the polyamines spermine and spermidine slightly decrease its stability. Finally, the pUG fold is sensitive to surrounding sequence context and complementary flanking sequences can stabilize pUG folds, while other sequences can interfere with folding. These data expand our understanding of RNA folding and provide a basis for understanding and predicting pUG folds.

biochemistry↗

bioRxiv2022

Identification of Transient Intermediates During Spliceosome Activation by Single Molecule Fluorescence Microscopy

Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA (snRNA) 3-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of yeast spliceosomes. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NineTeen Complex, NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both [Formula] and provide a kinetic framework for its formation and transformation during activation. Prior to [Formula] assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. Together our data suggest a Hfq-like function for Lsm2-8 in maintaining U2/U6 helix II integrity before it can be transferred to the NTC by transient formation of the [Formula] spliceosome. Significance StatementThe spliceosome active site is created de novo during activation and involves numerous conformational and compositional changes. Here, we define a kinetic pathway for yeast spliceosome activation using single molecule fluorescence that includes transient intermediates not previously identified. Real-time measurements allow us to uncover rapid, reversible sampling interactions of the NineTeen Complex (NTC) that may prevent its accumulation on defective spliceosomes. By analogy with bacterial Hfq, we propose that the homologous Lsm2-8 proteins stabilize U2/U6 helix II during activation before the helix is transferred to the NTC in a short-lived spliceosome containing both Lsm2-8 and the NTC. Our data demonstrate how single molecule studies of activation can reveal kinetically-competent intermediates and complement cryo-EM studies of stalled or inhibited complexes.

biochemistry↗