Stegemann Franziska, Marcus Erin, Neupert Savanah, Ostrowski Sarah, Mathews David H, Phizicky Eric M
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York 14642, USA.
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York 14642, USA
RNA. 2025 Mar 18;31(4):566-584. doi: 10.1261/rna.080315.124.
The pseudouridylase Pus1 catalyzes pseudouridine (Ψ) formation at multiple uridine residues in tRNAs, and in some snRNAs and mRNAs. Although Pus1 is highly conserved, and mutations are associated with human disease, little is known about eukaryotic Pus1 biology. Here, we show that Δ mutants are temperature sensitive due to decay of tRNA, as tRNA levels are reduced, and its overexpression suppresses the defect. We show that tRNA is degraded by the 5'-3' exonuclease Dhp1 (ortholog of Rat1), as each of four spontaneous Δ suppressors had mutations and restored tRNA levels, and two suppressors that also restored tRNA levels had mutations in ( ortholog), predicted to inhibit Dhp1. We show that Pus1 modifies U, U, and U of tRNA, raising the question about how these modifications prevent decay. Our results suggest that Dhp1 targets unspliced pre-tRNA, as a Δ strain in which the only copy of tRNA has no intron [Δ] is temperature resistant and undergoes no detectable decay, and the corresponding Δ strain accumulates unspliced pre-tRNA Moreover, the predicted exon-intron structure of pre-tRNA differs from the canonical bulge-helix-loop structure compatible with tRNA splicing, and a Δ strain with intron mutations predicted to improve exon-intron structure is temperature resistant and undergoes little decay. These results suggest that decay of tRNA by Dhp1 in Δ strains occurs at the level of unspliced pre-tRNA, implying a substantial role for one or more of the Ψ residues in stabilizing the pre-tRNA structure for splicing.
假尿苷酰化酶Pus1催化在tRNA以及一些snRNA和mRNA的多个尿苷残基处形成假尿苷（Ψ）。尽管Pus1高度保守，且其突变与人类疾病相关，但对真核生物Pus1生物学的了解却很少。在此，我们表明Δ突变体对温度敏感是由于tRNA的降解，因为tRNA水平降低，而其过表达可抑制该缺陷。我们表明tRNA被5'-3'核酸外切酶Dhp1（大鼠Rat1的直系同源物）降解，因为四个自发的Δ抑制子中的每一个都有突变并恢复了tRNA水平，并且两个也恢复了tRNA水平的抑制子在（直系同源物）中有突变，预计会抑制Dhp1。我们表明Pus1修饰tRNA的U、U和U，这就提出了这些修饰如何防止降解的问题。我们的结果表明Dhp1靶向未剪接的前体tRNA，因为其中tRNA的唯一拷贝没有内含子的Δ菌株[Δ]对温度有抗性且未发生可检测到的降解，并且相应的Δ菌株积累未剪接的前体tRNA。此外，前体tRNA的预测外显子-内含子结构不同于与tRNA剪接兼容的典型凸起-螺旋-环结构，并且具有预计可改善外显子-内含子结构的内含子突变的Δ菌株对温度有抗性且几乎不发生降解。这些结果表明在Δ菌株中Dhp1对tRNA的降解发生在未剪接的前体tRNA水平，这意味着一个或多个Ψ残基在稳定前体tRNA剪接结构中起重要作用。
