Butcher, S. E. | Search bioRxiv

bioRxiv2025

The structure, folding kinetics and dynamics of long poly(UG) RNA

Long poly(UG) or "pUG" dinucleotide repeats are abundant in eukaryotic transcriptomes. Thousands of human genes have pUGs longer than 24 repeats, including the cancer-associated lncRNA NEAT1. In C. elegans, enzymatic addition of long pUGs to RNA 3' ends (pUG tails) marks RNAs as vectors of gene silencing. Gene silencing requires at least one pUG fold, a left-handed quadruplex structure that incorporates 12 repeats, but longer pUG tails are more active. Here, we investigate the structure, folding kinetics and dynamics of long pUG RNAs. RNAs with 24 or more repeats slowly form compact, double pUG folds. The forward rate of pUG fold formation in vitro is length-dependent with a half-life (t1/2) of 13 minutes, while the unfolding rate is very slow (t1/2 [~]5 days). Long pUG RNAs display biphasic dynamics with an additional, faster unfolding phase (t1/2 [~]30 min). The amplitude of the faster phase indicates partial unfolding. From these data we propose a dynamic model for segmental register exchange and double pUG fold formation in long pUG RNAs. These data broaden our understanding of the structure and dynamics of long pUG RNAs and have implications for understanding the roles of pUG folds in biology and disease.

biochemistry↗

bioRxiv2020

Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.

biochemistry↗