前mRNA加工因子31(PRPF31)的突变,剪接体三snRNP复合物的核心蛋白,导致常染色体显性视网膜色素变性(adRP)。它仍然是一个谜,为什么广泛表达的三-snRNP蛋白的突变导致视网膜特异性疾病,到目前为止,剪接因子相关RP的潜在机制尚不清楚。
我们使用诱导多能干细胞(iPSC)技术从四名患有严重和非常严重PRPF31-adRP的患者中产生视网膜类器官和RPE模型,未受影响的个体和CRISPR/Cas9等基因对照。
为了全面评估PRPF31突变的影响,进行视网膜类器官和RPE细胞的定量蛋白质组学分析,显示RNA剪接,自噬和溶酶体,未折叠蛋白反应(UPR)和视觉周期相关通路受到显著影响。引人注目的是,患者来源的RPE和视网膜细胞的特征在于存在大量含有突变型PRPF31和错误折叠的细胞质聚集体,泛素结合蛋白,包括关键的视觉周期和其他RP连接的三snRNP蛋白,随着时间的推移而逐步积累。突变型PRPF31变体未整合到剪接复合物中,但是PRPF31野生型水平的降低导致PRPF31患者视网膜细胞Cajal体内的tri-snRNP装配缺陷,核斑点的形态改变和活性剪接体的形成减少,导致整体剪接失调。此外,受损的废物处理机制进一步加剧了聚集体的形成,并通过使用雷帕霉素激活自噬途径来靶向这些减少的细胞质聚集物,提高细胞存活率。
我们的数据表明,是渐进式的聚集使废物处理机械负担过重,而不是直接的PRPF31引发的错误拼接,因此,从不溶性细胞质聚集体中缓解RPE细胞提出了一种新的治疗策略,可以与基因治疗研究相结合,以完全恢复PRPF31-adRP患者的RPE和视网膜细胞功能。
Mutations in pre-mRNA processing factor 31 (
PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood.
We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe
PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control.
To fully assess the impacts of
PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant
PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant
PRPF31 variant was not incorporated into splicing complexes, but reduction of
PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival.
Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.