Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures

Abstract

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

“…The ability of m 1 Ψ and Ψ to change the decoding behavior of the ribosome while only modestly altering amino acid addition and termination has several implications. Given the emerging evidence that Ψ is commonly included into mRNA at increased levels under cellular stress conditions, these findings support the possibility that Ψ-derived modifications can provide the cells with a way to transiently increase the diversity of the proteome under stress to increase fitness (15, 30, 73, 74). Furthermore, this could potentially be advantageous for mRNA vaccines relative to traditional vaccine platforms; greater antigen diversity might provide broader protection against circulating virus populations than single-strain vaccine formulations, similar to the increased efficacy of multivalent vaccines.…”

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

“…Although our data do not report on the ability of the modifications that we uncover to control gene expression or identify the number of mRNAs that they are in, they do suggest that there will be consequences for translation when these modifications are encountered by the ribosome. It is also important to note that the levels and distributions of mRNA modifications (enzymatic and RNA damage) can change significantly in response to different environmental conditions, so the low levels of modification that we measure in healthy, rapidly growing yeast have the potential to significantly increase under stress 20,28,116,125 . The three modifications we investigated alter translation differently depending on their location within a codon.…”

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex)

“…For example, the Gm34 modification in tRNA Phe mediated by the Trm7-Trm734 complex [ 63 , 66 , 67 , 68 ] requires Cm32 modification by the Trm7-Trm732 complex [ 68 ] and the m 1 G37 modification by Trm5 [ 69 , 70 , 71 ]. In this view point, the Ψ13 modification, which is synthesized by Pus7 [ 72 , 73 ], may have an effect on the methylation by Trm11-Trm112 because various tRNAs containing Ψ13 do not possess the m 2 G10 modification. In addition, the anticodon-loop region contains various hyper modifications ( Figure 2 and Reference [ 1 ]).…”