yeast U6 3' RACE. Schizosaccharomyces pombe

Mpn1 proteins are evolutionarily conserved exonucleases that modify spliceosomal U6 small nuclear RNAs (snRNAs) post-transcriptionally. Mutations in the human MPN1 gene are associated to the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Mpn1 deficiency leads to aberrant U6 3’ end processing and accelerated U6 decay through unknown molecular mechanisms. Here we show that in mpn1Δ fission yeast cells U6 is barely bound by the protective Lsm2-8 complex, undergoes extensive oligoadenylation and is degraded by the nuclear RNA exonuclease Rrp6 independently of the poly(A) polymerase Cid14/Trf4. Mpn1 processes U6 in a spliceosome-dependent manner, as mutant U6 molecules that fail to join the spliceosome are not substrates for Mpn1. Moreover, human U6atac, the U6-like snRNA of the minor spliceosome, is a novel substrate for hMpn1. We unveil mechanistic details of a new U6 degradation pathway and further corroborate the notion that inefficient canonical and minor pre-mRNA splicing promotes PN. Overall design: the 3' termini of U6 or tagged-U6 species from the indicated mutant cells were compared to wt yeast strain

Mpn1蛋白是一类进化保守的核酸外切酶，可对剪接体相关的U6小核RNA（small nuclear RNAs，snRNAs）进行转录后修饰。人类MPN1基因的突变与遗传性皮肤病Clericuzio型中性粒细胞减少性皮肤异色症（Clericuzio-type poikiloderma with neutropenia，PN）密切相关。Mpn1缺陷会导致U6的3'端加工异常以及U6降解加速，但其具体分子机制此前尚未明确。本研究发现，在mpn1Δ裂殖酵母细胞中，保护性Lsm2-8复合物几乎无法结合U6；该RNA会发生广泛的寡腺苷酸化，并由核RNA核酸外切酶Rrp6介导降解，且该过程不依赖于poly(A)聚合酶（poly(A) polymerase）Cid14/Trf4。Mpn1对U6的加工依赖于剪接体，无法整合进入剪接体的突变型U6分子并非Mpn1的作用底物。此外，人类次要剪接体（minor spliceosome）的U6样snRNA U6atac是hMpn1的新型作用底物。本研究揭示了一条全新的U6降解通路的分子机制细节，并进一步证实了经典剪接与次要剪接的前体mRNA（pre-mRNA）剪接效率低下会促进PN发生这一观点。实验设计：将指定突变体细胞中U6或带标签U6分子的3'端与野生型酵母菌株进行对比分析。