Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

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.

Defining essential elements and genetic interactions of the yeast Lsm2–8 ring and demonstration that essentiality of Lsm2–8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24

“…Alternatively, the mutational effects on binding to an 8-mer U6 3 ′ RNA oligonucleotide in vitro are not directly applicable to binding to genuine U6 snRNA. For example, the RNA ligand used in the in vitro experiments has a "free" 3 ′ -OH, 2 ′ -OH end and does not have a 3 ′ -terminal monophosphate group found on native yeast U6 snRNA (Lund and Dahlberg 1992;Didychuk et al 2017). Yet another possibility is that mutational effects on U6 3 ′ -RNA binding are buffered by other interactions of the Lsm2-8 ring with the U6 snRNP, i.e., with Prp24 or other segments of the U6 snRNA.…”

“…Overexpression Prp24 suppresses lethality of usb1Δ S. cerevisiae Usb1 is an essential endoribonuclease/2 ′ ,3 ′ -cyclic phosphodiesterase enzyme that processes the 3 ′ end of the U6 transcript to generate the 3 ′ -monophosphate terminus found on the mature yeast U6 snRNA (Mroczek et al 2012;Didychuk et al 2017). Depletion of Usb1 results in destabilization of U6 snRNA in vivo.…”

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

“…In vitro transcription with T7 RNA polymerase was used to synthesize mature S. pombe U6 In order to generate a pentauridylate RNA with a 3′ cyclic phosphate for co-crystallization with S. pombe Lsm2-8, an RNA oligo with sequence 5′-UUUUUA-3′ was purchased from Integrated DNA Technologies (IDT) and treated with human Usb1, which rapidly removes 3′ adenosine residues from oligoU tracts and leaves a 3′ cyclic phosphate that is not subjected to ring opening as with S. cerevisiae Usb1 (35,42). Human Usb1 was prepared exactly as described elsewhere (42).…”

“…The C-terminal histidine residue of Lsm8 that stacks with the terminal syn uracil nucleobase is also strongly conserved in almost all eukaryotes ( Supplementary Data File 4). It is therefore clear that the C-terminus of Lsm8 and nearby residues are important for specifically recognizing post-transcriptionally modified U6 snRNA (25,35).…”

“…cerevisiae Lsm2-8 structure has been determined (25,26) but is unlikely to be representative of most eukaryotes due to significant differences in Lsm8 protein sequence and U6 snRNA posttranscriptional modification (35). Budding yeasts have Usb1 enzymes that open the 2′,3′ cyclic phosphate to produce a 3′ phosphate, and their Lsm8 proteins have evolved a unique Cterminal extension in order to interact with this 3′ phosphate through electrostatic interactions (25,35). In contrast, the Lsm8 proteins of most eukaryotes, from S. pombe to humans, do not have this Lsm8 extension and have U6 snRNAs with terminal 2′,3′ cyclic phosphates.…”