Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

Allison L Didychuk,Andrew T Delaitsch,Stefani E Lucarelli,Samuel E Butcher,David A Brow,William M Westler,Eric J Montemayor,Tucker J Carrocci,Aaron A Hoskins

doi:10.1038/s41467-017-00484-w

Abstract

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3′ phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2–8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.

Highlights

U6 small nuclear ribonucleoprotein biogenesis is essential for spliceosome assembly, but not well understood
Unlike the other small nuclear RNA (snRNA), U6 is synthesized by RNA polymerase III (Pol III) and, like other Pol III transcripts, its transcription is terminated stochastically when the polymerase encounters a stretch of adenines in the template strand (Fig. 1a)[2,3,4] with Saccharomyces cerevisiae requiring at least six sequential adenines in the template strand to terminate efficiently[5]
We demonstrate the importance of the identity of the 3′ end of U6 to small nuclear ribonucleoprotein (snRNP) formation and show how U6 RNA is involved in a series of protein-mediated handoffs prior to formation of the mature U6 snRNP

Summary

Introduction

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. We report structures of the U6 RNA processing enzyme Usb[1] from yeast and a substrate analog bound complex from humans. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp[1], Prp[24], Usb[1] and Lsm2–8) and anti-cooperative interactions between Prp[24] and Lhp[1]. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome. Splicing of precursor messenger RNA is an essential process in all eukaryotes and is catalyzed by the spliceosome. Loss-offunction mutations in human Usb[1] are associated with poikiloderma with neutropenia, a rare skin disease that is associated with loss of white blood cells[14]

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Conclusion